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Lipid phosphatases as regulators of immunological homeostasis Moody, Jennifer Lynn 2003

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Lipid Phosphatases as Regulators of Immunological Homeostasis  JENNIFER L Y N N MOODY I  B.Sc, York University, 1996  A THESIS [SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Biology, Genetics Graduate Program) We accept this thesis as conforming to the required standard:  THE UNIVERSITY OF BRITISH COLUMBIA December 2003 © Jennifer Lynn Moody, 2003  Abstract The objective of this thesis has been to investigate the results of combined deficiencies of the 5' inositol phosphatase SHIP and the 3' inositol phosphatase Pten in the murine immune and hematopoietic systems. SHIP and Pten act as negative regulators of the phosphatidylinositol 3-kinase (PI3K) pathway, by dephosphorylating the second messenger PIP to form PI(3,4)P and PI(4,5)P respectively. Their role in regulating a 3  2  2  common substrate while generating different products, suggests that Pten and SHIP may have both overlapping and distinct roles relating to cell signaling. The generation of mice deficient for these molecules previously revealed similarities and differences in phenotypes. These results represent the first examination of combined deficiencies of these molecules in an animal model system. The effects of combined deficiencies of these two molecules were examined in two ways. First, the generation and characterization of mice heterozygous for both Pten and SHIP revealed co-operativity between these molecules in the generation of autoimmune characteristics. Double heterozygotes showed increased immunoglobulin levels, higher autoantigen reactivity, and more severe immune-mediated kidney damage than did wild-type and singly-heterozygous controls. Additionally, further studies of heterozygous mice revealed heightened T cell responses to antigenic challenge in vivo, and increased cytokine production upon T cell stimulation in vitro. These results suggested the requirement for threshold levels of these phosphatases in the control of immune responses, and also illustrated that partial deficiencies in genes in the same pathway can play a role in the development of multigenic diseases such as autoimmunity. Secondly, the generation of Pten* 'SHIP' ' mice revealed a previously unappreciated role 1  1  for Pten in hematopoiesis, in that the phenotype of these mice manifested more severely than in SHIP  1  controls. Pten ~SHIP~'~ mice exhibited unique peripheral blood +I  abnormalities, altered functional behavior of committed progenitor cells, a more severe B cell developmental defect and a defect in multilineage repopulation of irradiated recipients, providing the first evidence that Pten and SHIP act cooperatively to maintain homeostasis in the hematopoietic system.  ii  Together, these results highlight the importance of regulation of the PI3K pathway in immune responses and hematopoiesis, and support cooperative roles for both Pten and SHIP in these processes.  iii  Table of Contents  Abstract  1 1  Table of Contents  '•  iv  List of Tables  vii  List of Figures  viii  List of Abbreviations  xi  Acknowledgements  xiii  Dedication  xiii  Thesis Format  xiv  Publications arising from this thesis  xiv  Chapter One: Introduction 1.1  The importance of lipids and lipid modification in cell signaling  1 1  Phosphatidylinositols as important signaling components of the membrane  1.2  1  The generation of 3-phosphoinositides  2  Biological effects ofPI3K activity  3  SHIP and PTEN as negative regulators of 3-phosphoinositides SHIP structure and expression  4 5  SHIP enzymatic activity and regulation PTEN structure and expression  6 7  PTEN enzymatic activity and regulation 1.3  The importance of PI3K signaling and regulation in the immune system The necessity of PI3K signaling in immune cells  8 9 10  The effects of over-expression of PI3K activity in vivo The in vivo role for SHIP in immune cells  11 12  The in vivo role of Pten in immune cells 1.4  Thesis goals  14 17  Chapter Two: Loss of a single allele of SHIP exacerbates the immunopathology of Pten heterozygous mice Author's contribution statement  18 18  2.1  Introduction  18  2.2  Materials and Methods  20  iv  2.3  Results and Discussion  22  Pteri' SHIP*' mice show augmented lymphadenopathy, splenomegaly, and a possible increase in thymic tumour frequency  22  Pteri' SHIP*' mice demonstrate severe kidney pathology  23  Elevated immunoglobulin levels in Pteri' SHIP*' mice  24  Increased anti-nuclear titres and reactivity against histone protein in Pteri' SHIP*' mice  25  Chapter Three: Combined heterozygosity for Pten and SHIP results in increased Tdependent response to antigenic challenge and increased cytokine production by CD4 T +  cells  28 Author's contribution statement  28  3.1  Introduction  28  3.2  Materials and Methods  32  3.3  Results  36  Pteri' SHIP*' mice demonstrate a significantly increased T-dependent antigenic response  36  Pten and SHIP protein levels are reduced in T cellsfromheterozygous and doubly heterozygous mice  36  Pteri' SHIP*' T cells do not show discernable increases in PIPj levels before or after stimulation with anti-CD3  37  Expanded CD4* T cells from Pten* 'SHIP*' mice show modest increases in Akt 1  phosphorylation  37  Pteri' SHIP*' T cells respond to anti-CD3 stimulation by producing greater amounts ofIL-4, IL-13 and IFN-y at the RNA and protein levels  38  Increased cytokine production is not attributable to increased survival or spontaneous altered polarization of the T cell population  39  Pteri' SHIP*' cells do not show differential phosphorylation ofSTAT6, 42I44MAPK P  3.4  or p38MAPK before or after stimulation with anti-CD3  40  Increased levels oflgGl and IgE but not IgG2a in young Pteri' SHIP*'' mice  41  Discussion  42  Chapter Four: The inositol phosphatases SHIP and Pten work co-operatively in maintaining hematopoietic homeostasis Author's contribution statement  47 47  v  4.1 Introduction  47  4.2 Materials and Methods  49  4.3 Results  51  Peripheral blood abnormalities and extramedullar hematopoiesis in Pteri' SHIP  1  mice  51  Pteri' SHIP marrow composition does not differ phenotypically from that of SHIP'' 1  marrow  53  Decreased clonogenic potential in the bone marrow, and increased liver CPU in Pteri'SHIP' mice  53  Pten heterozygosity confers increased sensitivity of SHIP' committed progenitors to GM-CSF  54  Reduction of immature bone marrow B cells in Pteri' SHIP' marrow  55  The stem cell compartment of Pteri 'SHIP' ' mice is not phenotypically altered and the 1  1  hematopoietic defects of Pteri' SHIP' mice are transplantable  56  Pteri ' SHIP' marrow cells are defective in their ability to carry out short-term 1  reconstitution of lethally-irradiated hosts  4.4 Discussion Chapter Five: Results summary and future directions  57  58 65  5.1 Results summary  65  5.2 Future Directions  67  References  72  vi  List of Tables  Table 1. Spleen and node weights are increased in Pten SHIP  mice at 24 weeks p. 98  Table 2. Pten SHIP spleens show increased numbers of B cells, T cells, +/  +/  macrophages and increased activation markers at 24 weeks  p. 98  Table 3. ANA titres of the studied cohort  p. 104  Table 4. Pten ' SHIP' mice have significant peripheral blood abnormalities  p. 125  +  Table 5. Increased WBC in Pten SHIP' mice is attributable to increased +I  neutrophils  p. 125  Table 6. FACS analysis of whole bone marrow reveals no differences between SHIP and Pten 'SHIP' mice 1  +/  p. 129  Table 7. Absolute number and percentage of Lin-Scal+c-kit cells in the bone marrow p. 135 Table 8. Recipients of Pten"' SHIP' ' cells are anemic and show decreased donor 1  reconstitution in the marrow  p. 136  vii  List of Figures Figure 1. The structure of phosphatidylinositol  p. 93  Figure 2. The interconversion of PI species is facilitated by the actions of kinases and phosphatases  p. 94  Figure 3. Cellular consequences of P I 3 K activation  p. 95  Figure 4. The structure of full length SHIP  p. 96  Figure 5. The structure of PTEN  p. 97  Figure 6. Severe kidney pathology in Pten '~SHIP +  +/  mice  p. 99  Figure 7. Electron microscopy reveals severity of kidney damage in Pten SHIP +,  kidney  +/  p. 100  Figure 8. Pten ' SHIP ~ mice have a greater than three fold increase in +  +/  circulating immunoglobulin levels Figure 9. Pten 'SHIP +/  p. 101  mice show increased circulating levels of most  +/  isotype subclasses  p. 102  Figure 10. Pten SHIP ~ mice have higher titres of anti-nuclear antibodies +/  +/  p. 103  Figure 11. PterP' SH1P '' mice have a higher reactivity against H I histone +  protein  p. 105  Figure 12. A simple schematic outlining T helper cell differentiation  p. 106  Figure 13. Pten SHIP '~ mice do not show significant differences in their +1  +  antigenic response to the T-independent antigen N P - F i c o l l Figure 14. Pten '~SHIP +  +/  p. 107  mice how an increased antigenic response to the  T-dependent antigen N P - K L H  p. 108  Figure 15. Reduced protein levels of Pten and SHIP in Pten SHIP +/  +/  T cells  Figure 16. Schematic of F R E T technique for PIP analysis Figure 17. Pten SHIP '~ T cells do not show significant increases in P I P +1  p. 110  +  3  levels  p. I l l  Figure 18. Pten 'SHIP ' +I  p. 109  +  T cells show modest increases in A k t phosphorylation  after stimulation with anti-CD3  p. 112  Figure 19. Increased cytokine R N A by Pten 'SHIP +  +/  Figure 20. Increased cytokine R N A by Pten 'SHIP +/  +/  T cells  p. 113  T cells  p. 114  viii  Figure 21. Increased cytokine protein by Pten*''SHIP* ' T cells  p. 115  1  Figure 22. Increased cytokine production cannot be attributed to increased survival by Pten*''SHIP*'' cells without stimulation  p. 116  Figure 23. Increased cytokine production cannot be attributed to increased survival by Pten*''SHIP*'' cells after stimulation  p. 117  Figure 24. Pten*' SHIP*' cells appear to make more IL-4 cytokine on a per cell basis  p. 118  Figure 25. Pten*''SHIP*'' cells do not show significantly increased levels of phosphorylated STAT6  p. 119  Figure 26. Pten*'SHIP*'' cells do not show increases in phosphorylated p42/44 MAPK before or after stimulation  p. 120  Figure 27. Pten*''SHIP*'' cells do not show increases in phosphorylated p38 MAPK before or after stimulation  p. 121  Figure 28. Young Pten*' SHIP*'' mice demonstrate significant increases in circulating IgGl, and IgE but not IgG2a  p. 122  Figure 29. Overview of hematopoiesis and the role of cytokines in lineage differentiation  p. 123  Figure 30. Pten* SHIP mice have comparable body weights to SHIP' mice  p. 124  Figure 31. Pten*'' SHIP' mice exhibit peripheral blood leukocytosis  p. 126  1  1  Figure 32. Pten*'SHIP' mice have splenomegaly and hepatomegaly at 4-5 weeks of age  p. 127  Figure 33. Extramedullary hematopoiesis in livers of 4-5 week old Pten*'SHIP mice  p. 128  7  Figure 34. Clonogenic assays for progenitor growth reveal differences between SHIP'' and Pten*'' SHIP' in marrow, liver, and peripheral blood  p. 130  Figure 35. Pten*'' SHIP' mice show increased sensitivity to low doses of GM-CSFover SHIP' littermates  p. 131  Figure 36. Pten*'SHIP' mice show subtle decreases over SHIP'' in the lymphoid compartment of the bone marrow  p. 132  Figure 37. Pten*'SHIP' mice show subtle decreases over SHIP' ' in the clonogenic 1  potential of pre-B progenitors  p. 133  Figure 38. Under-representation of mature B cells in the bone marrow of  ix  Pten 'SHIP mice  p. 134  +  Figure 39. Ability of PterC' SHIP' B M cells to repopulate irradiated recipients is compromised  p. 137  Figure 40. The proportion of donor derived B220 and Macl cells does not differ between recipients of SHIP' and PterP'SHIP' cells p. 138 +  +  x  List of Abbreviations Akt  oncogenefromAKR( mouse strain) thymoma  ANA ANOVA APC BAD  anti- nuclear antibody Analysis of variance antigen presenting cell Bcl-2 antagonist of apoptosis  Bcl-2 BCR BFU-E BSA Btk CRU CFA CFU ELISA EM ES cell FACS FAK FAS FASL FCS FITC FKHR-L FRET GAPDH, L32 GBM GEMM  B cell lymphoma-2 B-cell receptor Burst-forming unit- erythroid Bovine serum albumin Bruton's tyrosine kinase competitive repopulating unit Colony forming assay Colony forming unit Enzyme Linked Immunosorbant Assay electron microscopy embryonic stem cell Fluorescence Activated Cell Scanning/Sorting Focal adhesion kinase Fibroblast associated protein FAS ligand fetal calf serum Fluoresceine Isothiocyanate Forkhead Related transcription factor-1 Fluorescence Resonance Emission Tomography housekeeping genes used as loading controls glomerular basement membrane Colony arising from a progenitor capable of producing granulocyte,erythroid, macrophage and megakaryocyte cells Granulocyte-macrophage colony stimulating factor Colonies arising from progenitors capable of producing granulocytes and/or macrophages Golgin-160-related protein Glycogen Synthase Kinase-3 Hematopoietic stem cell Horseradish Peroxidase Interferon y Interleukin-12 Interleukin-3 Interleukin-2 Interleukin-3 Interleukin-4  GM-CSF GM/M/G Grpl GSK-3 HSC HRP IFNy IL-12 IL-13 IL-2 IL-3 IL-4  xi  IL-5 IL-6 IL-7 ILK  Interleukin-5 Interleukin-6 Interleukin-7 integrin linked kinase  Jak MAGI-3 MAPK min NP-Ficoll NP-KLH p27KIP, p21CIP PBS PDK-1 PDK-2 PE PEST  Janus kinase Membrane-Guanylate Kinase-3 Mitogen Activated Protein Kinase minute Nitrophenylacetyl-Ficoll Nitrophenylacetyl-Keyhole Limpet Hemocyanin cyclin dependent kinase inhibitors phosphate buffered saline Phosphoinostide Dependent Kinase-1 Phosphoinositide-Dependent Kinase-2 Phycoerytherin sequences enriched in Proline (P), glutamic acid (E), serine (S) and threonine (T) phosphatidylinositol phosphatidylinositol 3- kinase phosphatidylinositol 3-phosphate phosphatidylinositol(4,5) bisphosphate phosphatidylinositol(3,4) bisphosphate phosphatidylinositol (3,4,5) trisphosphate Phosphatase with tensin homology rotations per minute Stromal-Derived Factor-1 Standard Error of the Mean Steel factor SH2- containing inositol phosphatase Systemic Lupus Erythamatosus Signal Transducer and Activator of Transcription Tri-chloro acetic acid T-cell receptor 3,3',5,5' tetramethylbenzidine volts X-linked immunodeficiency protein 5-fluorouracil  PI PI3K PI3P PI(4,5)P PI(3,4)P PIP Pten/PTEN rpm SDF-1 SEM SF SHIP SLE STAT TCA TCR TMB V Xid 5-FU 2 2  3  Acknowledgements First and foremost I would like to thank Dr. Frank Jirik for his support, encouragement, optimism and his great sense of humor. Special thanks go to Cheryl Helgason for valuable advice throughout, and to Kamela Patel and Alex Gray for advice on PIP analysis. Many thanks go to all the members of the animal units in both Vancouver and Calgary, as their diligent work made possible the experiments of this thesis. Thank you to all members of the Jirik lab both past and present, especially Jackie Damen, Jim Peacock, Sue Hadjur, Linda Sandercock and Artee Luchman for the best support a PhD student could hope for scientifically, personally, (and in the ways of chocolate!). I would also like to thank my committee Dr. Elizabeth M . Simpson, Dr. Keith Humphries and Dr. Ann Rose for their advice along the way. Deepest thanks to Dr. Maria Sokolowski for early inspiration, Dr. John Heddle for his encouragement and faith in my abilities, and to Dr. Nina Josefowitz for showing me that I had wings. Finally I would like to acknowledge the support of my friends and family, especially my sisters. Special thoughts go to Brent for his never-ending encouragement and positive spirit. This work was supported by grants from the Canadian Institute for Health Research. Financially, I have been supported as a graduate student by University Graduate Fellowships from the University of British Columbia and by an Alberta Heritage Foundation for Medical Research (AHFMR) Studentship.  Dedication To Gerald and Mary Moody who gave me everything I needed to achieve this.  xiii  Thesis Format This thesis consists of five chapters. The first chapter provides an introduction to the PI3K signaling pathway and its negative regulators Pten and SHIP. This chapter also outlines the relevance of the PI3K pathway and these molecules in immune cells. Chapters 2, 3 and 4 are formatted as individual manuscripts that describe basic immunological and hematopoietic concepts, outline the hypothesis of the experiments, and present the results and discussion of the experiments of this thesis. Chapter 5 is a summary of the findings of this work as a whole, and deals with future directions that may be pursued in areas directly related to this work.  Publications arising from this thesis  Moody JL, Pereira CG, Magil A, Fritzler MJ, Jirik FR., 2003. Loss of a single allele of SHIP exacerbates the immunopathology of Pten heterozygous mice. Genes Immun. 2003 Jan;4(l):60-6. Moody, JL, Xu, L, Helgason, CD, Jirik, FR, 2003. Normal levels of both SHIP and Pten are required to maintain the hematopoietic system: Pten  +/  SHIP''' mice show anemia,  thrombocytopenia, leukocytosis, extramedullary hematopoiesis, and severely compromised reconstitution potential of progenitors, in revision. Moody, J.L and Jirik F.R Altered regulation of CD4 T-cell cytokine production and +  increased response to T-dependent antigens by Pten '~SHIP ' mice, 2003. submitted. +  +  xiv  Chapter One: Introduction 1.1  The importance of lipids and lipid modification in cell signaling  Phosphatidylinositols as important signaling components of the membrane  The contribution of phosphatidylinositol (PI) to cellular signaling was first alluded to in experiments by Hokin and Hokin in 1953 in which they observed the incorporation of P into the phospholipid fraction of brain tissue upon stimulation with 32  carbamlycholine or acetylcholine, indicating that phospholipid metabolism correlated with cellular stimulation (Hokin and Hokin, 1989). Later, the discovery that ligand induced cleavage of an inositol lipid could yield 1,2-diacylglycerol and a water soluble inositol lipid that regulated intracellular calcium levels (Michell and Allan, 1975) shed further light on the relationship between lipids and cellular signaling events. Much work since these early experiments have established the membrane as a critically dynamic location in the cell, one that houses not only lipid components but also receptors and a myriad of signaling molecules.  It is not surprising then, that the residence of  phosphatidylinositols in such an important location would eventually reveal a critical role for these lipid species in signaling events. PI comprises approximately 1% of the lipid species in the membrane and serves as a building block for all inositol lipids in eukaryotic cells. It is comprised of a myoinositol-1-phosphate linked via a phosphodiester bond to diacylglycerol that is embedded in the membrane via its fatty acid tails (Figure 1). The inositol ring has five free hydroxyl groups and up to three of these sites (3,4,5) have been observed to be phosphorylated in combinations that yield the 8 PI species documented in eukaryotic cells. These PI species are substrates for intracellular kinases and phosphatases that facilitate the inter-conversion of the different species within the cell membrane (Figure 2).  1  The generation of 3-phosphoinositides  Of the 8 PI species in eukaryotic cells, 4 species of 3-PIs have been identified: phosphatidylinositol 3-phosphate (POP), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P ), phosphatidylinositol 3,5-bisphosphate (PI(3,5)P ), and phosphatidylinositol 2  2  3,4,5-trisphosphate (PI(3,4,5)P ), hereafter referred to as PIP ). Under basal conditions 3  3  the most abundant species of these four is PI3P, and the concentrations of the other species rise abruptly after stimulation (Vanhaesebroeck et al., 2001). Phosphorylation on the 3-hydroxyl group of the inositol ring is facilitated by the activity of PI 3-kinase (PI3K), an enzyme belonging to a family that is conserved across species and expressed in all cell types. In mammalian cells there are three categories of PI3K family members that differ in their regulatory components but share a conserved catalytic domain. Class I PI3Ks are best characterized with respect to mammalian cells. They are primarily cytosolic and depending on the subunits involved and the mechanism of activation they can be further divided into subclasses 1A and IB. Class IA PI3Ks are activated by tyrosine-kinase receptors such as growth factor, cytokine and antigen receptors. They are heterodimeric enzymes that are comprised of a regulatory subunit (p85a, p85p\ or p55y) paired with a catalytic subunit (pi 10a, pi 10(3, or pi 105) and have wide tissue expression patterns. Class IB PI3Ks are activated by G-protein coupled receptors such as chemokine receptors, and are comprised of a 101 kDa regulatory protein (plOl) coupled with the pllOy catalytic subunit. This class of PI3K is restricted primarily to white blood cells. Both class IA and IB PI3Ks primarily utilize PI(4,5)P as an in vivo substrate to form PIP 2  3  (Vanhaesebroeck et al., 2001). The Class II and III PI3Ks are less well characterized. Three isoforms of Class II PI3Ks exist in mammalian cells, PI3K-C2a, (3 and y, and of these, a and (3 are ubiquitously expressed while y is expressed primarily in the liver. The in vivo regulation of the Class II PI3Ks is not well understood and while their preferred in vitro substrate is PI the in vivo substrate is not known (Vanhaesebroeck et al., 2001). Structurally, they contain a C-terminal C2 domain that allows binding to phospholipids, and thus are associated with the plasma membrane. Class III PI3Ks are homologues of the yeast-  2  vesicular-protein sorting protein (Vpsl5p) and are comprised of a Vpsl5p analogue complexed with a serine/threonine protein kinase pi50. Class III PI3Ks use PI as a substrate to generate PI3P. Their roles in membrane trafficking in yeast are well characterized (Odorizzi et al., 2000), and they are thought to be constitutively active and to play similar roles in mammalian cells. Biological effects of PI3K activity  PI3K is activated in many cell types by many different ligands and it is therefore capable of exerting diverse effects. Upon stimulation of a cell by a particular ligand, the activation of PI3K induces the phosphorylation of the PI species within the membrane. Molecules such as serine/threonine kinases, tyrosine kinases and exchange factors exist within the cytosol and can be induced upon stimulation to translocate to, and interact with the membrane via association with these reactive phospholipids.  The pleckstrin  homology domain (PH) facilitates these associations. There are over 100 molecules that contain PH domains and three dimensional structure analysis has revealed that the shape of domains can vary (reviewed in (Vanhaesebroeck et al., 2001). The specificity of PH domains also differs between molecules, for example, the domain of Btk displays a high affinity for PIP species (Salim et al., 1996), while that of TAPP1 shows a high affinity 3  for PI(3,4)P (Dowler et al., 2000). Translocation of these PH domain-containing 2  molecules to the membrane leads to their activation either by autophosphorylation or by phosphorylation by other molecules.  This activation facilitates the initiation of  downstream signaling cascades, which result in cellular processes such as growth, motility, survival, transcription, and translation (reviewed in (Cantley, 2002; Vanhaesebroeck et al., 2001)). Perhaps the best-characterized downstream effector of PI3K is Akt, (also known as protein kinase B (PKB)). Akt is activated via association with PI(3,4)P and PIP and 2  3  by its subsequent phosphorylation by the phosphoinositide-dependent kinase-1 (PDK-1) and a PDK-2 such as the integrin linked kinase (ILK). Activation of Akt, itself a serine/threonine kinase, leads to the phosphorylation of several molecules that are responsible for the downstream effects of PI3K. For example, phosphorylation of BAD  3  allows the association of this molecule with another protein known as 14-3-3. This association prevents the interaction of BAD with members of the Bcl-2 family thereby preventing apoptosis and promoting cell survival (Nunez and del Peso, 1998). There is however, some question as to the physiological relevance of the phosphorylation of BAD by Akt in all cell types (Scheid and Duronio, 1998). Additionally, Akt phosphorylation of human caspase-9, but not mouse caspase-9, inhibits this pro-apoptotic molecule also promoting survival (Cardone et al., 1998, Fujita, 1999 #220).  Furthermore,  phosphorylation of the Forkhead related transcription factor 1 (FKHR-L1) by Akt results in its sequestration in the cytosol preventing the transcription of genes involved in cell cycle inhibition and apoptosis (Brunet et al., 1999; Dijkers et ak, 2000). Glycogen synthase kinase-3 (GSK-3) is another downstream target of Akt whose functions include the negative regulation of cytosolic proteins and nuclear transcription factors (reviewed in (Ali et al., 2001)). Phosphorylation of GSK-3 by Akt results in its inactivation, thereby promoting cell cycle, transcription and translation. Additionally, PI3K is responsible for the activation of pathways through PDK-1 that lead to protein synthesis, and pathways through the exchange factors for GTP binding proteins Rho and Grpl that induce changes in the actin cytoskeleton. A schematic of some of the known effects of PI3K activity is shown in Figure 3. Additionally, an excellent resource by L.C. Cantley depicting the consequences and mechanisms of activation downstream of PI3K can be found in the 'Animations of PI3K Signaling.  Sci.  STKE  (Supplement  to  Connections  Maps)'  bttp://stke.sciencemag.org/cgi/content/full/$igtrans:CMP 6557/DCI.  1.2  SHIP and PTEN as negative regulators of 3-phosphoinositides It is clear that the activation of PI3K leads to an array of complex cellular  interactions and that its effects are vast and important to cell function. Equally important then, is the negative regulation of its activity, a function performed by the lipid phosphatases known as the S7/2-containing /nositol Phosphatases (SHIP and SHIP2) and the Phosphatase with TENsin homology (PTEN).  4  SHIP structure and expression  SHIP, also known as SHIP1 or Inpp5D, was originally identified as a 145-kDa protein that was phosphorylated and associated with the adaptor protein She in response to growth factor stimulation in hematopoietic cells (reviewed in (Huber et a l , 1999). Subsequently, the cDNA was independently cloned by several groups utilizing either cDNA library screening using degenerate probes obtained from peptides (Damen et al., 1996; Kavanaugh et al., 1996; Odai et al., 1997; Ono et a l , 1996) or by yeast two hybrid techniques using the PTB domain of She or the cytoplasmic sequence of an Fc receptor as bait (Lioubin et al., 1996; Osborne et al., 1996). The predicted amino acid sequence of 1190 amino acids spans a N-terminal SH2 domain, a central portion with two motifs highly conserved in inositol 5-phosphatases, and a carboxy-terminal domain with two NPXY sequences and proline rich sequences that could facilitate the binding of PTB or SH3 domain-containing proteins (Figure 4). While northern blot analysis found full length SHIP RNA expression in many tissues, this technique is easily contaminated with peripheral blood and in-situ analysis eventually revealed that SHIP was restricted to hematopoietic cells and testes (Liu et al., 1998b). Intriguingly, SHIP proteins have been found to exist at sizes of 145, 135, 125, 110, 104 kDa in various tissues, with the 104 kDa form, known as s-SHIP being expressed exclusively in embryonic and hematopoietic stem cells (Tu et al., 2001). These various forms of SHIP are thought to arise from alternative splicing, alternative transcriptional initiation, protein degradation or post-translational modification such as phosphorylation. It is important to note that in 1997 another 5'-inositol phosphatase was cloned that has significant homology to SHIP and was named SHIP2 (Pesesse et al., 1997). These two molecules are quite similar in structure but differ in the proline rich areas in the C terminal portion of the protein. They also differ in tissue expression. SHIP2 has overlapping expression with SHIP in B cells and T cells and platelets (Bruyns et al., 1999; Muraille et al., 1999), but is ubiquitously expressed in many other tissues. Despite overlapping expression in hematopoietic tissues, evidence that SHIP plays a more  5  dominant enzymatic role than SHIP2 in hematopoietic cells has been inferred from platelet studies (Giuriato et al., 2003) and also by the lack of hematopoietic defects in SHIP2 deficient mice (Clement et al., 2001). The remainder of the background and of the work described in this thesis deals solely with the effects of partial or complete deficiency of SHIP, although compensatory roles for SHIP2 are considered in the context of our studies. SHIP enzymatic activity and regulation  The central domain of SHIP shares sequence similarity to previously identified inositol phosphatases and has been found to be 96% identical between humans and mice (Ware et al., 1996). Its phosphatase activity in vitro was shown to be directed at the 5' position of PIP and the cytosolic 1,3,4,5-tetrakisphosphates (IP ) with the requirement of 3  4  prior phosphorylation at the 3' site of the inositol phospholipid. This requirement implied that SHIP would function sequentially after PI3K activation. Phospholipid analysis on cells lacking SHIP have reinforced an in vivo role for this phosphatase in dephosphorylating PIP species to form PI(3,4)P , (Damen et al., 1996; Lioubin et al., 1996; 3  2  Scheid et al., 2002). The in vivo evidence for de-phosphorylation of IP has been scant 4  and has only very recently been demonstrated in a study whereby TGF-(3 stimulation of a murine myeloma cell line resulted in the up-regulation of SHIP, a decrease in IP levels, 4  and concurrent increase in IP levels (Valderrama-Carvajal et al., 2002). The absence of 3  SHIP, at least in bone marrow derived mast cell lines, results in an approximate three fold increase in PIP levels over wild type immediately after stimulation with steel factor (SF) 3  with levels returning to wild type levels after 15 minutes (Huber et al., 1999; Scheid et al., 2002). Unstimulated levels of PIP and levels at late time points after stimulation 3  appear to be unaffected by the absence of SHIP and likely reflect the compensatory action of alternate 5'phosphatases such as SHIP2. Interestingly though, these experiments showed a significant decrease in the amount of PI(3,4)P produced after stimulation. This 2  implies that the enzymatic action of SHIP is primarily responsible for the generation of this species and also implies that SHIP may serve to shift the balance of reactive PI species in its ability to negatively regulate the product of PI3K activity.  6  While SHIP is phosphorylated in what has been shown to be a Src-kinase dependent manner after stimulation (Phee et al., 2000), its activity does not appear to be influenced by its tyrosine phosphorylation or by the interaction with adaptor proteins. Instead, it is thought that its activity is regulated by compartmentalization, in that cellular stimulation induces a translocation of SHIP to the membrane where it can act upon its lipid substrate. Structurally, studies looking at various mutant constructs have revealed that not only is the phosphatase domain essential for SHIP enzymatic function but so is the integrity of the C-terminal tail (Damen et al., 2001). A C-terminal mutant in this study was localized to but did not translocate to the membrane as did full length SHIP after stimulation. This suggests localization of SHIP in distinct compartments of the membrane and implies a level of regulation of SHIP activity that involves uncharacterized interactions with the tail portion of the protein. PTEN structure and expression  PTEN (also known as MMAC1, Mutated in Multiple Advanced Cancers or TEP1, TGF-beta-regulated and Epithelial cell-enriched Phosphatase) was originally cloned by two independent groups as a tumour suppressor gene residing in the chromosomal area 10q23, an area frequently lost in human tumours (Li et al., 1997; Steck et al., 1997). A third group identified the same gene and identified it as a phosphatase that was regulated by TGF-beta (Li and Sun, 1997). A multitude of studies have since identified loss or mutation of PTEN in a variety of human cancers, confirming and highlighting its role as a tumour suppressor in many tissues (Parsons and Simpson, 2003). PTEN's discovery was a long awaited "proof of theory" that phosphatases should act as tumour suppressors through their ability to antagonize the action of kinases, molecules whose activity was often implicated in carcinogenesis. Studies have revealed the ubiquitous expression of this protein in both human and mouse tissues (Gimm et al., 2000; Luukko et al., 1999). Structurally the gene encodes a 40-50 kDa protein, 403 amino acids in length. The N terminus contains a putative consensus PI(4,5)P -binding motif, along with a phosphatase 2  domain that includes a highly conserved Cx R active site motif that is characteristic of 5  protein tyrosine phosphatases (Figure 5). Within the C-terminus there is a C2 binding  7  domain that is thought to be involved in targeting PTEN to phospholipids in the membrane (Lee et al., 1999) as well as a PDZ-binding motif that facilitates the interaction of it with other proteins, for example the membrane-guanylate kinase MAGI-3 (Wu et ak, 2000). Additionally, the C-terminus contains two PEST homology regions that regulate PTEN stability (Georgescu et al., 1999). PTEN enzymatic activity and regulation  The discovery of the Cx R sequence motif within the phosphatase domain of 5  PTEN suggested that it would act as a dual-specificity protein tyrosine kinase. However, studies on its enzymatic activity revealed relatively poor phosphatase activity towards most protein substrates and a high enzymatic activity towards acidic substrates (Myers et al., 1997). Subsequent work revealed that the ability of PTEN to de-phosphorylate lipid substrates, particularly at the 3' site of PIP was the key to its tumour suppressor role 3  (Maehama and Dixon, 1998; Myers et ak, 1998). More recent analysis on the kinetics and enzymatic activity of PTEN have revealed that PI(3,4)P may also be a relevant substrate 2  (Downes et al., 2001). However, analysis of 3-PI levels in Pten'' ES cells have only revealed obvious increases in PIP levels in response to stimulation compared to wild3  type cells. While there has been evidence in vitro of PTEN's ability to de-phosphorylate protein substrates and alternate lipid substrates, the relevance of these findings in vivo is currently unclear (Caffrey et ak, 2001; Tamura et ak, 1999; Tamura et ak, 1998). The regulation of PTEN activity has become the focus of much work in recent years and it is proving to be complex. Studies have revealed that the integrity of the C2 domain, PDZ domain and C-terminal tail are all important in either PTEN activity or stability (Campbell et al., 2003; Georgescu et ak, 1999; Georgescu et al., 2000). This is consistent with the fact that mutations in various regions throughout the gene have been observed in humans with Cowden's syndrome, a disorder resulting from germline autosomal dominant mutations in PTEN (Waite and Eng, 2002). Phosphorylation of the C-terminus of PTEN by casein kinase-2 was found to increase PTEN stability, and phosphorylation mutants of PTEN showed increased proteosomal degradation (Torres and Pulido, 2001). Other work has shown that membrane recruitment of PTEN is  8  inhibited by phosphorylation of the tail, and that membrane recruitment leads to rapid PTEN degradation (Das et al., 2003). Furthermore, another study revealed that inhibiting the generation of phosphoinositides decreased phosphorylation at the C-terminus of PTEN and lead to its degredation (Birle et al., 2002). While the mechanisms are not yet completely clear, these studies imply that PTEN stability and localisation to the membrane can be regulated by phosphorylation at the C-terminus, and that its stability at the membrane may be subject to a feedback loop that relies on phosphoinositide levels. Additionally, very recent work has demonstrated that Src-kinase activity results in tyrosine phosphorylation of PTEN and that this inhibits its enzymatic activity, and decreases PTEN protein (Lu et al., 2003). This work also revealed for the first time a relationship with an alternate phosphatase SHP-1, wherein SHP-1 can de-phosphorylate PTEN reversing the effects induced by Src kinase. This implies that a network of kinases and phosphatases may be involved in the regulation of this phosphatase. It has been shown that the actual degredation of PTEN protein can be induced by caspase-3 cleavage (Torres et al., 2003) and can be inhibited by signaling through the bone morphogenic pathway (Waite and Eng, 2003). Finally, PTEN has been shown to be transcriptionally regulated by the peroxisome proliferator-activated receptor-y (Patel et al., 2001) and also by the tumour suppressor p53 (Stambolic et al., 2001). These studies have revealed multiple levels of regulation for PTEN that will likely be clarified with more work.  1.3  The importance of PI3K signaling and regulation in the immune system A vast array of data on the roles of PI3K mediated signaling in immune cells was  originally gleaned from experiments using transformed leukemic cell lines. The realization that the Jurkat T cell line was deficient for Pten and SHIP (Freeburn et al., 2002; Shan et al., 2000) and thus displayed constitutive PI3K signaling, revealed a great caveat in drawing conclusions strictly from in vitro work on cell lines. As such, the generation of in vivo models, animals deficient for components of the PI3K signaling  9  pathway or transgenic for constitutive pathway activation, has provided valuable information regarding the roles of PI3K in immune cells. The necessity of PI3K signaling in immune cells  Deletion of the p85a regulatory subunit in mice has been achieved by eliminating only the p85a transcript allowing for alternative splicing of the p55a and p50a transcripts, or by preventing the generation of all three regulatory proteins (Fruman et al., 1999; Suzuki et ak, 1999). The first resulted in a viable phenotype, whereas the complete loss resulted in death shortly after birth therefore necessitating the use of the RAG complementation system to study the effects in B and T cell lineages. In this system, the knock-out ES cells are injected into blastocysts from mice deficient for the recombinase activating gene (RAG). Without this gene, mice are unable to develop mature B or T cells, and thus the injection of knockout cells into RAG' blastocysts yields mice with B and T cells originating from the knockout cells. The resulting phenotype in both studies revealed a block in B cell development at the pro-B cell stage, a decrease in splenic B cells, as well as impaired T-independent antibody responses.  Interestingly, this  phenotype is quite similar to the Xid phenotype in mice that is caused by a point mutation in Btk, a downstream effector of PI3K signals (Rawlings et al., 1993) suggesting that impaired signaling through the PI3K pathway was necessary for proper B cell development. There were no apparent defects in T cell development or T cell signaling in p85a'~ mice, which was attributed to possible compensation by the p85(3 subunit. There is evidence that p85 is a signaling molecule independent of the catalytic subunit of PI3K (Kang et ak, 2002), and thus it was important to investigate the effect of deletion of the catalytic subunits of PI3K to further evaluate the role of PI3K ablation. Deletion of pi 10a and pllOBfi were found to be embryonic lethal (Bi et ak, 2002; Bi et ak, 1999), and conditional knockouts of these molecules in immune cells have not been investigated. Deletion of pi 106, the isoform predominantly expressed in leukocytes, resulted in a block in B cell development reminiscent of that which was attributable to deletion of p85a and also revealed impaired B and T cell signaling (Clayton et ak, 2002; Jou et ak, 2002; Okkenhaug et ak, 2002). Studies examining the deletion of pllOy  10  revealed that B cells were seemingly unaffected by the absence of this subunit, but various defects in T cell, mast cell, and platelet function, as well as defective migration by neutrophils were revealed by numerous groups (Hirsch et al., 2001; Hirsch et al., 2000; Laffargue et al., 2002; Sasaki et al., 2000). Together these studies provided compelling evidence that PI3K plays a vital role in various immune cell types and that immune cell function is generally impaired in the absence of proper PI3K signaling. The effects of over-expression ofPBK activity in vivo  The generation of transgenic animals using promoters that drive the cell specific expression of genes can provide valuable information of the effects of over-expression of a molecule on that cell type. This approach has been used to look at the effects of overexpression of PI3K or Akt specifically in T cells. Expression of an oncogenic regulatory subunit of PI3K--p65, under the control of the Lck promoter, directed expression of this molecule throughout T cell development (Borlado et al., 2000). The result was an infiltrative,  lymphoproliferative phenotype  accompanied  by  autoimmune  glomerulonephritis. Additionally, this transgene together with an absence of the p53 tumour suppressor gene, contributed to an early onset of T cell lymphomas. This indicated that over-expression of PI3K could manifest in autoimmunity and contribute to oncogenic transformation. Other studies examined the effects of over-expression of a constitutively active form of Akt using a transgene driven by the CD2 promoter (Jones et al., 2000; Parsons et al., 2001).  These mice developed splenomegaly and  lymphadenopathy as a result of increases in T cells and B cells. The T cells in these mice had higher levels of activation markers than control cells, showed enhanced viability in culture and were resistant to several pro-apoptotic stimuli. Additionally, the transgenic mice had increased serum IgG/IgA levels and autoantibody production compared to controls. These studies reinforced the idea that uncontrolled PI3K signaling can result in autoimmune characteristics.  11  The in vivo role for SHIP in immune cells  Much has been learned about the role of SHIP in immune cells owing to the fact that the SHIP'' mice generated by two independent groups were found to be viable (Helgason et al., 1998; Liu et al., 1999). However, the SHIP'' mice had a shortened lifespan compared to littermates due to a progressive expansion and accumulation of myeloid cells. As a result, these mice suffered from particularly severe myeloid cell accumulation in the lungs and also in the spleen, liver and kidney. The overrepresentation of myeloid cells compared to lymphoid cells was evident in both the bone marrow and peripheral blood of these mice. An assessment of progenitor growth in cells derived from the marrow of SHIP'' mice revealed an increase in the number of granulocyte-macrophage progenitors with a concomitant decrease in pre-B and erythroid progenitor number. Furthermore, the granulocyte-macrophage progenitors showed increased sensitivity when exposed to limiting amounts of various growth factors including GM-CSF, IL-3 and SF compared to that of wild-type cells (Helgason et al., 1998). Experiments using bone marrow-derived macrophages revealed that these cells were resistant to apoptosis induced by several pro-apoptotic stimuli, including cytokine withdrawal, and showed increased PIP levels and sustained phosphorylation of Akt after 3  growth factor stimulation (Liu et ak, 1999). More detailed analysis on myeloid lineage cells has revealed more specific functions for SHIP in these cell types. It has been proposed that SHIP may actually restrain the differentiation of myeloid cells owing to the observation that SHIP' bone marrow-derived mast cells and macrophages differentiate in vitro more quickly in vitro than cells from wild-type mice (Rauh et ak, 2003). SHIP has been shown to negativelyregulate degranulation of mast cells and also to negatively-regulate the production of IL6, a pro-inflammatory cytokine through the activity of NF-kB (Kalesnikoff et al., 2002). Interestingly, SHIP has been shown to be a positive regulator of NO production in macrophages as SHIP' cells produce less NO than wild-type cells after LPS stimulation. This was found to be the result of alterations in the biochemical synthesis of NO and may suggest a role for SHIP in determining the cytokines produced in end stage macrophage differentiation (Rauh et ak, 2003). Finally a role for SHIP in the negative regulation of  12  macrophage phagocytosis was demonstrated in experiments that showed that SHIP' macrophages were more efficient at phagocytosis initiated through Fc and complement receptors (Cox et al., 2001). Thus, SHIP has a demonstrated role, both negative and positive, in the regulation of various aspects of myeloid cell behaviour. The decrease in lymphocytes and in pre-B cell clonogenic growth prompted further studies into the role for SHIP in B cells. This was achieved by studying B cells directly from SHIP' mice (Brauweiler et al., 2000; Helgason et al., 2000), or by the use 1  of the RAG complementation system to bypass the myeloproliferative disorder (Liu et al., 1998a). Detailed FACS analysis revealed decreases in mature bone marrow B cells and normal percentages of early pro-B cells implying a defect in intermediate stage B cell development. One study demonstrated an acceleration of B cell development in the marrow, reminiscent of the recently described accelerated differentiation observed in vitro for the myeloid lineage cells (Brauweiler et al., 2000; Rauh et al., 2003). Furthermore, increases in the absolute numbers of IgM'° IgD subset of B cells in the hl  spleens of SHIP mice were observed, representing an enhancement of B cell maturation 1  in the spleen. SHIP' ' mice showed increased levels of circulating immunoglobulins and 1  enhanced response to defined antigenic challenge with T-independent antigen (Helgason et al., 2000). There was also evidence that SHIP mice showed characteristics of 1  autoimmunity, namely antibodies against single and double stranded DNA and immune complex mediated glomerulonephritis ( C D . Helgason, personal communication). Additionally, SHIP ' B cells showed increased proliferation in response to stimulation 1  with IgM with or without crosslinking of the inhibitory FcyRIIB receptor, confirming a role for SHIP in inhibitory signaling in B cells (Helgason et al., 2000). Stimulation of SHIP' B cells demonstrated increased PIP levels over wild-type cells, increased Ca  2+  3  flux and increased phosphorylation of Akt and Erk demonstrating the negative role of SHIP in BCR mediated signaling events. Finally, studies revealed a resistance to BCRinduced apoptosis in B cells from SHIP' mice (Brauweiler et al., 2000), all together demonstrating that SHIP plays a critical role in the development, proliferation, Ig production and survival of B cells.  13  The role for SHIP in T cells has only ever been reported from the R A G complementation study (Liu et a l , 1998a). Here it was reported that SHIP'' reconstituted RAG'' mice had slight increases in the number of CD4 T cells compared to SHIP ' +  +/  reconstituted RAG' controls. However, the SHIP' cells had comparable proliferative responses, IL-2 production, Ca mobilization, downstream molecule phosphorylation, 2+  and cytokine production to SHIP*' cells, suggesting a minor role for SHIP in regulation of T cell development and signaling. A role for SHIP in control of PIP levels in thrombin stimulated platelets has very 3  recently been described (Giuriato et ak, 2003). In this study, platelets isolated from wild type, SHIP*', SHIP' or SHIP2*' mice, were stimulated with thrombin and PIP and 3  PI(3,4)P levels were measured. Significant increases in PIP levels as well as decreases 2  3  in PI(3,4)P were evident in SHIP'' cells after stimulation. Interestingly, the PI(3,4)P 2  2  levels in SHIP*'' mice were also decreased over wild type and SHIP2*' levels, though not as significantly as in the SHIP'''' cells. Given the difference in PI(3,4)P levels between 2  SHIP*'' versus SHIP2*' mice, the authors suggest that SHIP plays a greater role in PIP  3  metabolism in platelets than SHIP2. The role for SHIP in cell migration has also been addressed in a study that evaluated the chemotactic ability of progenitor cells, B cells and mature T cells and thymocytes from SHIP' mice (Kim et al., 1999). All of these cells showed an increase chemotactic ability towards gradients of stromal derived factor-1 (SDF-1) that was not attributable to altered chemokine receptor expression but rather was inferred to result from altered cytosolic signaling through chemokine receptors. The in vivo role of Pten in immune cells  The embryonic lethality associated with Pten deficiency precluded the immediate study of its in vivo role in immune cell development and behaviour. However, an important observation was made in that mice heterozygous for the Pten gene developed a progressive lymphoproliferative phenotype and features of autoimmunity (Di Cristofano et ak, 1999; Podsypanina et al., 1999). The accumulation of lymphocytes in the lymph nodes and spleen was determined to be a polyclonal expansion of cells, and these cells  14  retained one intact Pten allele (Podsypanina et al., 1999). Pten ~ mice, particularly the +/  females, were found to have increased numbers of both B and T cells in their lymph nodes and spleen as well as increased activation markers on these cells. Increased levels of circulating IgG were reported, as was the presence of antibodies directed against antinuclear antigens (ANA) and single stranded D N A . Furthermore, the kidneys of these mice showed increased immunoglobulin deposition and evidence of glomerulonephritis. A l l of these features were reminiscent of the phenotype seen in the well-characterized mouse model for lupus, the Ipr mice that result from a mutation in the Fas receptor (Takahashi et al., 1994). Indeed, functional experiments on cells isolated from Pteri ' 1  mice revealed a resistance to Fas induced apoptosis by both T cells and B cells (Di Cristofano et al., 1999). These experiments were of key importance in that they suggested that the development of a lymphoproliferative, autoimmune disorder in the Pteri' ' mice 1  was the result of haploinsufficiency for Pten.  This also implied that even partial  reduction in the regulation of PI3K activity was sufficient to perturb the immune system. Recent work in generating conditional knockouts of Pten has revealed critical roles in the T and B cell lineages. Complete deficiency for Pten (Pteri " ') in T cells was 10  1  achieved by cre-mediated excision driven by the Lck promoter (Suzuki et al., 2001). Pteri *'' mice had expanded numbers of C D 4 and B220 cells in the periphery and 10  +  +  developed CD4 lymphomas by the age of 17 weeks. Additionally, these mice showed +  increased  reactivity  towards  single  stranded  DNA,  more  severe  hypergammaglobulinemia, and an increase in both T h l and Th2 cytokine production compared to Pten  +,+  mice. Pteri " ' T cells were hyper-proliferative to various stimuli, 0  1  and more resistant to apoptosis compared to controls. Using an additional transgenic cross to a strain which expresses a T cell receptor (TCR) specific for the male Hy antigen, the authors were able to demonstrate a 10-fold greater increase in thymic cells expressing the transgenic T C R in HyPteri ""'' male mice compared to HyPten 1  +/+  male  mice, implying defective thymic negative selection. Finally, lysates from the Pteri "'' T 10  cells showed increased phosphorylation of both Akt and Erk indicating a hyper-activation of these signaling pathways. These results clearly demonstrate that Pten has tumour  15  suppressor function in T cells and is essential in regulating their development, the maintenance of tolerance, and their response to both stimulatory and apoptotic stimuli. Two groups have reported the conditional deletion of Pten in B cells (Anzelon et al., 2003; Suzuki et ak, 2003), and both utilized a CD19-Cre transgenic cross to facilitate Pten deletion. Both studies reveal that B cell specific deletion of Pten results in an expansion of the B1 a sub-population in the peritoneal cavity and the spleen as well as an increase in the marginal zone B cells of the spleen. This is suggestive of the necessity of PI3K signals in the differentiation of these B cell subsets, and is in keeping with the fact that these populations are diminished in pi 106' mice (Clayton et ak, 2002; Okkenhaug et ak, 2002). Additionally both studies agree that B cells deficient for Pten are hyperproliferative towards various stimuli and that stimulation results in increased phosphorylation of Akt. One group reported that deletion of Pten in B cells was able to rescue the defects in germinal center formation observed in CD19 mice (Anzelon et ak, 7  2003). This implied that it was an impairment in PI3K recruitment and activation by this co-stimulatory molecule that was responsible for the defects previously reported in CD19 mice (Engel et ak, 1995). The other group reported defects in the class switching A  ability of Pten deficient B cells, such that levels of IgM were elevated and IgG and IgA subtypes were decreased (Suzuki et ak, 2003). Finally, there were discrepancies between the two studies that included either the increase or decrease in chemotactic ability of Pten deficient B cells and resistance to apoptosis versus increased apoptosis in response to stimulation. The reasons for these discrepancies are unknown, but from these studies it is evident that Pten is important in B cell response to stimulation, cell differentiation and function. Curiously, autoimmunity was not addressed in either study. It has since been reported that the Pten B cell knockout mice live to a normal lifespan free from autoimmune disease (Suzuki et ak, 2003). This may imply that Pten expression in T cells, rather than in B cells, is critical to maintaining peripheral immune tolerance. To date, the effects of deletion of Pten in cells of the myeloid lineage have not been addressed and no reported bone marrow defects have been reported in mice deficient for Pten in any cell type.  16  1.4  Thesis goals Both Pten and SHIP have demonstrated roles as negative regulators of PI3K  activity and together they share a common substrate, PIP . 3  The similarities and  differences in the immune phenotypes of the mice suggest both common and disparate roles for these molecules in immune cell signaling. However, no in vivo studies to date have addressed the effects of combined deficiencies of these molecules. This thesis set out to look at the effects of combined deficiencies of these molecules on the phenotype of the mice and on the behavior of various immune cell types. Specifically the goals were: 1) To determine the effect of SHIP heterozygosity on the autoimmune phenotype of Pten* ' mice. 1  2) To investigate the effects of combined heterozygosity for SHIP and Pten specifically in CD4 T cells. +  3) To investigate the effects of partial Pten deficiency on the hematopoietic defect seen in SHIP mice and to reveal any role for Pten in hematopoiesis. 1  17  Chapter Two: Loss of a single allele of SHIP  exacerbates the  immunopathology of Pten heterozygous mice.  Author's contribution statement  These experiments were performed by Jennifer Moody at the University of British Columbia and the University of Calgary. Joan Miller and Cheryl Hanson performed the blind scoring of the ANA slides. 2.1  Introduction  The immune system must balance the ability to mount brisk immune responses against foreign antigens with the ability to minimize the inappropriate recognition of selfantigens. Chronic autoimmune diseases such as systemic lupus erythamatosus (SLE) likely result from the failure of a variety of different immunoregulatory mechanisms (reviewed in (Kotzin, 1996)). While the primary immunological defects responsible for the progression of human SLE are poorly understood, susceptibility appears to be influenced by complex genetic inheritance, resulting from the combined effects of alleles at multiple genetic loci (Roberton and Vyse, 2000; Vyse and Todd, 1996). Identification of candidate susceptibility genes for human autoimmunity has been facilitated by studying mouse strains prone to autoimmune diseases, and also by generating transgenic mice having disruptions of genes involved in the regulation of the immune system (reviewed in (Foster, 1999)). Some of the genes implicated in tolerance include those encoding proteins that regulate antigen receptor signaling. For example, an autoimmune phenotype results from the over-expression of CD 19, a co-receptor that augments signals from the B cell receptor (BCR), and also from the deletion of the FcyRIIB inhibitory receptor responsible for the recruitment of molecules that attenuate BCR signaling (Bolland and Ravetch, 2000; Inaoki et ak, 1997). It is well established that the strength of antigen receptor signals is a key determinant in tolerance induction, as it dictates the positive and negative selection of B and T cells (Goodnow, 1996). As the proteins involved in antigen receptor signaling must operate within complex biochemical  18  pathways, it is plausible that quantitative or qualitative perturbations owing to heterozygosity of multiple genes might have additive or synergistic effects with respect to immune regulation. In keeping with this notion, studies of compound heterozygote mice have revealed the importance of threshold levels of proteins in controlling immune responses (Cornall et al., 1998; Satterthwaite et al., 2000). The PI3K pathway which is activated following BCR and TCR stimulation (Campbell, 1999; Ward et al., 1996), is well poised to regulate immune responses through antigen receptor signaling via the generation of phosphatidylinositol (3,4) bisphosphate (PIP ) and phosphatidylinositol (3,4,5) trisphosphate (PIP ). These reaction 2  3  products allow translocation of multiple pleckstrin homology (PH) domain-containing proteins to the membrane, and these in turn regulate downstream events such as proliferation, apoptosis, protein synthesis, and cell shape (reviewed in (Chan et al., 1999; Martin, 1998). The levels of PIP and PIP are regulated via the actions of PTEN, a 3' 3  2  inositol phosphatase, and SHIP, a 5' inositol phosphatase (Huber et al., 1998; Maehama and Dixon, 1998) and recent studies have provided evidence of a role for negative regulators of PIP in the maintenance of immune tolerance. While complete deletion of 3  Pten in mice results in embryonic lethality, (Di Cristofano et al., 1998; Podsypanina et al., 1999; Suzuki et al., 1998), Pteri''  mice develop an autoimmune phenotype  characterized by lymphadenopathy and splenomegaly that was attributed to defective B and T cell apoptosis (Di Cristofano et al., 1999). These mice also demonstrated autoimmunity, developing antibodies against nuclear antigens as well as immune complex-mediated glomerular disease. Immunopathology of Pteri' ' mice was worsened 1  when both alleles of Pten were deleted in the T cell compartment (Suzuki et al., 2001). Additionally, mice expressing a constitutively active form of PI3K specifically in T cells developed a phenotype similar to that of Pten '~ mice (Borlado et al., 2000) providing +  further evidence that the deregulation of phosphatidylinositol levels in this cell type can promote the development of autoimmunity. Assuming that it is the altered level of inositol phosphatase activity that promotes the autoimmune phenotype in Pteri' ' mice, we hypothesized that reduced expression of 1  SHIP, achieved by generating mice doubly heterozygous for both Pten and SHIP, might  19  lead to a worsening of the Pten* ' phenotype. Based on total and subtype-specific 1  immunoglobulin levels, lymphoproliferation, autoantibody titres, and kidney pathology, we demonstrate that heterozygosity for both genes aggravates the lymphopathology of Pten* ' mice. 1  2.2  Materials and Methods  Mice  Pten* ' and SHIP*'' mice were generated as previously described (Helgason et ak, 1  1998; Podsypanina et ak, 1999) and backcrossed to C57B1/6 strain for 4-6 generations. Both the Pten and SHIP strains were maintained at n=6 by intercrosses of wild-type and heterozygous mice. Pten*'SHIP*'', Pten*'', SHIP*', and wild-type littermates were generated by crossing SHIP*'' females with Pten*'' males. Mice were maintained in double barrier facilities located at both the Center for Molecular Medicine and Therapeutics and the University of Calgary Health Sciences Center. Serum Immunoglobulin Analysis  For determination of total serum IgM, IgGl, IgG2b, IgG2a, IgE, IgA, Nunc Maxisorp™ immuno plates (VWR Canlab) were coated with 2 u,g/ml capture antibody (BD Pharmingen). IgG capture antibody (Cedarlane) was used at a concentration of 10 3  p:g/ml. Plates were blocked with PBS/10% FCS for 2 hr at room temperature and serially diluted serum samples were incubated on the plates overnight. Plates were washed in PBS/0.05% Tween-20 and then incubated with 2 u.g/ml biotin conjugated antibody, (HRP conjugated IgG at 1/10,000 dilution) for 1 hour. After washing, streptavidin HRP (BD 3  Pharmingen) at 1/1000 dilution was applied for 30 min to the biotin-conjugated antibodies. All plates were developed using 3,3',5,5'-tetramethylbenzidine (TMB) (Sigma, St. Louis, MO) and read at 405/490nm on a Biotek ELISA reader using DeltaSoft™ software. Concentrations were determined from curves generated from mouse lg standards (BD Pharmingen and Cedarlane).  20  Autoantibody Analysis  Anti-nuclear antibody titres were determined by incubating serial dilutions (1/40, 1/80, 1/160, 1/320, 1/640, 1/1280) of serum in PBS on HEp-2 cells (ImmunoConcepts) for 30 min at room temperature. After washing in PBS, slides were incubated with a 1/80 dilution of goat anti-mouse FITC-conjugated polyspecific IgM/IgG antibody (Jackson Immunoresearch) for 30 min. After thorough washing in PBS, the slides were viewed on a fluorescence microscope. The antibody titre was defined as the maximum dilution at which nuclear staining was indistinguishable from that of the cytoplasm (or a "1+" stain on a scale of 1-4). Antibodies directed against dsDNA were assessed using Crithidia lucilliae slides (ImmunoConcepts). Sera was diluted 1/20 in PBS and incubated on Crithidia slides for 30 min at room temperature. Slides were washed in PBS and then incubated with a 1/80 dilution of goat anti-mouse FITC-conjugated polyspecific IgM/IgG antibody (Jackson Immunoresearch) for 30 min. Western blot analysis of purified calf thymus histone protein (Boehringer Mannheim) was performed to determine the presence of anti-histone antibodies.  Strips were cut from the nitrocellulose filter, blocked in  PBS/0.05% Tween-20 + 5% milk powder (PBST/milk) for 30 min, and were incubated with mouse sera (1/100 dilution in PBST/milk) for 1 hr. Strips were then washed in PBS, and incubated for 1 hr with a 1/500 dilution of peroxidase-conjugated goat anti-mouse IgG in PBST/milk (ICN Pharmaceuticals, Costa Mesa, CA). After three 10 min washes in PBS, strips were developed with ECL reagent (Amersham Pharmacia Biotech) and immediately visualized on film.  Densitometric analysis was performed using  ImageQuant software and arbitrary densitometric units (AU) were assigned based on the intensity of the signal. Flow Cytometry  Spleens were passed through a wire mesh in RPMI media with 2% FCS. Cells were suspended in PBS/2% FCS, blocked with anti-FcyRIIB (24G2, BD Pharmingen) for 30 min and then stained with FITC or PE conjugated anti-CD4, CD8, B220, C D l l b , CD69 (BD Pharmingen) for 45 min on ice. Cells were washed three times in PBS before  21  analysis on a FACScan using CellQuest™ software. Live cells were gated based on forward and side scatter profiles and 10,000 events were collected for analysis.  Histology  Tissues were fixed in 10% formalin, paraffin embedded and sectioned at 2-3 um, and stained with either hematoxylin and eosin or periodic acid-Schiff reagent. For electron microscopy (EM), 1-2 mm thick tissue pieces were fixed and stored in 2.5% gluteraldehyde, then fixed in 1% osmium tetroxide for 1.5 hrs. After a 1.5 hr rinse in water, tissues were stained with 5% aqueous uranyl acetate for 1 hr and 20 min and dehydrated through a series of alcohol changes before being embedded in Effapoxy and sectioned at 60-100nm for grid analysis. Statistical Analysis  Comparison of lymph node weight between Pten '~SHIP ' and Pten ~ mice was +  +  +/  performed using the Student's t test. Determination of p values between all four genotypes for spleen weights, spleen cellularity and Ig analysis was performed using a one way ANOVA and the Tukey test for multiple comparisons.  2.3  Results and Discussion  Pten SHIP +/  +I  mice show augmented lymphadenopathy, splenomegaly, and a possible  increase in thymic tumour frequency.  Breeding heterozygotes for Pten and SHIP yielded Pten SHIP ~ mice according +/  +/  to the expected Mendelian ratios. The majority of Pten SH1P mice developed striking +I  +I  enlargement of cervical and axillary nodes, between the ages of 18 and 26 weeks. This was generally before the onset of any signs of disease in Pten ' littermates. Of mice +I  between 8 and 40 weeks, we observed thymic tumors in 4/34 (11.7%) Pten ' SHIP mice +  +I  compared to 1/27 (3.7%) of Pten ~ mice and 0/81 wild-type and SHIP*' mice. Previous +/  reports cite the frequency of lymphoid tumors in Pten '~ mice at 2.7% and 12%, with the +  22  former sharing the same genetic background as our colony (Podsypanina et ak, 1999; Suzuki et ak, 1998). It is possible that heterozygosity for SHIP could promote the tendency of Pten mice to develop these tumors by further compromising the ability of T +/  cells to de-phosphorylate PIP . However a more comprehensive study with greater 3  numbers of aging mice of each genotype would be required to determine if the difference between Pten ~ and Pten 'SHIP +/  +  +/  thymic tumour frequency differs significantly.  Based on pilot studies an endpoint of 24 weeks was selected to evaluate autoantibody production, serum lg levels and pathology. This was also before the general onset of tumors that occurs in Pten* ' mice. Twenty-four week old Pten 'SHIP 1  +,  +I  mice displayed cervical and axillary lymphadenopathy with significantly increased lymph node weight compared to Pten ~ mice (Table 1). Lymphadenopathy was not present in +/  wild-type littermates or SHIP ~ mice. P t e n SHIP*' +/  +  spleen weights were also  significantly increased in comparison to the control groups (Table 1). The relative percentages of splenic B cells, T cells and macrophages were generally comparable between genotypes (data not shown), with the increased cellularity in Pten ' SHIP +  +/  spleens resulting from the expansion of the B, T and macrophage/granulocyte lineages (Table 2). The higher percentage of CD69 cells in the spleens of Pten SHIP +  +/  +/  mice  was consistent with the presence of an increased number of activated T-cells, B-cells or NK-cells (Table 2). Similar to the Pten ' mice (Podsypanina et ak, 1999), histological +/  sections of secondary lymphoid organs revealed aggregates of cells interspersed by areas of fibrosis as well as loss of the normal cortical and medullary distinction, and a total absence of germinal centers (data not shown). Pten '~SHIP '~ mice demonstrate severe kidney pathology +  +  Analysis of kidney sections revealed normal glomeruli or very mild mesangial proliferation in the glomeruli of wild-type and SHIP  +/  mice (Figure 6a,b), and only a few  SHIP '~ mice exhibited mild interstitial infiltrates. Pten ~ mice showed mild to moderate +  +I  mesangial proliferation with the occasional female showing increased endocapillary cellularity in some glomeruli (Figure 6c). The most severe kidney pathology was limited to Pten 'SHIP +  +/  mice. As a group, these mice demonstrated moderate mesangial  23  proliferation, and three females exhibited severe diffuse proliferative glomerulonephritis (Figure 6d). The majority of glomeruli in these females were hypercellular and lobular in appearance, and were surrounded by interstitial inflammatory infiltrates. Examination at the E M level revealed small, mesangial, electron dense deposits and normal glomerular basement membrane (GBM) architecture in the wild-type (Figure 7a) and SHIP*' kidneys (not shown). Pten* ' kidneys showed a moderate number of medium sized electron dense 1  deposits in the mesangium and minor G B M irregularities (not shown), while Pten* ' 1  SHIP*' glomeruli contained small and medium sized deposits in the mesangium, as well as scattered small subepithelial, intramembranous and subendothelial deposits (Figure 7b). Additionally, in the latter, there was fusion of the foot processes, and the glomerular capillaries contained occasional infiltrating monocytes, lymphocytes and neutrophils. The distribution of electron dense deposits throughout the structure of the glomeruli in these mice was consistent with immune complex mediated damage, as was the presence of inflammatory infiltrates within and surrounding the glomeruli. Elevated immunoglobulin levels in Pteri''SHIP*' mice.  As B cell hyper-responsiveness and hypergammaglobulinemia appear to be contributing factors in murine lupus (Mohan et al., 1997) we examined circulating Ig levels. Analysis of serum Ig levels at 24 weeks, revealed that Pten*' SHIP*' mice produced on average ~325% of wild type Ig compared to ~194% for Pten *'~ mice, and ~132% for SHIP* ' mice (Figure 8). This was the result of statistically significant 1  increases in IgG2b, IgM, IgGl, IgG3 and IgE subclasses (Figure 9a-e), with no significant difference in the IgG2a subclass, and a curious significant decrease in the IgA subclass (Figure 9f,g). Notably, the highest levels of IgM, IgG2b, and IgG3 were generated in the Pten*'~SHIP*' females (data not shown), consistent with the gender bias seen in the Pten*'' mice (Di Cristofano et al., 1999). These increases correlated well with the severity of the kidney pathology seen in the female Pten* 'SHIP*'' mice, where 1  complement fixation by IgG2b and the nephritogenic potential of IgG3 is thought to play a role (Fulpius et al., 1993). Thus Pten*'SHIP*' mice spontaneously show increased levels of IgM and class switched antibodies. This could be a consequence of a number of  24  factors: the hyper-responsiveness of T cells producing cytokines that induce switching, the hyper-responsiveness of B cells, either inherently or in response to T cell help, or the increased survival of antibody secreting plasma cells. Increased anti-nuclear titres and reactivity against histone protein in Pten SHIP ' mice. +/  +/  We next determined whether Pten SHIP ~ mice would generate autoantibodies +I  +I  against nuclear antigens. Evaluation of anti-nuclear antibody (ANA) binding to HEp-2 cells revealed various degrees of reactivity within the serum of all four genotypes, with the majority of the Pten SHIP +/  +/  (7/9) scoring for intense nuclear fluorescence (or '4 ' +  staining) at 1/40 dilution (Figure 10a), as compared to wild-type (1/11), SHIP  +/  (3/11),  Pten ' (3/10) littermates. Sera were then serially diluted to determine the titre, which +I  was defined as the dilution at which nuclear staining becomes indistinguishable from cytoplasmic staining, (or ' 1 ' staining, Figure 10b). The titres for all the mice are +  presented in Table 3 and the percentage of mice for each genotype that titred out at the 1/320 dilution or greater are presented in Figure 10c. 80% of Pten*' SHIP  +/  mice had  titres greater than or equal to 1/320 with lower percentages of positivity seen in the other groups. Reactivity was age dependent, as serum taken at 16 weeks showed markedly reduced reactivity in all the genotypes (data not shown), indicating that autoreactive antibodies detectable by indirect immunofluorescence developed between 16 and 24 weeks. The identity of these anti-nuclear antibodies was investigated by assessing the reactivity towards dsDNA and core histone proteins. As with the ANA test, various degrees of reactivity against histone HI were seen in all four groups (Figure 11a). In order to quantitate the reactivity of each animal, densitometric analysis was performed on the western blot films.  Approximately 55% (5/9) of the Pten ~SHIP ' mice had high +/  +  reactivity against HI compared to 30% (3/10) of Pten \ 18% (2/11) SHIP +,  +/  and 18%  (2/11) of wild-type littermates (Figure 1 lb). A similar pattern of higher titre reactivity in Pten 'SH1P +I  +I  serum samples against H3, H2B, H2A, and H4 was also seen (data not  shown). To examine whether antibodies to dsDNA were present, we screened the serum for reactivity to the kinetoplast of C. lucilliae. All 41 mice tested were negative (data not  25  shown), indicating an absence of dsDNA antibodies at 24 weeks in all the genotypes. Positive kinetoplast staining was seen in two older mouse samples (>10 month old Pten* ' 1  and Pten* 'SHIP* ) from the colony (data not shown), suggesting that anti-dsDNA 1  1  antibodies might also arise over time. The reactivity and specificity of autoantibodies observed in Pten* SHIP*' mice 1  are of interest.  In both human and animal models of SLE, it is well known that  autoantibodies to chromatin components such as DNA and histones, commonly accompany the development of glomerulonephritis and flares of the disease (Berden, 1997; Burlingame et al., 1994; von Muhlen and Tan, 1995). Indeed, there is evidence that antibodies to histones and dsDNA participate in the pathogenesis of glomerulonephritis (reviewed in (Berden, 1997)). The presence of anti-histone antibodies in these mice in the absence of antibodies to dsDNA is consistent with the observation that histone autoantibodies precede the appearance of anti-dsDNA in MRL/lpr mice (Burlingame et al., 1993). Thus it appears that the Pten*'' and Pten*''SHIP*' mice develop a pattern of anti-chromatin antibodies similar to that of this spontaneous model of SLE and glomerulonephritis. The presence of high levels of various isotypes in the absence of germinal centers is also noteworthy, given the requirement for effective B cell-T cell interactions in isotype switching. This is a feature of other autoimmune strains such as MRL/lpr (Theofilopoulos and Dixon, 1985) and may reflect relative B cell independence from T cell help, the existence of germinal centers in less affected nodes, or the persistence of plasma cells generated prior to nodal architecture disruption. In as much as Pten and SHIP share the property of negatively regulating PIP  3  levels, targeted disruption of these genes results in very different phenotypes. Unlike Pteri'mice, SHIP' mice are viable. They do however have reported defects in B cell development, enhanced B cell function in the periphery and develop a progressive and fatal myeloproliferative disorder (Brauweiler et al., 2000; Helgason et al., 1998; Helgason et al., 2000). In contrast with the lymphoproliferative and autoimmune disorder seen in the Pten*'' mice, SHIP*' mice have no reported phenotype. This may imply a more important role for Pten activity in the immune system, and may be attributable to a number of factors including kinetics, subcellular localization or  26  expression levels of these proteins, or the efficacy of compensatory proteins such as SHIP2 and the recently reported TPIP (Walker et al., 2001). Indeed, a recent study evaluating suppressor and enhancer genes that alter the phenotype of Btk transgenic B cells supports a greater role for Pten in the regulation of proliferative pathways over that of SHIP (Satterthwaite et al., 2000). In this study, Pten heterozygosity was able to suppress a proliferative defect in B cells expressing reduced Btk protein levels, and this effect was comparable to that achieved by the complete absence of SHIP. The study of compound heterozygotes of genes within the same pathway has the potential to reveal what proteins regulate threshold levels for immune system activation. This approach was successfully used to study the dosage effects of various proteins downstream of the BCR, leading to the conclusion that limiting amounts of proteins within the same pathway have the potential to alter B cell responses both in vivo and in vitro (Cornall et al., 1998; Satterthwaite et al., 2000). We have utilized a compound heterozygote to address the role of changes in phosphatidylinositol phosphatase levels in vivo. The exacerbation of the Pten*'' autoimmune phenotype by the introduction of SHIP heterozygosity lends further support to the importance of PIP levels in immune system 3  function. It is intriguing that some of the mouse susceptibility genes for SLE are syntenic with human SLE susceptibility genes identified by linkage analysis (Roberton and Vyse, 2000). Cowden syndrome, a cancer predisposition syndrome in which patients carry mutations in PTEN, has not been reported to be associated with autoimmunity. Interestingly however, there has been a report of an individual with a mutation in PTEN suffering from both Cowden and Sjogren syndrome, the latter an inflammatory autoimmune disorder that is associated with a benign infiltrative lymphoproliferation and increased lymphoma risk (Raizis et al., 1998). Also of potential relevance, another study has identified a human SLE locus in 2q37 (Lindqvist et al., 2000), a region containing human SHIP.  It remains to be determined whether mutations in components of PI3K  pathways will be of any relevance to human SLE.  27  Chapter Three: Combined heterozygosity for Pten and SHIP results in increased T-dependent response to antigenic challenge and increased cytokine production by CD4 T cells. +  Author's contribution statement  This work was completed at the University of Calgary by Jennifer Moody with the exception of the PIP analysis. For those experiments T cells were isolated, 3  expanded, stimulated and flash frozen in Calgary. Samples were then shipped to Dundee, Scotland to Dr. Alex Gray in the lab of CP Downes. Lipid analysis was performed by Dr. Gray. Data analysis and statistics were performed by Jennifer Moody in Calgary.  3.1  Introduction The previous data in Chapter Two revealed that the introduction of a partial  deficiency SHIP heterozygosity onto the Pten heterozygous background resulted in quantifiable increases in the severity of the autoimmune characteristics of the Pten* ' 1  mice. The worsened immunopathology of the spleen, nodes and kidneys as well as the increased serum immunoglobulin levels and autoantibody titres provided convincing phenotypic evidence that tolerance mechanisms were more severely compromised in the compound heterozygotes than in either single heterozygous strain alone. Those studies did not however implicate a particular cell type or a specific biochemical defect that could be responsible for the increased severity of the autoimmune characteristics. Peripheral immune tolerance is maintained by several mechanisms, primarily in B cells and T cells.  Emigration of these cells from the bone marrow or thymus  respectively, throughout the body and into the peripheral lymphoid organs allows these cells to come into contact with professional antigen presenting cells (APC) such as dendritic cells and macrophages. Both B cells and T cells must be able to discern selfantigens from non-self antigens in order to mount an appropriate immune response.  28  Accordingly, these lymphocytes must proceed through selection processes during development to eliminate self-recognition. B cell development begins at the common lymphocyte progenitor stage in the bone marrow and reaches a critical checkpoint at the pro-B to pre-B stage (Janeway, 1999). At this point recombination of the antigen receptor loci results in the expression of the pre-B receptor.  A signal through this receptor in combination with IL-7  stimulation confirms a successful rearrangement and transmits a survival signal to the cell. It then progresses through further rearrangement of the antigen receptor producing a functional IgM antigen receptor also known as the BCR. The cell reaches the next checkpoint in the marrow at the transition from the IgM IgD'° to the IgM'°IgD ' hi  h  phenotype. At this stage the strength of signals through the BCR determines the degree of self-recognition, and high avidity signals result in negative selection. Interestingly, proper signaling through PI3K is critical for both of the above checkpoints. Mice deficient for the p85a regulatory subunit of PI3K suffer from a block in B cell development at the pro to pre-B cell stage implicating PI3K in the signals required for positive selection (Suzuki et al., 1999). However, mice transgenic for a constitutively active form of Btk, a known downstream effector of PI3K, suffer from a block at the IgM IgD'° to IgM IgD transition (Maas et al., 1999). The constitutive Btk signal was hi  l0  hi  proposed to have mimicked receptor occupancy with self-antigen at the negative selection checkpoint and accordingly resulted in apoptosis. Thus it seems that B cells must have appropriately strong PI3K signals to proceed through positive selection and appropriate attenuation of PI3K signals at the stage of negative selection. T cell development occurs in the thymus subsequent to the migration of T cell progenitors from the bone marrow. Early in T cell development cells are CD4CD8 (double negative). As antigen receptor rearrangement leads to the formation of the pre-T cell receptor the cells become CD4 CD8 (double positive). It is at this stage that the +  +  processes of positive and negative selection occur and it is the interaction between the mature T cell receptor (TCR) and major histocompatibility complex (MHC) molecules on the surface of thymic epithelial cells that is key to both of these processes (Janeway, 1999). Signals resulting from weak MHC:self recognition drive the positive selection of  29  T cells and lead to further differentiation into CD4 or CD8 T cells while strong +  +  recognition of MHC:self results in negative selection and apoptosis. While the methods of positive and negative lymphocyte selection are quite efficient, a fundamental defect exists in the fact that not all self-antigens are present, or at high enough concentrations, to evoke negative selection in the bone marrow or the thymus. Cells which are stimulated just enough to pass positive selection, but not enough to be eliminated by negative selection will enter the periphery as self reactive cells. Peripheral mechanisms of tolerance induction are therefore in place to compensate for this defect (Janeway, 1999). In T cells, stimulation through the TCR and co-stimulatory molecules on APC are required to initiate a T cell response. Stimulation through the TCR alone does not result in sufficient IL-2 production and leads to an unresponsive state known as anergy. This mechanism prevents the activation of T cells by MHC alone bearing self-peptides. Interestingly, it was recently demonstrated that deficiency for Cblb, a negative regulator of TCR signaling, resulted in an uncoupling of T cell activation from co-stimulation and resulted in autoimmunity in mice (Bachmaier et ak, 2000). This suggested a mechanism by which unrestrained cellular signaling could compromise peripheral tolerance mechanisms in T cells. Alternatively, B cells can become anergic when exposed to soluble circulating antigen. This response involves the down-regulation of surface IgM, thereby inhibiting BCR signals. Upon interaction with a corresponding self reactive T cell, these anergic B cells become highly susceptible to the apoptotic effects of Fas ligand:Fas receptor binding on their surface due to diminished antiapoptotic signals transmitted through the BCR. Self-reactive B cell clones can thus be eliminated in the periphery by Fas mediated apoptosis. A defect in this mechanism of tolerance induction has been clearly demonstrated in mice with mutations in either the Fas receptor (Ipr) or Fas ligand (gld), which both result in accumulation of lymphocytes and autoimmunity. Additionally, the lymphoproliferative syndrome and autoimmune characteristics seen in Pten '~ mice were in part attributed to defects in Fas mediated +  apoptosis (Di Cristofano et ak, 1999). In systemic autoimmunity such as lupus, end organ damage is the result of antibodies or immune complexes directed against self-antigens.  Generally these  30  autoantibodies show a high degree of somatic hypermutation and the B cells that produce them have been shown to have undergone clonal expansion (Janeway, 1999). These characteristics are indicative of the stimulation and expansion of B cell populations by auto-antigen specific CD4 T cells. CD4 T cells are known as helper T cells as they +  +  function to elicit B cell help in fighting pathogens. CD4 cells are born through positive +  selection in the thymus and leave the thymus as naive T cells. They circulate throughout the body and through the lymph nodes and spleen where they encounter APCs. Recognition of specific antigen together with a co-stimulatory molecule on the APC results in progression of the T cell into cycle, the production of IL-2 and the expansion and differentiation into armed effector T cells. CD4 cells can follow two differentiation +  pathways, to that of either a Thl or a Th2 effector cell. The direction of this differentiation is influenced by a number of factors including cytokine milieu and transcription factor expression (Figure 12 and reviewed in (Murphy and Reiner, 2002)). These effectors differ in the cytokines they ultimately produce and the cellular responses that these cytokines elicit. Thl cells produce IFN-y and TNFa, that can induce the production of opsonising antibodies by B cells and the activation of macrophages eventually leading to cell mediated immunity. Th2 cells produce IL-4, IL-13, and IL-5. These cytokines lead to B cell activation and the secretion of IgM, IgGl, IgG3, and IgE antibodies ultimately resulting in humoral immunity. By differentiation into either of these pathways, T helper cells influence the response of B cells. This is an important point because while a breakdown of tolerance in systemic autoimmunity requires selfantigen specific B and T cells, it is the B cells that are directed by T cell instruction. As described earlier, signal strength downstream of antigen receptors can dictate the outcome of T cell selection, activation and the downstream signaling consequences that result in effector function. Since it is well recognized that lymphocyte reactivity and tolerance induction is regulated by a balance of positive and negative signals (Goodnow, 1996), then it is not surprising that there is increasing evidence that a loss of negative regulation of TCR signaling can play a role in the initiation of autoimmune disorders (reviewed in (Ohashi, 2002)). In support of this hypothesis, alterations in PI3K signaling specifically in murine T cells, was shown to favor the emergence of autoimmune  31  phenotypes (Borlado et al., 2000; Parsons et al., 2001; Suzuki et ak, 2001). It was therefore of interest to investigate alterations in CD4 T cells from Pten*' SHIP*' mice for +  defects that could ultimately lead to the exacerbation of the autoimmune phenotype. In this chapter we examine the effects of antigenic challenge on young mice before the onset of overt pathology, and also examine biochemical differences in T cells isolated from mice at this age. The results of these studies indicate that partially reduced levels of the lipid phosphatases responsible for regulating PI3K activity are capable of altering T lymphocyte responses to stimulation.  3.2  Materials and Methods  Mice  Pten 'SHIP*' mice on a C57BL/6 background (N=7-8) were generated as +  previously described (Moody et ak, 2003), and were pathogenic virus antibody-free. All experiments were performed in accordance with CCAC guidelines for animal care in the double barrier facility at the University of Calgary.  Splenic CD4 T-cell isolation +  CD4 T cells were positively selected using the Miltenyi Biotech magnetic +  separation system as directed by the manufacturer. Briefly, splenocytes were isolated as above, depleted of red blood cells by ammonium cloride lysis and incubated with antiCD4-conjugated magnetic beads in MiniMACS buffer (PBS, 2 mM EDTA, 0.5% BSA) for 15 minutes at 4°C. Suspensions were passed through MS columns attached to the Miltenyi magnet to retain bead-bound cells. Columns were removed from the magnet and cells were eluted with 1 ml of MiniMACS buffer. Cells were washed and plated at a density of 3-3.5 x 10 cells/ml in either RPMI + 10% FCS or AIMV synthetic media, 6  with 3-6% IL-2 conditioned media (Gibco-BRL) in 24 well plates pre-coated overnight with 1.0 u.g/ml anti-CD3 antibody (2C11, BD Pharmingen). After 48 hours, cells were washed and plated in the absence of CD3 but presence of IL-2 for 5 days, with a 1:2  32  feeding of fresh media/IL-2 approximately every two days. Cells were used on day 8 after isolation.  Immunoblotting  Expanded CD4 T cells on day 8 after isolation were enumerated and re+  suspended in fresh AIMV media. 1 x 10 cells were stimulated with 10 u.g/ml soluble 7  anti-CD3 for the indicated times and were then lysed in phosphorylation solubilization buffer (PSB; 50 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), 100 mM NaF, 10 mM Na P 0 , 2 mM Na V0 , 2 mM EDTA (ethylenediaminetetraacetic 4  2  7  3  4  acid), 2 mM NaMo0 , 1% Triton X) in the presence of a protease inhibitor cocktail 4  (Roche Diagnostics). Protein concentrations were determined by Bradford method-based assay (Biorad). 80 pg total protein was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) at 150V and transferred to polyvinylidine difluoride (PVDF) membranes by electroblotting using a semidry transfer method. Filters were blocked then probed over night at 4°C with antibodies against Phospho(473)-Akt, Akt, Phospho-p42/44 MAPK, p42/44MAPK, Phospho-p38MAPK, p38MAPK, Phospho-STAT6, STAT6 (Cell Signaling), Pten (Cell signaling), SHIP (Santa Cruz, sc-8425) and subsequently incubated with HRP-conjugated secondary antibody (Dako). Proteins were detected by chemiluminescence (Amersham), using a Fluor-S Max Multi Imager equipped with densitometry software (Bio-Rad Laboratories).  Antigen specific immunizations  Six to seven female mice of each genotype between the ages of 8-10 weeks were selected for this study. Blood samples were taken on day 0 before immunization by saphenous puncture. Mice were injected intra-peritoneally with 20 u.g NP-Ficoll or lOug NP-KLH (Biosearch Technologies Inc). Mice injected with NP-Ficoll were boosted with an additional dose of 20 u.g NP-Ficoll on Day 7. A final blood sample was obtained from these mice on day 17 by vena puncture post sacrifice. Mice injected with NP-KLH were  33  boosted with an additional 10 u.g of NP-KLH on day 10. A final blood sample was taken from these mice on day 21. ELISAfor serum immunoglobulin, cytokine and NP-specific antibody production  ELISA quantitation of serum immunoglobulin levels was performed as previously described using rat-capture and biotinylated detection antibodies for IgGl, IgG2a and IgE (Moody et al., 2003)(BD Pharmingen). Concentrations were determined from curves generated from mouse Ig standards (BD Pharmingen). Cytokine ELISA on cell supernatants were performed in an analogous manner using rat-capture and detection antibodies to IL-4 and IFN-y along with recombinant standards (BD Pharmingen) and IL13 (R&D Systems). For NP-specific antibody detection, 96 well plates were coated with 1 u.g/ml NP-BSA (Biosearch Technologies Inc) in PBS overnight at 4°C. Serial dilutions of sera using PBS+10%FCS were performed and plated in duplicate. Dilutions ranged from 1/100- 1/1000 for Day 0 samples, to 1/20,000-1/2,560,000 for day 21 samples. Plates were washed and then incubated with 2 u-g/ml of biotin conjugated secondary antiIgM (BD Pharmingen), or with a 1/10,000 dilution of pan-anti-IgG (Jackson Immunoresearch). Plates were developed with TMB substrate and were stopped with 2M H S0 and read at 450nm using a Multiscan ELISA reader and Multiscan software. Each 2  4  plate contained blank wells, and the NP titres were defined as the dilution that gave an OD  450  twice that of the background of the plate.  RNAse protection Assay  CD4 positively selected and expanded T cells were plated in fresh AIMV media +  at a concentration of 1 x 10 cells/ml, 0.5 ml/well in a 24 well plate. Cells were either left 7  unstimulated or were stimulated from 2-8 hours with 10 u.g/ml soluble anti-CD3. After various time points, cells were harvested and the supernatants were stored at -20°C and reserved for ELISA analysis. RNA was extracted as per TRIZOL manufacturer instructions (Ambion). Approximately 2 u,g RNA was utilized in the RNAse protection assay kit (BD Pharmingen, Kit mCk-1), as per manufacturer's instructions and using P 33  34  as a radiolabel. Developed film was analyzed densitometrically using the F l u o r - S - M a x system using Quantity One software. Control for loading was achieved by normalizing to the L 3 2 or G A P D H internal control bands and values were expressed as fold increase over wild type levels at each time point.  Intracellular staining and apoptosis For assessments o f cell viability and apoptosis, expanded T cells prior to and after 12 hours stimulation with soluble anti-CD3 were washed in ice cold P B S and stained with 0.5 u.g propidium iodide and 5 u l A n n e x i n V ( B D Pharmingen) for 15 m i n i n the dark. Un-gated samples were collected on a B D F A C S c a n and analyzed with F l o w J o software.  For intracellular staining, expanded T cells were stimulated with 10 p,g/ml  soluble a n t i - C D 3 for 5 hours in the presence of G o l g i Stop ( B D Pharmingen). Intracellular staining was performed using the B D Pharmingen kit as per manufacturer's instructions, and permeablised cells were stained with PE-anti-IL-4 or FITC-anti-IFN-y ( B D Pharmingen). In each experiment 10000 ' l i v e events', based on forward and side scatter pattern, were collected for analysis.  PIP analysis 3  Expanded T cells were starved for 3 hours in R P M I media + 2% F C S and were then stimulated with 10p.g/ml soluble anti-CD3 for various times using 10 cells/100 (al 7  for each time point. Reactions were stopped with 300 u.1 of 0 . 5 M trichloroacetic acid ( T C A ) and flash frozen on dry ice. Samples were shipped on dry ice to D r . A l e x Gray in the laboratory of Dr. C P . Downes, where whole lipids were extracted and the samples were analyzed for P I P levels according to a published F R E T technique (Gray et ak, 3  2003). P I P levels were extrapolated from a standard curve generated for each experiment 3  and values were normalized to the total extracted lipid per sample.  35  3.3  Results  Pten* 'SHIP* ' mice demonstrate a significantly increased T-dependent antigenic 1  1  response.  Our previous studies demonstrated  spontaneous  elevations of serum  immunoglobulin levels in older Pten* 'SHIP* mice with lymphadenopathy (Moody et al., 1  1  2003). Searching for evidence of an intrinsic abnormality that might have a bearing on the eventual development of hypergammaglobulinemia, we therefore assessed humoral responses of young mice in response to defined antigenic challenges. Day 0 baseline titres for both K L H and Ficoll immunized groups showed no significant reactivity directed against NP-BSA in serum from any of the groups of pre-immunized mice (Figures 13,14). Titres of NP-specific antibodies in the serum of mice immunized and boosted with NP-Ficoll showed no significant differences in the IgM or IgG response between genotypes (Figure 13a,b). In contrast, a comparison of the different groups immunized with the T-dependent antigen NP-KLH, revealed a statistically significant increase in the IgM responses of Pten* 'SHIP* ' mice over the other genotypes (p<0.05, 1  1  Figure 14a). Pten*'SHIP*' IgG responses to NP-KLH were also significantly elevated over those of wild-type and SHIP* mice (p<0.005), but these elevations were not 1  significant when compared to Pten* ' samples. The latter were statistically increased over 1  wild type and SHIP* ' (p<0.05) (Figure 14b). These results demonstrated that Pten* ', and 1  1  Pten* SHIP* mice in particular, were able to mount more vigorous humoral immune 1  1  responses after immunization with a T-dependent antigen. Pten and SHIP protein levels are reduced in T cells from heterozygous and doubly heterozygous mice.  Both Pten and SHIP are known to be expressed endogenously in T cells (GjorloffWingren et ak, 2000; Liu et ak, 1998b), however, a reduced level of these proteins in cells from the heterozygous mice has not been demonstrated. While loss of one codominant allele can lead to an ~50% reduction levels of the encoded protein, the possibility of allelic compensation remains. Thus, before carrying out a phenotypic  36  analysis of in v/fro-expanded CD4 T-cell populations, it was important to assess Pten +  and SHIP protein levels in lysates prepared from these cells. Figure 15 demonstrates reduced levels of Pten, and SHIP, in lysates prepared from Pten* ' and Pten*' SHIP* ', and 1  1  SHIP*' and Pteri' SHIP*' cells, respectively. Furthermore, levels of Pten and SHIP, both before and after anti-CD3 stimulation, revealed no change in the levels of either protein as a result of anti-TCR activation (data not shown). Pten*' SHIP*' T cells do not show discernable increases in PIP levels before or after 3  stimulation with anti-CD3.  As Pten and SHIP share the same substrate PIP and elevated levels of this species 3  are hypothesized to be responsible for the phenotypic alterations, we sought biochemical evidence for this increase in the purified, expanded T cell populations. To achieve this, we used a novel fluorescence resonance emission tomography (FRET) technique that has been recently published (Gray et al., 2003). It is an assay based on the competitive interaction of the PIP specific PH domain of GRP1 with lipid bound to an 3  allophycocyanin (APC) molecule versus unlabelled lipid in the sample being tested. A diagrammatic representation of how the assay works is presented in Figure 16. T cells were stimulated with anti-CD3 for various time points. The results of the combined data from 3 independent experiments expressed as fold increase over wild type levels are shown in Figure 17. These experiments failed to demonstrate statistically significant results between genotypes at any time point due to variability within and between experiments. There were however trends beyond the five minute point which suggested that increases over wild type PIP levels may exist after TCR stimulation in the Pteri*'' 3  and Pten*'SHIP*' T cells. Expanded CD4* T cells from Pten*' SHIP* ' mice show modest increases in Akt 1  phosphorylation.  Since Akt is a well-known downstream target of PIP and PI3K activation 3  (reviewed in (Nicholson and Anderson, 2002)) and since Akt phosphorylation at serine473 (P-Akt) can be directly correlated to its activity (Jones et al., 2000; Scheid et  37  al., 2002), we examined Pten*'SHIP* T cells for differences in the phosphorylation of 1  Akt. Figure 18(a) is a representative blot of these results, showing no significant difference in phospho-Akt (P-Akt) levels in unstimulated cells, and an increase in P-Akt in Pten*'' and Pten*''SHIP* ' lysates after 10 and 20 minutes of stimulation with anti-CD3 1  antibody. Figure 18(b) represents the combined densitometric results of four experiments that revealed consistent increases in the ratio of P-Akt/total Akt in the Pten*'SHIP*' cells compared to the other genotypes after 20 minutes of anti-CD3 stimulation. When the data from the four experiments were pooled together, the differences in phosphorylation of Akt in Pten*'SHlP*' lysates were not dramatic, especially compared to that seen in Pten* ' lysates. However, these results did indicate that signaling pathways downstream 1  of PI3K could be subtly affected by double heterozygosity for Pten and SHIP.  Pten*'SHIP*' T cells respond to anti-CD3 stimulation by producing greater amounts of IL-4, IL-13 and IFN-y at the RNA and protein levels.  The augmented humoral response to a T-dependent antigenic challenge suggested that T-helper cell function might be abnormal in Pten*'SHlP*'  mice.  We thus  hypothesized that T helper cell cytokine production may be altered as a result of compound heterozygosity for Pten and SHIP. Using an RNAse protection assay template that allowed quantitation of 9 individual cytokines (IL-4, IL-5, IL-13, IL-10, IL-2, IL-6, IL-15, IL-9, and IFN-y), we examined the pattern of cytokines produced by expanded Tcell populations following anti-CD3 stimulation. In three independent experiments we observed increases in IL-4, IL-13 and IFN-y transcripts in RNA samples from Pten* ' 1  SHIP*' cells, as compared to cells from the other genotypes (Figure 19 and Figure 20). The data shown represent typical results obtained 8 hrs post-stimulation, and the relative increases are over wild-type cytokine RNA levels. While there was some variation in the time point at which cytokines reached their maximum level after stimulation (data not shown), increases were most often observed at the 8 hr point. No consistent differences in the levels of transcripts of any of the other cytokines were observed. To determine whether increases in transcripts would be reflected at the protein level, cytokine secretion into the supernatant was quantitated by ELISA. Analysis of  38  supernatants from 4 independent experiments revealed consistent and significant increases in the amounts of IL-4 and IL-13 protein in the supernatant of Pten*' SHIP* ' 1  cells (Figure 21a,b, p<0.001 over all genotypes). These increases over wild type averaged to approximately 14-fold, and 3-fold, for IL-4, and IL-13 secretion, respectively. We detected a 1.5 fold increase in IFN-y secretion compared to wild-type cell supernatants, and this increased production was statistically significant over that of wild-type and Pten*' supernatants (Figure 21c, p<0.05, not significant over SHIP*'). These results demonstrated at both the transcript and protein levels that cultures of Pten*' SHIP*'' cells produced greater quantities of three key T-helper cell cytokines following anti-CD3 stimulation, with the increase in IL-4 production being particularly impressive. Increased cytokine production is not attributable to increased survival or spontaneous altered polarization of the T cell population.  The ability of PI3K pathway activity to increase cellular survival has been well characterized, primarily as a function of Akt activation (reviewed in (Cantley, 2002)). Having previously demonstrated modest increases in Akt phosphorylation in T cells from Pten*'SHIP*' mice (Moody et al., 2003), it was possible that increased survival of cells might be a factor contributing to the differences in cytokine concentrations in supernatants. To investigate this, we examined the viability of expanded T cells from each of the different genotypes, both with and without anti-CD3 stimulation, using annexinV/PI staining and flow cytometry. We found that cell viability did not differ between the genotypes neither without stimulation (Figure 22a-d), nor after 12 hours of stimulation (Figure 23a-d). Thus it was not possible to attribute increased cytokine production to an increased survival of Pteri''SHIP*' cells. As the increases in IL-4 and IL-13 secretion were of greater significance over all genotypes than the increases seen in IFN-y, we postulated that the T cell population might have been skewed towards a greater proportion of Th2 cells. Although these T cells were expanded under neutral conditions, the question of whether genotype had effected spontaneous polarization needed to be addressed. We thus examined cytokine production using an intracellular staining method for IL-4 and IFN-y. Figure 24 (a-d) illustrates the  39  results typical of at least four independent experiments and reveals no significant differences in the percentage of IL-4 or IFN-y producing cells between genotypes. Thus, the increased production of cytokines by Pten SHIP ~ cells could not be accounted for +/  +/  by an abnormality of T-cell polarization. Instead, it indicated an increase in cytokine production by cells of this genotype on a 'per cell' basis. However, since our FACS analysis did not reveal an upward shift in intracellular IL-4 staining intensity in Pten ' 1  SHIP ~ cells, increased duration of secretion by these cells might account for the higher +/  levels of IL-4 seen in the ELISA. Pten' SHIP 1  +/  cells do not show differential phosphorylation ofSTAT6, p42/44MAPK or  p38MAPK before or after stimulation with anti-CD3.  As our ELISA data had shown a 14-fold increase in protein levels of IL-4, and our ICS data suggested that this may be a function of increased production on a per cell basis, we postulated that this increase may be the result of over-activation or prolonged activation of signaling pathways.  We therefore initiated studies to investigate the  activation of a number of signaling pathways that have been implicated in IL-4 production. STAT6 plays a key role in the production of IL-4 by T cells (reviewed in (Murphy and Reiner, 2002). It is phosphorylated and activated by Jak2 in response to an initial source of IL-4 and is responsible for the initiation and maintenance of IL-4 production. We thus examined T cell lysates from our mice for indications that STAT6 may be differentially phosphorylated in Pten ' SHIP +  +/  cells. Preliminary experiments  revealed that the levels of phosphorylated STAT6 did not change with anti-CD3 stimulation (not shown). Figure 25a therefore illustrates the levels of phosphorylated and total STAT6 seen in unstimulated expanded T cells. Four independent sets of lysates were evaluated in this manner and no consistent increases in STAT6 phosphorylation could be deduced in Pten 'SHIP 'T +I  +/  cell lysates (Figure 25b). This suggested that the  increases in IL-4 production were not a result of augmented phosphorylation of STAT6. Recent work implicating the p42/44 MAPK pathway in TCR induced production of IL-4 in T cells (Jorritsma et ak, 2003) suggested that altered activation of this pathway  40  may be responsible for the increases in IL-4 production seen in our  Pten 'SHIP 'cells. +/  +  We thus examined the phosphorylation status of p42/44MAPK (P-MAPK) in T cell lysates before and after stimulation with the same dose of anti-CD3 used in the RPA and ELISA experiments. Figure 26(a) is a representative western blot showing no significant difference in p42/44 MAPK phosphorylation before or after 30 minutes of stimulation with 10 (xg/ml soluble anti-CD3. The results of four independent western blots are shown in Figure 26(b) which demonstrates the combined densitometric ratios of PMAPK:MAPK and illustrates no difference between genotypes. There is also evidence in the literature that p38 M A P K can influence IL-4 secretion by mouse T cells in response to TCR or TCR/CD28 stimulation, and that a specific inhibitor of p38 MAPK, SB203580, inhibits IL-4 production in a dose dependent manner (Zhang et al., 1999). We therefore examined the phosphorylation of p38 MAPK (P-p38MAPK) in response to this anti-CD3 stimulation. Figure 27(a) is a representative western blot that demonstrates no significant difference in the phosphorylation kinetics of p38MAPK before stimulation or over a time course of 20 minutes of anti-CD3 stimulation.  Figure 27(b) are the results of combined densitometry ratios of P-  p38MAPK:p38MAPK and illustrates no differences between genotypes.  These  experiments indicated that combined heterozygosity for Pten and SHIP did not result in obvious alterations in the above signaling pathways and that the increases seen in IL-4 production were likely the result of an alternate mechanism. Increased levels oflgGl and IgE but not IgGla in young Pten SHIP ' mice. +/  +/  Given the ability of Pten* SHIP* T-cells to generate increased levels of IL-4 and 1  1  IL-13, we sought evidence for this phenotype in vivo. IL-4 is involved in class switching to the IgGl and IgE isotypes (Kuhn et ak, 1991), and IL-13 may play a role in IgE switching in mice (McKenzie et ak, 1998) while IFN-y is responsible for the induction of IgG2a. By ELISA, we examined immunoglobulin isotypes in serum from 8 wk old mice, a time well in advance of any clinical signs of lymphoproliferative disease. There were increases in serum IgGl in Pten 'SHIP ~ mice that were significant over all genotypes +I  +I  (Figure 28a). Increases in IgE that were significant over SHIP* ' and wild-type mice, were 1  41  also seen, although these were not significant over Pten* ' samples (Figure 28b). Serum 1  IgG2a levels were quite variable within each group and did not show any significant differences between genotypes (Figure 28c). The pattern of spontaneous Ig isotypes in young mice was thus in keeping with the ability of the stimulated Pten" SHIP* ' T-cells to 1  produce greater amounts of IL-4 and IL-13 following in vitro activation with anti-CD3.  3.4  Discussion The finding that Pten heterozygosity augmented the IgG response to a T-  dependent antigen is novel, and likely plays a role in the age-dependent hypergammaglobulinemia seen in these mice (Di Cristofano et al., 1999).  The  heightened IgG and IgM response seen in the Pten*' SHIP* ' mice to the same antigen is 1  indicative of an even greater response by these mice, and is in keeping with the spontaneous increases in both IgM and class switched IgG isotypes that we previously observed (Moody et al., 2003). It is interesting to contemplate these results in light of the experiments that showed an increased IgG response to a T-independent antigen in mice transplanted with SHIP ' cells (Helgason et al., 2000), and implied a heightened B cell 1  responsiveness independent of T cell help. Those experiments were done in the context of a different mouse background; however, the discrepancy is consistent with many differences seen between mice deficient or conditionally deficient for SHIP or Pten and likely is due to currently unclear factors affecting antigenic responsiveness that may be independent of PIP . 3  The possibility of increased B cell response to T cell help was not examined in this study, and cannot be excluded. In fact, given the known co-operativity between B cells and T cells in the development of other models of murine lupus (Sobel et al., 2002), intrinsic defects in the B cells of Pten*' SHIP*'' mice may exist. However, the increased response of Pten*', and especially Pten*'SHIP*' mice towards the NP-KLH in the absence of a differential response in T-independent responses suggests that reduced levels of inositol phosphatases have a greater impact on T cell help than on B-cell responses. This lead us to hypothesize that biochemical defects specifically in Pten*'' SHIP*' T cells may be detectable and may contribute to the manifestation of the more  42  severe autoimmune characteristics described in Chapter two. The case for the importance of T cells in the development of autoimmunity is supported by the autoimmune characteristics described in the T cell specific knockout of Pten (Suzuki et al., 2001). In this model, T cells were found to be autoreactive and produced greater amounts of cytokines.  Furthermore, the mice developed autoantibodies  and hyper-  gammaglobulinemia. One caveat of the Suzuki study was that it was done in the context of B cells that were heterozygous for Pten, and for this reason the potential contribution of B-cell dysfunction to this process could not be excluded. Additionally though, there is accumulating evidence that heightened T cell responses induced by the de-regulation of PI3K signaling in T cells alone can result in the development of autoimmune characteristics. For instance, constitutive active PI3K expressed in transgenic mice by the T cell specific L C K promoter resulted in lymphadenopathy and autoimmune mediated glomerulonephritis (Borlado et al., 2000). Likewise, a transgenic model in which constitutively active PKB was placed under the control of the CD2 promoter resulted in lymphadenopathy and autoantibody production (Parsons et al., 2001). We thus focussed our attention on detectable biochemical defects in T cells from Pten* ' 1  SHIP*' mice. Given our hypothesis that it was the decreased regulation of PIP that was 3  responsible for the phenotypic changes, differences in the levels of this inositol species before and after stimulation was of great interest. We attempted to analyze the levels of PIP in T cells in these studies and were unsuccessful in demonstrating significant 3  differences. While trends of increased PIP levels were observed, the variability in our 3  samples precluded statistically relevant results. It is likely, given the lack of a phenotype in SHIP*' mice, that the effects on PIP levels would have been subtle and difficult to 3  detect. While the use of sensitive non-radioactive techniques that do not require metabolic labeling have improved phospholipid analysis for primary cells recently (Gray et al., 2003), the detection of what was anticipated to be subtle changes in PIP levels has 3  remained a challenge in primary cells from these mice. It is also important to consider that these experiments were based on the analysis of whole lipid extracts from the cells. A recent study utilizing the transgenic expression of the Akt PH domain conjugated to  43  GFP has demonstrated that PIP levels in primary T cells are distinctly localized to the 3  area of interaction with an APC and curiously at the opposite pole from this synapse (Costello et al., 2002). Thus, differences in PIP levels may be critical in localized 3  microdomains that are unappreciated in the approach we have taken in these experiments. As an indirect measurement of PIP levels, we analyzed the phosphorylation of 3  Akt, a molecule that depends on PIP and PI(3,4)P for its activation (Scheid et ak, 2002). 3  2  Using the same stimulation as was used for the PI analysis, we were also only able to observe subtle increases in the phosphorylation of Akt in Pten '~SHIP '~ T cell lysates +  +  particularly compared to Pten* ' lysates. Again, in anticipating subtle changes induced by 1  the introduction of SHIP heterozygosity, this was not incomprehensible. One might expect that chronic amplification of signals in immune cells could lead to immune disregulation over time in our mice. It is also possible that subtle, hard to detect changes in immediate early signaling events could translate into amplified signals downstream. In keeping with this hypothesis, we were able to reveal significant differences in our analysis of downstream events, namely in cytokine RNA and secretion amounts. These experiments were performed using the same stimulation as the PI and Akt experiments and suggest that while differences in early events were difficult to detect with this stimulation, downstream events were much more obvious. The increases seen in cytokine production at the RNA and protein level could not be attributed to increased survival of Pten 'SHIP +/  +/  cells as the measurement of cell death  by AnnexinV/PI staining, either with or without stimulation, revealed no differences between genotypes. An increased survival capacity was almost expected in these experiments in light of previous experiments describing a resistance to activation induced cell death in Pten ~ T cells (Di Cristofano et ak, 1999). However, these previous +/  experiments were performed on CD3-enriched splenocytes and not on T cells that had been expanded over time in the presence of IL-2. Our attempts to study Fas induced apoptosis in our expanded cells resulted in no significant differences between genotypes either (data not shown), implying that our in vitro expansion process may not recapitulate conditions by which early data was obtained by other groups.  44  Results from these experiments, at least with respect to IL-4, also implied that the greater amounts of cytokine produced may be a function of increases on a per cell basis or in the duration of secretion. We hypothesized that this increase could arise from more intense or prolonged activation of signaling pathways implicated in IL-4 generation. An examination of a number of pathways known to have roles in IL-4 production did not reveal significant differences in their activation. Biochemical analysis of other pathways implicated in IL-4 production remain to be investigated as does the hypothesis that subtle signaling changes may converge on a number of transcription factors involved in IL-4 transcription such as API, NFAT, and c-Maf. A future focus would be to assess the phosphorylation status of GSK-3, a signaling molecule that is a downstream target of Akt. The results of these experiments would be of particular interest with respect to IL-4 transcription, as GSK-3 is responsible for the re-phosphorylation of the transcription factor NFAT. NFAT is permitted to enter the nucleus upon its de-phosphorylation by calcineurin and its re-phosphorylation by GSK-3 serves to shuttle NFAT out of the nucleus (Beals et al., 1997), negatively regulating its transcriptional activity. As GSK-3 function is inhibited by phosphorylation by activated Akt, mechanisms downstream of PI3K could feasibly result in increased nuclear activity of NFAT. An alternate explanation for the increased cytokine production lies in the possibility of altered mRNA stability. This was recently shown to be responsible for the strain differences in IL-4 expression between C57B1/6 and DBA/2 mice (Butler et al., 2002). Although the mechanism for the increased mRNA stability was not determined in this study, it would be interesting to see if message stability is a factor in our results. In this regard, despite the detection of increases in IL-4, IL-13 and IFN-y at earlier time points in our RNAse protection assays, the 8 hr time point was found to be the most consistent, suggesting that message stability may be important to examine. In addition to underscoring the importance of tight regulation of PI3K activity in controlling T cell responses, this work also sheds light on a role for SHIP in T cell responses, a role that has been elusive. There is evidence that SHIP is able to regulate phosphoinositol metabolism in T-cells, in that Jurkat cells deficient for both Pten and SHIP have much higher PIP levels than cells deficient only for Pten (Freeburn et al., 3  45  2002). Furthermore, re-introduction of SHIP in Jurkat cells increased the levels of PI(3,4)P and could decrease PKB localization to the membrane. 2  The in vivo role of SHIP was also studied via the generation of SHIP' RAG1' mice, which yielded B- and T-cells deficient for SHIP (Liu et ak, 1998b). All assays of Tcell function in this study were found to be similar to those of wild-type cells, save for a slight increase in the numbers of CD4 cells in the SHIP' mice. In contrast, a study of +  primary cells from SHIP knockout mice revealed increased chemotaxis by CD4 and +  CD8 thymocytes, as well as CD4 splenic T cells, indicating that the ability to detect +  +  phenotypic differences was dependent on the assay used (Kim et ak, 1999). Our study reveals that partial deficiency for SHIP alone has little effect on T cell behaviour, however, in combination with a decrease in Pten expression it can alter the threshold required for cytokine production in response to stimulation. We propose this as evidence that Pten and SHIP work co-operatively in these processes. The generation and comprehensive study of mice conditionally deficient in SHIP specifically in T cells will likely more conclusively define SHIP'S role in this cell type. Furthermore, a comparison of these cells with T cells from the conditional Pten deficient mice (Suzuki et ak, 2001) might allow dissection of functional differences stemming resulting from alterations in PIP versus PI(3,4)P intracellular pools. 3  2  46  Chapter Four: The inositol phosphatases SHIP and Pten work cooperatively in maintaining hematopoietic homeostasis.  Author's contribution statement  These experiments were carried out by Jennifer Moody at the University of Calgary with the exception of the reconstitution experiments. For these experiments the mice were bred and sacrificed in Calgary, femurs were shipped to Vancouver where cells were transplanted into irradiated hosts at the BC Cancer Agency under the supervision of Dr. Cheryl Helgason. All FACS data and histology on reconstituted mice were collected in Vancouver. The statistical analysis of the data and examination of the histology slides were performed by Jennifer Moody in Calgary.  4.1  Introduction  Hematopoiesis is a hierarchical process that is sustained in adult marrow by a population of pluripotent stem cells. These cells are capable of long term reconstitution of all hematopoietic cell lineages through their ability to self-renew, proliferate and eventually differentiate into progeny with defined potential. Differentiation and proliferation are driven by various cytokines and growth factors that are produced by marrow stromal cells and include IL-3, GM-CSF, IL-6, and IL-1. As differentiation progresses the cells become committed to a particular lineage and differentiation from these committed progenitor cells, influenced again by various cytokines and growth factors, leads to the formation of all blood cells including erythrocytes, platelets, granulocytes, monocytes, B cells, T cells and NK cells (Figure 29). The ability of bone marrow stem cells to respond to cytokine and growth factor stimulation is a critical aspect in maintaining steady state hematopoiesis as well as in the swift response to hematopoietic crisis. The PI3K pathway has been shown to be critically important in the development of certain hematopoietic cell types inasmuch as mice deficient for the p85a regulatory subunit of PI3K show developmental defects in both the B cell and erythroid lineages  47  (Fruman et al., 1999; Huddleston et al., 2003; Suzuki et ak, 1999). Moreover, PI3K is well poised to play a critical role in hematopoietic processes by virtue of its activation downstream of various cytokine receptors. Cytokines can use distinct mechanisms to initiate PI3K activation. For instance, SF and M-CSF are receptor tyrosine kinases that are capable of direct interaction with the SH2 domain of the regulatory subunit of PI3K. Alternatively, the IL-3 and GM-CSF receptors activate PI3K through a complex mechanism initiated by phosphorylation of their common (3 receptor chain by Jak kinase (reviewed in (Fruman and Cantley, 2002)). The importance of negative regulation of PI3K signaling in hematopoiesis has been suggested by the myeloproliferative disorder and by the B cell and erythroid developmental defects in mice completely deficient for SHIP (Brauweiler et ak, 2000; Helgason et ak, 1998; Helgason et ak, 2000; Huber et ak, 1999). Furthermore, committed progenitor cells from SHIP ' mice demonstrated an increased clonogenic sensitivity to 1  growth factors such as GM-CSF, SF and IL-3 (Helgason et ak, 1998). Additionally, the absence of this lipid phosphatase had a significant impact on hematopoietic stem cell regeneration as the SHIP ' stem cell compartment was shown to have a higher fraction of 1  proliferating cells and a defective ability to regenerate in vivo (Helgason et ak, 2003). Given the effects of Pten haploinsufficiency on cells in the peripheral immune system (Di Cristofano et ak, 1999; Podsypanina et ak, 1999), we hypothesized that partial deficiency for Pten might similarly disturb some aspect of hematopoiesis, a presumption that had not been previously described or examined in any study of Pten ~ mice. As it +/  was possible that in sharing the same substrate, PIP , that SHIP might compensate for a 3  partial deficiency of Pten, we also examined the effects of Pten heterozygosity in the context of complete deficiency for SHIP. In this instance it was hypothesized that PIP  3  metabolism would be further dysregulated, perhaps exacerbating the SHIP' phenotype. Lastly, given the potent tumour suppressor function of Pten, the myeloproliferative-like syndrome of SHIP' mice, and the possible role for SHIP in leukemic transformation (Jiang et ak, 2003; Luo et ak, 2003), we hypothesized that the combination of Pten heterozygosity with a complete deficiency for SHIP might promote.leukemogenesis.  48  4.2  Materials and Methods  Mice  Pten* ' and SHIP' mice were generated as previously described (Helgason et al., 1  1998; Podsypanina et al., 1999) and have been backcrossed to the C57BL/6 background (n=6-7) in our laboratory. Pten SHIP' mice were generated by intercrosses of Pten* ' +/  1  SHIP*' (Moody et a l , 2003) and SHIP* ' mice. Locus specific polymerase chain reaction 1  (PCR) was used to genotype the mice. The congenic C57BL/6-Ly-Pep3b (Pep3b; The Jackson Laboratory) strain was used as transplant recipients.  The strains are  phenotypically distinguishable on the basis of allelic differences at the Ly5 locus: C57BL/6 mice express Ly5.2 whereas Pep3b express Ly5.1. Irradiated animals were provided with acidified water (pH 3.0) to prevent bacterial growth. Strains were bred and maintained at either the University of Calgary double barrier facility or at the British Columbia Cancer Research Center animal facility in accordance with protocols approved by the Animal Care committee at the University of Calgary and University of British Columbia. Blood analysis  For the Calgary experiments, mice were killed by an overdose of Avertin, and blood samples obtained by cardiac puncture were transferred to microtainer tubes containing EDTA. Samples were analyzed for RBC, WBC, platelets, hemoglobin and hematocrit on a Coulter counter. Peripheral blood smears were stained with WrightGiemsa and scored morphologically (100 cells/slide). For the data obtained in the transplant experiments in Vancouver, the mice were killed and blood was obtained by cardiac puncture. The blood was transferred to heparinized tubes to prevent clotting. WBC counts were enumerated by dilution of whole blood in 3% acetic acid/PBS. RBC counts were enumerated by dilution in PBS. Clonogenic assays for committed hematopoietic progenitor growth  Single cell suspensions were obtained by flushing femurs with ct-MEM media/2% FCS or by pressing spleen or liver segments in a-MEM media/2% FCS through a wire  49  mesh. Un-fractionated B M cells, spleen or liver cells were plated in 1.1 ml of complete methylcellulose media (in duplicate) supplemented with EPO, IL-3, IL-6, and SCF (M3434, StemCell Technologies). Dishes were incubated for 10-12 days at 37°C, 5 % C 0 , 95% humidity. For measurement of Pre-B progenitor growth, un-fractionated bone 2  marrow cells were plated in 1.1 ml of 1% methylcellulose media supplemented with rlL7 (M3430, StemCell Technologies), in duplicate and were incubated for 6 days at the above conditions.  Growth factor dose responses were performed using a 1%  methylcellulose media (M3234, StemCell Technologies) supplemented with various concentrations of either GM-CSF, IL-3, SF (R&D) and incubated as above for 10-12 days. All colonies >20 cells were scored morphologically using a gridded stage on an inverted microscope. FACS analysis and lineage depletion  Single cell suspensions were obtained by flushing femurs with a-MEM media/2% FCS. Cells were washed with PBS, blocked with 2 u.g/ml anti-FcyRIIB antibody (2.4G2, BD Pharmingen) and subsequently stained with FITC, PE, or PerCP-conjugated antibodies against C D l l b , CD41, NK1.1, B220, CD43, and IgM (BD Pharmingen). Terl 19 was obtained as a biotin-conjugate and was visualized using a Streptavidin-FITC antibody (BD Pharmingen). A minimum of 10,000 events were analyzed using a FACScan (Becton Dickinson, Mountain View, CA) flow cytometer equipped with CellQuest software (Becton Dickinson). Data was analyzed using FlowJo software. Lineage-depleted samples were collected from both femurs per mouse using the StemSep isolation protocol and reagents (StemCell Technologies). The cells in the flow-through were enumerated, blocked with anti FcyRIIB antibody and then stained with PE or FITC-conjugated antibodies against c-kit and Seal (BD Pharmingen). Cells were analyzed as above. Multilineage Repopulation analysis  Lethally irradiated Pep3b (Ly5.L) recipients (900 cGy of Cs y irradiation) were l37  injected with lx 10 cells per mouse of wild type, Pten '\ SHIP' or Pten"-SHIP' bone 6  +  50  marrow cells (2-4 independent donors per genotype, 4-8 recipients per donor). Donorderived contributions were evaluated by monitoring the percentage of Ly5.2 -B-lymphoid +  (B220 ), T-lymphoid (CD-3 ),or myeloid cells (Macl ) in the periphery at 4 weeks post +  +  +  transplant by FACS. Statistical Analysis  Statistical significance was performed using a one-way ANOVA combined with a Tukey test for multiple comparisons in all experiments comparing 4 genotypes. A Student's t-test was utilized in the transplantation data that only compared recipients of wild-type and Pten*'SHIP' cells (weights, WBC, RBC and femoral Ly5.2 cells). +  4.3  Results  Peripheral blood abnormalities and extramedullary hematopoiesis in Pten*'SHIP' mice  Previously, it was reported that the SHIP'' mice having a mixed 129J-C57BL/6 background survived approximately 14 weeks, and occasionally up to a year (Helgason et al., 1998). Subsequent back-crossing onto the C57B1/6 background resulted in a more severe phenotype, such that by the n=6 backcross, SHIP'' mice were dying by 6 to 12 weeks of age ((Helgason et al., 2003) and our unpublished observations). Although showing runting and decreased body weight similar to that of SHIP' littermates (Figure 30), our colony of Pten*'SHIP' mice rarely survived beyond 5 weeks of age. In an examination of the peripheral blood of all four genotypes, it was apparent that Pten haploinsufficiency did not have an effect on the circulating blood counts as Pten*'' mice did not differ from wild-type mice in any parameter measured (Table 4). The SHIP'' mice on the other hand showed significant anemia compared to wild-type and Pten*'' mice, and showed a correspondingly significant decrease in their hemoglobin and hematocrit levels (Table 4, p<0.05). This contrasts with data previously reported for SHIP'' mice on a mixed background (Helgason et al., 1998), and may reflect an increased severity of the in vitro erythroid defect seen in those studies with subsequent backcrossing to.the C57BL/6 background. However, and perhaps as a contributing factor  51  in their early demise, the peripheral blood analysis of Pten*' SHIP  1  mice between the  ages of 4 and 5 weeks revealed significant normocytic anemia as well as thrombocytopenia compared to all genotypes including SHIP' (Table 4). This was accompanied by a significant leukocytosis (Table 4). Analysis of peripheral blood smears revealed that neutrophils accounted for the major fraction of the leukocyte elevation seen in Pteri' SHIP ' peripheral blood smears (Table 5 and Figure 31). One 1  Pten*' SHIP' mouse not showing increased neutrophils instead had high levels of basophils. Despite a decreased percentage of lymphocytes within the WBC category (Table 4), Pten*' SHIP'' mice had increased absolute numbers of lymphocytes (Figure 31), and showed a trend towards increased numbers of monocytes, although this was not significant over that of SHIP'' mice in terms of either percentage or absolute numbers. No immature forms, such as blast cells, more than the occasional metamyelocyte, or nucleated erythrocytes, were observed in the peripheral blood or marrow of any of the genotypes (data not shown). As such, according to the recent Bethesda Proposals for classification of nonlymphoid hematopoietic neoplasms, the phenotype of the Pten*'' SHIP''mice could be classified as myelodysplastic in nature (Kogan et al., 2002). On necropsy, spleens and livers from Pten*'SHIP' mice revealed splenomegaly and hepatomegaly that was significantly increased over that of the littermate controls (p^0.005 over all genotypes, Figure 32). Lung weights (Figure 32), as well as pulmonary infiltrates of myeloid cells and disruption of splenic architecture, were comparable to those of aged-matched SHIP' littermates (data not shown). SHIP' and  Pteri'SHIP'  mice demonstrated no evidence of overt myelofibrosis on hematoxylin and eosin staining of marrow sections (data not shown). Despite the striking peripheral blood thrombocytopenia, estimates of megakaryocyte numbers in the bone marrows of Pten " +/  SHIP'' mice were similar to those of controls as assessed by examination of marrow sections as well as flow cytometry of B M samples using the anti-CD41 antibody (see +  below and Table 6). The livers of Pten*'' SHIP' mice, in contrast, revealed multiple foci 1  of extramedullary hematopoiesis (Figure 33), with evidence of myelo-, megakaryo-, and erythro-poiesis that was absent from the control mouse livers. Although extramedullary hematopoiesis had been previously reported in SHIP' mice (Liu et al., 1999), this was  52  not evident in age-matched C57BL/6 SHIP ' controls from our colony. Furthermore, the 1  level of liver involvement in Pten SHIP mice increased between 1 and 5 weeks (data +I  1  not shown), suggesting that the presence of hematopoietic cells in the liver was not due to the persistence of fetal hematopoiesis, but instead was the result of progressive migration of these cells to the liver. Pten"'SHIP' marrow composition does not differ phenotypically from that of SHIP' marrow  Phenotypic analysis of whole bone marrow was undertaken to examine the cellularity and composition of the bone marrow in these mice. Overall the cellularity of Pten ' SHIP ' marrow was significantly decreased when compared to wild-type and +/  1  Pten"' mice (psO.005), however, this did not differ significantly from that of SHIP' mice, despite a modest downward trend in the Pten"' SHIP'' values (Table 6). Flow cytometric analysis of whole bone marrow using antibodies against B220, CD1 lb, Ly6G, CD14, CD41, NK1.1, CD34 and Terl 19 did not show significant differences between the wild-type and Pten" mice. Our analysis also failed to demonstrate a difference between .Pten"'SHIP' ' mice and SHIP' 1  littermates (Table 6) indicating a lack of qualitative  differences between the marrow of these two genotypes. Decreased clonogenic potential in the bone marrow, and increased liver CFU in Pten"' SHIP'' mice.  Given that the phenotypic analysis of bone marrow did not show differences between the SHIP' and Pten"SHIP',  yet the peripheral blood phenotype of the Pten"'  SHIP ' mice was suggestive of a hematopoietic defect more severe than that previously 1  documented for SHIP' mice, colony forming assays were used to assess the number and growth ability of committed progenitors in the bone marrow, spleen, liver and peripheral blood.  On a per femur basis, there was a reduction in total committed myeloid  progenitors in the bone marrow of Pten" SHIP' mice compared to controls (Figure 34a, p<0.05 over all genotypes). Providing a plausible explanation for the anemia, a decrease in primitive BFU-E was observed  (p^O.Ol over wild type and Pten"', but not  53  significantly over SHIP ). Similarly, GEMM subpopulations from Pteri SHIP marrows 1  1  1  were reduced (Figure 34a, p<0.05 over all genotypes). Splenic CFU numbers from Pten*'SHIP'' mice did not differ significantly from SHIP' controls, but similar to SHIP''' 1  splenocytes, they were significantly increased over wild-type and Pten*'' controls particularly in the GM/G/M sub-population (Figure 34b, p<0.05). Likely owing to the presence of extramedullary hematopoietic activity, colony forming assays of Pten* SHIP' liver samples yielded a significantly higher frequency of 1  CFU (primarily of the granulocytic and monocytic lineages) than did littermate controls of all the relevant genotypes (Figure 34c, psO.Ol). To investigate the possibility of peripheral blood progenitor contamination of liver samples, circulating CFU were quantitiated. As shown in Figure 34d, SHIP'' mice demonstrated statistically significant increases in the frequency of peripheral blood, granulocyte-macrophage CFU (p<0.005 over all genotypes). Curiously, this affect was seemingly repressed with the introduction of Pten heterozygosity as Pten* SHIP 1  1  did not differ from wild type and Pten* ' 1  peripheral blood CFU frequency. Thus, the increased CFU seen in Pten* ' SHIP' liver 1  samples was attributed to extramedullary hematopoiesis, and not simply to high levels of CFU circulating in the peripheral blood. Pten heterozygosity confers increased sensitivity of SHIP'' committed progenitors to GMCSF.  Lack of SHIP was reported to confer an increased sensitivity of committed progenitors toward growth factor stimulation (Helgason et al., 1998). Hypothesizing that Pten heterozygosity might further deregulate such responses, we assessed the sensitivity of Pteri' SHIP'' progenitors to titrations of growth factors. Consistent with previous observations (Helgason et al., 1998), we saw increased sensitivity of both SHIP' and Pten*'SHIP'' cells to low doses of SF, IL-3 and GM-CSF over wild-type and Pten  +/  controls. Additionally we observed a significantly increased sensitivity of Pteri' SHIP  1  cells over SHIP ' cells at one dose of GM-CSF (Figure 35a, p<0.05 over all genotypes). 1  Although there were trends of increased sensitivity of Pten*' SHIP'' cells to IL-3 and SF, there was no statistical difference in the growth response of committed progenitor cells  54  towards various doses of these cytokines over the SHIP' response (Figure 35b,c). Similar to previous results obtained in mice of a mixed background (Helgason et ak, 1998) factor-independent growth was occasionally seen in both SHIP' and  Pten"'SHIP'  cultures lacking any of the above cytokines (data not shown). These small clusters of predominately macrophage lineage cells very rarely met the 20 cell criteria defining a colony and were likely the result of heightened sensitivity to serum factors contained in the methylcellulose. Although there was a modest increase in the sensitivity of Pten"' SHIP' cells to GM-CSF, the physiological relevance of this result, especially within the context of the complex pathology of the mice, is unclear.  Furthermore, Pten  haploinsufficiency did not appear to alter the responses of the cells to growth factors. Reduction of immature bone marrow B cells in Pten SHIP'' marrow +/  In keeping with a previous report (Liu et ak, 1999), FACS analysis of SHIP' bone marrow revealed an increase in the larger forward scatter 'myeloid' compartment, and a decrease in the small forward scatter 'lymphoid' compartment in SHIP' and Pten ' +/  SHIP' marrow (Figure 36a-d). The further reduction in the lymphoid compartment in Pten"SHIP'' marrow over SHIP' marrow was consistent (Pten"'SHIP' 10.15% ± 1.42, SHIP' 13.28% ± 0.84, average of 5 mice ± SEM) but was subtle and did not reach statistical significance. As the decreased lymphoid compartment in SHIP ' mice has been attributed to 1  decreased pre-B progenitor growth and a defect in B cell maturation (Helgason et ak, 1998; Helgason et ak, 2000), we analyzed the Pten" SHIP'' cells for differences in pre-B progenitor growth and cell surface markers. Examination of the ability of bone marrow B cell precursors to form colonies in methylcellulose confirmed the previously reported defect seen in SHIP' mice (Helgason et ak, 1998)(ps0.01 for SHIP'' and Pten"SHIP' over wild type and Pten") but revealed a subtle and not statistically significant reduction with the addition of Pten heterozygosity (Figure 37). FACS analysis within the lymphoid compartment of Pten" SHIP'' mice and all controls was performed to assess B220 staining. Analysis of B220 cells within the 'myeloid' compartment in all mice revealed +  negligible staining in large forward scatter cells, however restricting our analysis to the  55  'lymphoid' compartment yielded cleaner results. These experiments revealed that Pten ' +I  SHIP' marrow contained a significantly reduced percentage of B220 cells over all +  controls (p<0.05 over all genotypes) (Figure 38a). Although FACS analysis revealed no difference in the percentage of the early pro-B (CD437B220 ) population between +  genotypes, it did reveal a significant decrease in the percentage of IgM7B220 B cells in +  Pten"SHIP'  mice (p<0.05 over all genotypes) (Figure 38b,c). This suggested the  possibility of a more severe defect in intermediate to late stage bone marrow B cell development associated with the addition of Pten heterozygosity on the SHIP' background. The stem cell compartment of Pten" SHIP' ' mice is not phenotypically altered and the 1  hematopoietic defects of Pten" SHIP'' mice are transplantable.  The decreased clonogenic potential of committed Pten"' SHIP' B M progenitors, especially given either an equivalent, or greater, sensitivity to growth factors, combined with the finding of peripheral blood cytopenias, raised the possibility of a stem cell defect in Pten"'SHIP' mice. Analysis for Lin"Scal c-kit cells by flow cytometry however, +  +  revealed no significant differences in either the percentages or absolute numbers of this population amongst genotypes although the Pten"'SHIP'' mice showed a downward trend in the absolute numbers of lineage-negative cells (Table 7). However, as phenotype is not indicative of potential, it was also important to carry out an analysis of B M progenitor function across the genotypes, and we thus initiated studies to evaluate the reconstitution ability of Pten"SHIP'  marrow in irradiated hosts. In these experiments, 10 cells of 6  whole, unfractionated marrow from 4-5 week-old donors of each genotype, were transplanted into lethally irradiated C57BL/6-Ly-Pep3b recipients (4-8 recipients per donor).  Over the course of four independent experiments, we found that mice  reconstituted with Pten" SHIP marrow cells regularly failed to survive beyond 4 weeks 1  post-transplant. Analysis of wild-type and Pten"'SHlP'  mice at this time point revealed  a significant decrease in body weight in recipients of Pten"'SHIP  1  cells compared to  recipients of wild-type cells (Table 8). Peripheral blood analysis also revealed that the anemia seen in the Pten" SHIP ' mice was transplantable as the recipients of these cells 1  56  were significantly anemic (Table 8). Blood smears from Pten*'SHIP' mice revealed an increase in circulating monocytes and a decrease in circulating lymphocytes consistent with what was seen in the original mice (data not shown).  Furthermore, the  myeloproliferative disorder and extramedullary hematopoiesis as phenotypes also appeared to be transplantable as histological evidence of both could be found in the livers and lungs of the primary recipients of Pten* 'SHIP' cells (not shown). 1  Pten*'' SHIP ' marrow cells are defective in their ability to carry out short-term 1  reconstitution of lethally-irradiated hosts  The inability of many of the Pten*'SHIP' mice to survive through 4 week post transplant is suggestive of an inability of the donor cells to radio-protect these mice as these experiments were performed without the aid of competing Ly5.1 cells. We assessed recipients of both wild-type and Pten*''SHIP ' cells for Ly5.2 contribution to the marrow 1  at 4 weeks post-transplant and found a decreased re-population by donor cells and significant reductions in overall Ly5.2 cells, Ly5.2 B220 cells, and Ly5.2 Macl cells +  +  +  +  +  (Table 8). Additionally, examination of the Ly5.2 contribution to the peripheral blood compartment was performed on all four genotypes at this time point. The results of these experiments are summarized in Figure 39. Both wild-type and Pten* ' mice showed a 1  donor-derived repopulation of 91.3% and 90.2% respectively at 4 weeks post transplant, while SHIP' marrow cells proved significantly less effective at repopulating the recipients. Percentages of Ly5.2 cells in the peripheral blood of SHIP' recipients were +  significantly decreased compared to those in recipients of wild-type and Pten*'' cells (p<;0.005), as were the percentages of Ly5.27B220 cells (psO.OOl). These results, in +  addition to the significant increases in the percentages of Ly5.27Macr (over all genotypes, psO.01), is in keeping with recently published results (Helgason et al., 2003). Strikingly, peripheral blood samples of Pten*'SHIP' marrow cell recipients revealed a marked defect in re-population by donor derived Ly5.2 cells (p<0.001 over all +  genotypes).  Percentages of Ly5.2 B220 cells were significantly reduced over all +  +  genotypes (p<0.05), as were the percentages of Ly5.2 /Macl cells (p<0.001 over SHIP' +  +  alone). The latter were in contrast to the increase in myeloid (Macl ) cells seen in SHIP' +  57  cell recipients. Analysis of percentage Ly5.2 /CD3 cells in the peripheral blood on the +  +  other hand, showed no significant differences amoungst the various genotypes. Interestingly, the proportion of Ly5.2 cells that was B220 versus MacT in mice +  +  receiving Pten"'SHIP' cells was unchanged from the proportion seen in recipients of SHIP'' cells, while the proportion of donor T cells was significantly increased (p<0.025 over all genotypes) in recipients of Pten" SHIP ' cells (Figure 40). Taken together, these 1  results indicate that Pten heterozygosity exacerbates the repopulating defect of SHIP'' B M cells, perhaps providing a partial explanation for the cytopenias seen in Pten"'SHIP' mice.  4.4  Discussion  Despite the spontaneous lymphoproliferative phenotype of Pten"' mice, these animals did not reveal abnormalities in any of the hematological assays we employed. However, the introduction of this deficiency onto a SHIP' background was able to exacerbate the hematopoietic phenotype from that seen in SHIP' mice. We suggest this as evidence that Pten can play a role in regulating hematopoiesis in the absence of SHIP, and that it implies that Pten and SHIP can act co-operatively during hematopoiesis. While SHIP has a demonstrated role as a negative regulator of PI3K, its additional role as an adaptor molecule has precluded the association of the mouse phenotype resulting from its loss directly to increased PI3K signaling. The induction of an exacerbated phenotype by compound deficiency of two negative regulators of PI3K argues for the importance of threshold levels of PI3K regulation, and implicates the PI3K pathway in a number of processes that may be critical in maintaining normal hematopoiesis. It has been presumed that what leads to the eventual demise of SHIP' mice is the accumulation of monocytic and granulocytic cells in the lungs. The addition of Pten heterozygosity onto the SHIP' background in this study resulted in a failure of these mice to survive past 5 weeks of age. However, we did not observe a worsening of the pathology in the lungs or of the bone marrow of Pten" SHIP'' mice compared to SHIP' mice. The hematological phenotype of Pten" SHIP ' mice, in contrast, was clearly 1  58  distinguishable from that of SHIP ' mice. The previous characterization of the SHIP 1  knockout on a mixed genetic background (Helgason et ak, 1998) had noted an increased percentage of neutrophils in the peripheral blood of 4-5 wk old mice, however, no leukocytosis, anemia, or thrombocytopenia was observed, even in mice up to 8-10 wks of age. Similar results were obtained for mice from our predominantly C57BL/6 SHIP' colony, with the exception of a modest but significant anemia (Table 4). The anemia seen in the Pten" SHIP'' mice is supported by the significant decreases in the clonogenic potential of primitive BFU-E seen in the marrow of these mice and may be indicative of an exacerbation of the erythroid developmental defects originally reported for SHIP' ' mice (Helgason et ak, 1998). Furthermore, the anemia seen 1  in the primary recipients of Pten" SHIP ' cells supports an intrinsic RBC progenitor 1  defect and argues against extrinsic factors that may have suppressed erythropoiesis in the original animal. Megakaryocytic function was not assessed in this study, and functional defects in this lineage could be responsible for the thrombocytopenia. However it is likely that this was a secondary event to the anemia. When anemia occurs, erythropoietin (EPO) produced peripherally provides feedback to progenitor cells in the marrow in an attempt to generate more RBC. Since megakaryocytes and erythrocytes share a common precursor, chronic anemia can lead to a shunting towards erythrocyte production at the expense of megakaryocyte and platelet production (McDonald and Sullivan, 1993). Given the significant increase in spleen size even over the SHIP' spleens, hypersplenism may also be a contributing factor in this phenotype. Compared to SHIP ' mice and the other genotypes, there was a large increase in 1  peripheral blood granulocytes in Pten" SHIP' mice. Although this seems paradoxical given the decreased clonogenic potential of Pten" SHIP' marrow GM/M/G progenitors, it might be accounted for by an increase in peripheral survival of the differentiated cells. This is an interesting observation given very recent work that has coupled phospholipid metabolism with apoptosis in hematopoietic cells (Valderrama-Carvajal et ak, 2002). This study revealed that TGF-p" is able to induce apoptosis in hematopoietic cells through the up-regulation of SHIP, and that the resulting decrease in PIP levels also decreases 3  phosphorylation of the downstream anti-apoptotic molecule PKB. Additionally, it  59  demonstrated that bone marrow-derived macrophages from SHIP'' mice were resistant to TGF-(3 induced apoptosis. While Pten levels were not changed by TGF-f3 stimulation in these experiments, the implication was that signals downstream of PIP regulate 3  hematopoietic survival. Additional experiments examining the survival capabilities of Pten 'SHIP'' cells are required to determine any threshold effects of lipid phosphatases +  on cellular survival. Myeloproliferative disorders and cytopenias can precede the development of myeloid leukemias and indeed there is intriguing evidence in the literature that suggests that PI3K and/or its negative regulators may have roles in the progression of this disease. For example, not only is PI3K activity required for the proliferation of Philadelphia chromosome positive cells (Skorski et al., 1995) but it has been shown that PI3K inhibitors can enhance the effects of the BCR/Abl inhibitor STI571 (Klejman et al., 2002). Furthermore, BCR/Abl has been shown to down-regulate molecules that can act as negative regulators of PI3K, namely SHIP, Cbl-b and c-Abl (Issa et al., 1999; Sattler et al., 2002; Sattler et al., 1999). However, while there is some evidence that SHIP mutations in humans may contribute to the development of A M L (Luo et al., 2003), and that SHIP is downregulated in mature leukemic cells in CML patient samples (Jiang et al., 2003; Sattler et al., 1999), a progression to a leukemic state has not been reported in a SHIP' mouse model. Despite the increased severity of the peripheral blood phenotype in the Pten+'SHIP' ' mice, we did not detect any evidence of blast cells in the peripheral 1  blood or histological sections from these mice or in the peripheral blood or tissues of the primary recipient mice of these cells. Thus, while the phenotype of the Pten 'SHIP~' +  mice fulfills several defining criteria for non-lymphoid leukemia, the absence of any immature forms/blasts indicates a diagnosis that is more accurately described as "myelodysplastic" (Kogan et al., 2002). This suggests that loss of negative regulation of PI3K is not an initiating factor in the development of myeloid leukemias, but likely acts as a promoting factor in the progression of the disease. This idea fits with data that reports the downregulation of SHIP in mature leukemic cells as opposed to primitive ones (Jiang et al., 2003) and is also in keeping with the idea that loss of Pten heterozygosity in tumour samples is often found as a late event in tumour progression  60  (Zhou et al., 1999). It is possible that if our Pten"'SHlP' mice could live longer than 5 weeks, then additional genetic alterations may allow the progression of this disease to a leukemic state. Complete deletion of SHIP results in a previously reported release of CFU-S into the peripheral blood and a significant increase in peripheral blood CFU in this study (Helgason et ak, 2003). However, in our study the introduction of Pten heterozygosity seemed to suppress this phenomenon. This may be explained by an overall decrease in the numbers of committed progenitors or alternatively by increased migration of these cells into tissues. The liver for example, is a site of SDF-1 production (Muller et ak, 2001) and both histologically and using colony forming assays we were able to demonstrate a significant presence of progenitor cells in this tissue. In support of this, there is evidence in the literature that SHIP' progenitors, B cells and T cells as well as Pten"' B cells show increased sensitivity to this chemokine (Fox et ak, 2002; Kim et ak, 1999). Indeed, experiments examining the sensitivity of whole bone marrow to SDF-1 chemotactic gradients have revealed trends towards an increased sensitivity of Pten"' SHIP ' cells (J. Moody and F.R. Jirik, unpublished observations). 1  The significant decrease in the percentages of peripheral blood lymphocytes in Pten" SHIP ' mice were supported by the FACS analysis showing a tendency for a 1  decreased lymphoid compartment size, as well as by a trend showing decreased clonogenic potential of Pten"'SHIP'' pre-B progenitors. FACS analysis of IgM7B220 cells in the bone marrow from Pten"'SHlP'  +  suggested a more severe defect in  intermediate-to-late stage B cell development compared to SHIP' ' and the other controls. 1  An unanswered question with regard to the B cell developmental defect in previous SHIP studies was whether the defect was mediated through increased levels of PIP , or by 3  decreased levels of PI(3,4)P resulting from the absence of SHIP. Very recent work has 2  identified PH domain-containing molecules in B cells that are specifically activated by PI(3,4)P (Marshall et ak, 2002). While the role of these molecules in B cell development 2  are unclear, it is possible that an absence of PI(3,4)P resulting from SHIP deficiency 2  might be responsible for impeding B cell development. The apparent worsening of this defect observed following the addition of Pten heterozygosity on the other hand, suggests  61  that regulation of PIP levels may be the critical factor. In keeping with this concept, 3  transgenic mice expressing the E41K constitutively active Btk mutation in B cells suffer from a block at the late IgM to IgM stage (Dingjan et al., 1998; Maas et al., 1999). It 10  hi  has been postulated that this over-activation of Btk, whose PH domain is specific for PIP , may mimic strong receptor signaling by self-ligands, and result in increased 3  negative-selection of developing B cells. We hypothesize that increased receptor signaling caused by decreased negative regulation of the PI3K pathway in our study may achieve the same effect. Curiously, very recent work describing conditional B cell knockouts of Pten do not describe alterations in bone marrow B cell populations (Anzelon et al., 2003; Suzuki et al., 2003) indicating that this phenomenon may be more complicated than just increased PIP levels, and suggests that the ratios of PI(3,4)P and 3  2  PIP may be critical. These studies emphasize the need for extensive and direct 3  comparisons between SHIP ' B cells and conditional B cell knockouts of Pten. 1  The reconstitution experiments provided an opportunity to determine whether the reduced B cell percentages were due to cell intrinsic defects or were a function of microenvironment. While the percentages of donor Ly5.27B220 cells in the peripheral +  blood of the recipients of Pten ' SHIP'' cells were significantly decreased even over +  SHIP'' recipients, the proportion of the donor cells that were myeloid and lymphoid were similar in their respective recipients. While this does not preclude an intrinsic B cell defect in Pten+'SHIP'' cells, as decreased output from the marrow can be accompanied by an expansion in the periphery (Helgason et al., 2000), it may suggest that the decrease seen in the B cells in Pten+'SHIP' marrow may be in part due to microenvironment. Curiously however, the increased proportion Pten+'SHIP' ' T cells in the periphery may 1  suggest an intrinsic advantage in the T lymphocyte lineage. Further experiments examining the long term reconstituting ability of lymphocyte precursors are warranted to clarify these results. PI3K is activated downstream of multiple cytokine receptors (reviewed in (Fruman and Cantley, 2002)), so not surprisingly then, SHIP'' progenitor cells demonstrated an increased sensitivity to growth factors (Helgason et al., 1998; Liu et al., 1999). The introduction of Pten heterozygosity in this study increased this sensitivity  62  significantly in the case of a low dose of GM-CSF and very marginally in the case of SF and IL-3. This may be indicative that a further compromise in the ability to regulate PIP  3  levels in response to these cytokines can contribute to an increased clonogenic potential of progenitor cells. What is particularly interesting is that Pten '~SHIP'~ mice show +  decreased numbers of committed progenitors in the bone marrow despite similar bone marrow cellularity to SHIP'' mice and despite a similar or increased sensitivity of these cells over SHIP'' cells to growth factors. This suggested a potential stem cell defect in these mice. While these experiments were in progress, a study characterizing the stem cell compartment of SHIP' mice was published revealing a repopulating defect of SHIP'' cells and suggested an impairment of HSC self-renewal (Helgason et ak, 2003). 5-FU treatment and subsequent CRU analysis of the stem cell compartment of these mice also indicated that a greater proportion of the SHIP'' stem cell compartment was cycling compared to wild-type cells. It is interesting that the absence of SHIP, despite the persistence of expression of s-SHIP in embryonic and hematopoietic cells in SHIP' mice (Kalesnikoff et ak, 2003), had dramatic effects on the stem cell compartment and raises the question of the relative roles of these two SHIP isoforms in these cell populations. Reduced capacity for serial transplantation has been described in mice deficient for the p21 cell cycle inhibitor (Cheng and Scadden, 2002) as well as for mice transplanted with dominant-negative TGF-(3 type II receptor-transduced bone marrow cells (Shah et ak, 2002). In the latter study, where the myeloid expansion and inflammatory phenotype of the mice is reminiscent of that of the SHIP' mice, cells are rendered insensitive to the effects of TGF-beta. It has been suggested that TGF-p" acts as a regulator of HSC growth and potential via its up-regulation of cell cycle inhibitors (Ducos et ak, 2000; Pierelli et ak, 2000) although which inhibitor is disputed (Cheng and Scadden, 2002). Our shortterm reconstitution study has demonstrated that the defect in multi-lineage repopulating ability of SHIP' B M is worsened by Pten heterozygosity. Given the effects of PI3K in the promotion of cell cycle (Chang et ak, 2003) it is an intriguing possibility that the loss of negative regulation of this pathway may have effects akin to the absence or inhibition of cell cycle inhibitors. Studies regarding the long term repopulating ability, renewal capacity and cycling status of the stem cell compartment of these mice will be the focus  63  of future studies, and will provide valuable insight into the role of PI3K regulators in stem cell biology.  64  Chapter Five: Results summary and future directions 5.1  Results summary  The unifying theme in this thesis is the concept that partial and combined deficiencies for Pten and SHIP can exert phenotypic results on the immune and hematopoietic systems in mice. The basis for this work was founded by previous studies that described the haploinsufficiency of Pten in the peripheral immune system (Di Cristofano et al., 1999; Podsypanina et a l , 1999). This suggested that threshold levels of regulation for PI3K were required for the maintenance of peripheral immune tolerance. Additionally, unpublished work by Dr. Cheryl Helgason (personal communication) had also revealed that SHIP' mice on a mixed background developed characteristics of autoimmunity, and that subtle indications of kidney disease were present in aging SHIP '~ +  mice. This suggested that Pten and SHIP, in addition to sharing a common substrate, shared a similar phenotype with respect to peripheral immune tolerance. We thus hypothesized that if a threshold had been crossed in the Pten mice with respect to PI3K +I  regulation, then the addition of SHIP heterozygosity may alter that threshold further and result in an exacerbation of the Pten phenotype. +/  The results in Chapter two confirmed this hypothesis and demonstrated that the introduction of SHIP heterozygosity onto the Pten ~ background could manifest +/  phenotypic changes that were more severe than either single heterozygote alone. Changes included an increased severity of pathology in secondary lymphoid organs and kidneys, increased circulating serum immunoglobulins of both IgM and class switched isotypes, and increased titres and reactivity against nuclear self-antigens. These results were particularly interesting because prior to this work, Pten and SHIP had only been examined individually and this was the first in vivo study to look at simultaneous deficiencies of both of these lipid phosphatases in a murine model. Furthermore, these results implied that the regulation of PI3K in immune cells was exquisitely sensitive to changes in the levels of these phosphatases. In an effort to ascertain the degree of dysfunction in lymphocytes from young mice prior to the development of immunopathology, we initiated experiments to examine  65  the in vivo response to defined antigenic challenge. Immunization of young Pten"'SHIP" mice revealed an increased response to T-dependent antigens and no difference in their response to T-independent antigen stimulation, suggesting that T helper cell function may be augmented in these mice. The data described in chapter three involved examining expanded CD4 T cells from these young mice for functional and biochemical differences +  in Pten" SHIP" cells. We were unable to detect significant differences in PIP species in 3  Pten SHIP" +  T cells before of after stimulation with anti-CD3. As an indirect readout of  PIP species, we were able to demonstrate modest increases in the phosphorylation of Akt 3  at Serine 473 in Pten" SHIP"' T cell lysates. This increased activation of an antiapoptotic molecule did not however translate into an increased survival capability of the expanded T cells. We were able to show that upon in vitro stimulation with anti-CD3, T cells from Pten" SHIP" mice produced increased levels of IL-4, IL-13 and to a lesser extent IFN-y both at the RNA and protein levels. These increases could not be attributed to increased survival of these cells compared to the other genotypes, nor to a tendency for spontaneous polarization of the T cell population to the Th2 phenotype. Examination of three signaling pathways that could potentially lead to increased IL-4 cytokine production on a per cell basis did not reveal any significant differences in Pten" SHIP*' T cells. Taken together, the results of this chapter reveal that the introduction of SHIP heterozygosity onto the Pten"' background increases the immune response to Tdependent antigenic stimulation and is capable of altering T cell cytokine production in response to stimulation, albeit by a mechanism that is still unclear. There had previously been no descriptions of the effects of Pten heterozygosity on cells of the myeloid lineage. The experiments described in chapter four revealed that a partial reduction in Pten alone did not have an effect on general hematopoiesis, on the clonogenic potential or growth factor response of committed progenitors, or on the ability of marrow cells to multilineage reconstitute irradiated recipients. As haploinsufficiency had previously been described in cells of the peripheral immune system, this suggested a possible compensatory mechanism that we hypothesized might involve SHIP. Indeed when Pten heterozygosity was combined with complete deficiency for SHIP, the resulting peripheral blood abnormalities, the decreased numbers of femoral committed  66  progenitors, the increased sensitivity of committed progenitors to growth factor and the decreased ability to multilineage reconstitute recipients implied that Pten could play a role together with SHIP in hematopoietic processes. The decrease in IgM7B220 B cells +  in the marrow of Pten+'SHIP''' mice also implied that Pten and SHIP may both contribute to mechanisms involved in B cell development.  Despite our hypothesis that a  consequence of further compromising the ability of cells to regulate PIP levels may be a 3  predisposition to leukemia, we did not see any evidence of this in Pten+'SHIP' mice or primary recipients of their cells. The results of the thesis as a whole have shown that combined deficiencies for these molecules, be it in the lymphoid or in the myeloid compartments, resulted in measurable differences from that resulting from single gene deficiencies. The fact that SHIP heterozygosity leads to no measurable defects in peripheral immune tolerance or T cell behavior until combined with Pten deficiency, and that Pten heterozygosity has no effects on hematopoietic processes until combined with SHIP deficiency has revealed previously unrecognized roles for these molecules in the processes. Given the common substrate shared by Pten and SHIP, these experiments also suggest that the ability to titrate PI3K pathway activity is critical to both hematopoiesis and peripheral immune tolerance.  5.2  Future Directions  The study of lymphocyte reactivity using genes in the heterozygous state has been previously used to demonstrate the significant effects on immune cell function that can arise from compound heterozygosity of molecules in the same signaling pathway (Cornall et al., 1998). Its use here, to examine altered thresholds of negative regulation of the PI3K pathway is novel. Studying the impact of gene heterozygosity on T-cell function and autoimmunity represents an important approach when contemplating the relevance of mouse models to human disease, since mutations that lead to decreased function are more common than biallelic gene inactivations. While the latter can reveal critical functions of the encoded molecules, the absence of a protein can potentially alter developmental and differentiation programs of cells, and may even select for the up-  67  regulation of compensatory proteins or pathways. Additionally, transgenic approaches to over-expression of a molecule can result in expression levels that vary from slight to greater than anything ever seen physiologically. The potential importance of synergistic heterozygosity has been recently recognized for human metabolic disorders (Vockley et ak, 2000) in that an increasing number of human patients with metabolic disorders are being found to have multiple heterozygous mutations in proteins involved in energy metabolism instead of complete gene deletions. It is likely that this phenomenon represents a clinically relevant mechanism by which multigenic disorders arise. While the direct relevance of the effects of mutations in Pten and SHIP in human autoimmunity is currently uncertain, the value of this approach in evaluating dosage effects of genes implicated in multigenic disorders is clear. In chapters two and three, we expected to induce subtle changes by introducing SHIP heterozygosity onto the Pten*'' background since partial deficiency for SHIP alone did not have any appreciable effects. While the changes that were induced resulted in measurable phenotypic differences in the mice and in their response to antigenic stimulation, the detection of biochemical differences at the level of PIP and protein 3  phosphorylation in primary cells from these mice proved more difficult.  We were  successful in showing significant differences at the transcriptional and translational levels that may represent the culmination of subtle differences upstream. In keeping with the idea that nuclear events may show detectable differences, it would be interesting to look at the nuclear presence and binding of transcription factors involved in IL-4 and IL-13 transcription. Specifically, GATA3, NFAT, c-Maf and AP-1 have been implicated in the production of IL-4 in T cells (Murphy and Reiner, 2002). Interestingly, NFAT has also been implicated in transcriptional control of IFN-y, and given the increases we saw in this cytokine, the investigation of NFAT activity in the nucleus would be of great interest. The examination of message stability as a potential factor to account for differences in cytokine production would also be warranted. Cell numbers were an issue in some of these experiments, particularly for the PIP  3  analysis where total lipid from 10 cells per stimulation may have been insufficient. 7  68  Pooling together mice to obtain greater cell numbers could have alleviated this, however, pooling would have been better suited to experiments in which the mice were fully backcrossed and littermate controls were not required. An alternative method for visualizing PIP levels, such as the one employed by the Cantrell group, may allow for 3  visual quantitation of PIP dynamics and would require fewer cells (Costello et al., 2002). 3  This approach would involve crossing our mice with a mouse transgenic for a PIP  3  specific PH domain fused with GFP. These experiments did not address issues of thymic selection and how they might be altered in Pten+'SHIP+ mice. It would be useful to take the approach used previously for the conditional knockouts of Pten to address this issue (Suzuki et al., 2001). This would involve an additional cross to a transgenic mouse expressing the Hy antigen and assessment of negative selection in male animals. Differences in this regard would provide a mechanism and a possible explanation for how tolerance may be compromised in these mice. Additionally, a search for potential B cell abnormalities in Pten+ SHIP+ mice is warranted. Given the results of the antigenic challenge experiments, it is possible that the B cells in these mice are able to generate a more vigorous response to T cell help than B cells from the other genotypes. In vitro stimulation of B cells to measure proliferative response to titrated stimuli such as IgM and LPS might be informative. Given our data showing increased IL-4 production by Pten+'SHIP+T cells, and the fact that IL-4 is a potent activator of B cells, experiments measuring the proliferative effects of IL-4 on B cells and the effect of IL-4 on in vitro class switching ability would also be worth pursuing. With respect to the experiments in chapter four, perhaps the most compelling line of research that logically follows the experiments described here are ones to address the long term reconstituting potential of Pten+'SHIP' stem cells. 1  Our reconstitution  experiments were done in the absence of recipient-derived helper cells. As such, the inability of the Pteri SHIP' ' cells to properly reconstitute the recipient marrow lead to the 1  demise of these mice at approximately 4 weeks post-transplant, a time point that only addressed the short-term repopulating ability of the donor cells. With the aid of helper  69  cells, the mice would be radio-protected beyond the 4-week time point and the long-term reconstituting potential of the mutant cells could be evaluated at 16 weeks posttransplant. Additionally, experiments to evaluate the number of stem cells in the marrow compartment, such as the competitive repopulating unit (CRU) assays employed previously with the SHIP'' model (Helgason et ak, 2003) could address the differences between the Pten"SHIP ' and SHIP' stem cell compartments. Also, given the role for 1  PI3K in cell cycle progression (Chang et ak, 2003) it would also be interesting to assess the cycling profile of the stem cell compartment in Pten 'SHIP' +I  mice using 5-  fluorouracil (5-FU) treatment and subsequent CRU analysis. Both arms of this thesis have dealt with the hypothetical increase of PIP species 3  predicted to result from combined deficiencies of these genes. However, there are very interesting questions regarding precise nature of the PI species and the differences between Pten and SHIP that were not addressed by these experiments. While T cell conditional knockouts of SHIP have not been generated, the RAG'SHIP'  experimental  results (Liu et ak, 1998a) indicate, at least in vitro, that SHIP deficient T cells behave normally. Why is it that Pten deficiency in T cells causes such a dramatic autoimmune phenotype while SHIP deficiency does not? This may involve compensation by an alternate 5' phosphatase such as SHIP2 in these cells. However, given the described substrate specificities, deficiency for Pten should increase both PIP levels and PI(3,4)P 3  2  levels, whereas a deficiency for SHIP should increase PIP levels, and if the findings in 3  mast cells (Scheid et ak, 2002) extends to other cell types, a decrease in PI(3,4)P levels. 2  Does the difference in PI(3,4)P levels and the possible downstream effectors activated 2  by this species account for the difference in T cell behaviour? Also, this question extends to the disparate B cell phenotypes seen in the mice. SHIP deficient B cells are developmentally impaired in the bone marrow, but Pten deficient B cells have no reported developmental defect. Does this allude to the function of molecules such as TAPP1 that are activated specifically by PI(3,4)P levels and are potentially impaired by 2  the lack of SHIP? Side by side experiments of T cells and B cells completely deficient for these molecules and analysis of signaling pathways in these cells will shed light on the differences that result from deficiencies of Pten and SHIP.  70  Another very curious question arises when one considers the consequences of complete deletion of Pten and SHIP in cells. Would the resulting increases of PIP result 3  in oncogenic transformation as has been described for many conditional knockouts of Pten (Kishimoto et ak, 2003)? Or are PI(3,4)P increases that also result from Pten 2  deficiency important for transformation? In the latter case, the elimination of SHIP and the potential decrease of PI(3,4)P levels might actually suppress transformation events. 2  Complex breeding strategies of conditional knockouts could reveal the answers to these questions. On the other hand, RNA interference techniques, whereby double stranded RNA corresponding to the gene(s) of interest is introduced into cells effectively eliminating the endogenous RNA, could be used. Pren-specific interfering RNA could be delivered to cell populations by lentiviruses or retroviruses to knock-down Pten expression in cultures of SHIP' ' cells. Alternatively, transgenic approaches using this 1  technology have recently been developed (Kunath et ak, 2003; Rubinson et ak, 2003) that may allow for the tissue specific knockdown of one or more genes depending on the construct. 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Int J Cancer 84, 150154.  92  diacylglycerol  fatty acid tails lie within Inner leaflet of lipid bilayer  J  phosphodiester link  OH /  nositol head  cytosolic  group  OH  Figure 1. The structure of phosphatidylinositol. From Vanhaesebroek, 2001.  93  (PI5K)  (PI4K)  (3  A (4-phosphatase)  PTEN  PI3K Class I Class II Class III  A  r  SHIP PI3K Class I Class II  PTEN  (PI4K)  PI(3,4,5)P.  '?\(2A)P  Y  PTEN  (PI5K)  t PI(4,5)P  PI(4)P (4-phosphatase)  PI3K Class  2  (5-phosphatase)  Figure 2. The interconversion of PI species is facilitated by the actions of kinases and phosphatases. From Koyasu, 2003.  94  Figure 3. Cellular consquences of PI3K activation. Receptor engagement leads to the activation of PI3K, the generation of PIP3 species and the translocation of PH domain containing proteins. This triggers a cascade of activation that ultimately results in diverse cellular consequences. From Cantley, 2002.  95  SH2 domain: Interacts with phosphorylated ITIMs, ITAMs. and possibly cytoplasmic proteins  C-terminus: Phosphotyrosines mediate interactions with PTB and SH2 domains. Proline-rich regions interact with SH3 domains.  5 IPase Domain: Catalyzes the conversion of PI(3,4,5)P, to PI(3,4)P, and l(l,3,4,5)P toI(l,3,4)P, 4  SHIP1 T Y917 Y1020  Figure 4. The structure of full length SHIP. From March and Ravichandran, 2002.  96  Membrane  Membrane PI PJ?-Binding Motif  [KxxxxRxKR) (6-14)  PDZ protein(s)  Active Site  PDZ-Binding MbtH  (124-130)  (401 -403)  rrxv-oooH]  [CxxxxxRJ  PTPase Domain  C2 Domain  Figure 5. The structure of PTEN. From Maehama et al, 2001.  97  Table 1. Spleen and node weights are increased in Pteri' SHIP* ' mice at 24 weeks 1  Genotype wildtype SHIP* ' Pten*'' Pten+'SHIP*  Spleen weight (g) 0.11 ±0.01 0.15 ±0.02 0.13 ±0.01 *0.30 ± 0.06  1  1  8  Node weight (g) NM NM 0.23 ±0.05 *0.67 ± 0.12  "All values represent the mean ± the S E M , NM= not measured. *p< 0.02 for Pten* 'SHIP* ' weights over Pten*'-, SHIP*', and wildtype weights n= 13 to 20 mice per group. 1  1  Table 2. Pten*'SHIP*''spleens show increased numbers of B cells, T cells, macrophages and increased activation markers at 24 weeks 1  7  Absolute cell numbers x 10 B220  +  Genotype wildtype SHIP*'' Pten *'' Pteri'SHIP* '  8.41 ± 1.05 *10.65± 1.30 8.52 ± 1.17 16.72 ± 3.07  1  CDllb 3.97 4.66 3.87 9.75  +  ± 0.64 ± 0.26 ± 0.48 ± 1.44  CD4 2.70 3.19 2.94 5.77  CD8  +  ±0.71 ±0.34 ± 0.26 ± 0.68  §  +  1.89±0.45 1.94 ±0.19 1.53 ±0.08 3.17 ±0.44  "All values represent the mean ± the S E M . p< 0.05 for Pten* 'SHIP*'' over all genotypes for all cellular analyses except for * and where p<0.07. n= 4 to 6 mice per group. 1  00  §  CD69  +  2.88 ±0.30 3.03 ± 0.37 2.88 ± 0.33 7.84 ± 1.95  Figure 6. Severe kidney pathology in Pten ~SH1P ~ mice. H & E stained kidney sections showing representative glomeruli from wildtype (a), SHIP ~ (b), Pten" ' (c) and Pten SHIP ~ mice (d). Note the mild mesangial cell proliferation in the SHIP ^~, more moderate mesangial proliferation in Pten ', and the hypercellular appearance o f the Pten ^~SHIP ^~ glomerulus due to mesangial, endocapillary, and peri-glomerular infdtration. Magnification 4 0 0 X . +/  +I  1  +/  +I  +/  +  +  +  +  99  Figure 7. Electron microscopy reveals the severity of damage in Pten I-SHIP I- kidney. + Electron micrographs of wildtype are shown in (a) and Pten /-SHIP+I- kidney in (b). For orientationred blood cells can be seen in capillaries of both sections (RBC in Cap). Podocytes (podo) are fused in (b). Regions of cellular infiltration in (b) are marked with * and immunoglobulin depostion with lg. Magnification 6750X. 100 +  +  Figure 8. Pten+I-SHIP+I- mice have a greater than three fold increase in circulating immunoglobulin levels. Serum immunoglobulin levels were measured by ELISA and the above values were determined by the sum of the average of individual subtypes and expressed as a percentage of wild-type levels.  101  b)  3000  I I SH1P+/-  d)  Pten+/-  SH1P+/-  Pten+/-  Pten+/SHIP+/-  P t e n + /  8000 <§• 7000 "a 6000 % 5000 •S 4000 e •g 3000 § 2000 u  1000 SHiP+A  e) g,  250  +/+  SHIP+/-  Pten+/-  Pten+/SHIP+/-  g)  ftcnH-  Plcn+/-SHIP+/-  Figure 9. Pten+I-SHIP+I- mice show increased circulating levels of most isotype subclasses. Pten I'SHIP ' mice show significant increases in IgG2b (a), IgM (b), IgGl (c), and IgG3 (d), *p < 0.05 compared to wildtype, SH1P+ - and Pten+I- mice. The IgG2a class was not significantly increased (f) and levels of IgA were decreased in Pten+I'SHIP+I' serum (#p<0.05). Values represent the average of duplicate samples, assessed in triplicate, and error bars represent ± S E M of the triplicate readings. +  +/  1  102  Figure 10. Pten /-SHIP /- mice have higher titres of anti-nuclear antibodies. Hep-2 cells were used to titre out reactivity of anti-nuclear antibodies in serum from mice of all four genotypes, (a) represents an example of high reactivity or 4+ staining by the serum of a Pten /-SHIP /- mouse at 1/40 dilution, while (b) illustrates staining by the same serum at a 1/640 dilution approximating the titre point, or 1+ reactivity, (c) is a graph representing the percentage of mice in each genotype that titred out at a greater than 1/320 dilution. +  +  +  +  103  Table 3. A N A titres of the studied cohort. Titre at which staining was scored as 1+  1/40 wildtype SHIP +/  Pten*''  Pten 'SHIP +  +/  1/80  1/160  1/320  1/640  1/1280  1 2 1 3  0 1  0 1 0 2  8 2  0 2  2 3  2  3  3  0  0  2  1 2  Sera were serially diluted in P B S the titre of antinuclear antibodies was determined by reactivity with Hep-2 cells. The dilution which yielded 1+ staining was defined as the titre for the animal.  104  % of animals AU>185  Figure 11. Pten+/SHIP+/- mice have a higher reactivity against H I histone protein. Densitometric analysis of western blots was performed using ImageQuant software and arbitrary densitometric units (AU) were assigned based on the intensity of the signal, a) raw scores of all mice in the cohort b) the percentage of mice of each genotype with A U scores greater than 185. 105  Figure 12. A simple schematic outlining T helper cell differentiation. Naive T cells capable of making IL-2, can differentiate into T h l or Th2 cells through signals initiated by IL-12 or IL-4 respectively. Key to commitment to these lineages is the expression of the transcription factors T-bet or GATA3. Differentiated T h l cells secrete IFN-y leading to cell mediated immunity and Th2 cells secrete IL-4 that initiates humoral immunity through B cell help.  106  a)  130000 160000  A  140000  DayO  A  Day  ll'OOOO  A  1?  A  A  100000  30000  A  60000  A  40000  T A A  30000  A A  Q wl  SH  PH D H  wt  SH P H D H  b)  70000 -r 60000 -  DavO  50000 •  Day  1:  40000 • 30000 20000 A  A A  10000 A  0 Wt  SH  PH  DH  wt  SH  PH  DH  Figure 13. Pten /-SHIP /- mice do not show significant differences in their antigenic response to the T-independent antigen NP-Ficoll. Titres of antibodies generated against NP were determined by ELISA for 6-7 mice per genotype. No significant differences were determined in either the IgM response(a) or the IgG response (b). Red bars represent the average titre in each group. wt= wildtype, SH= SHIP+/-, PH= Pten+/; DH=Pten+/-SHIP+/-+  +  107  a)  POOOO 30000 •0000 -  DavO  Dav 2 1  60000 50000 40000 30000 20000  T t*  10000 0 art  SH  PH  DH  wt  SH  PH  DH  b)  *  2000000 "_» *J .' 1S00000 -  **  A  1600000 -  Day 0  uooooo -  Day 21  1200000 |  _4_  1000000 • 800000 600000 -  A  •100000 i  200000 -  i  o  wt  SH  PH  DH  wt  —  T •  SH  i —  PH  DH  Figure 14. Pten SHIP mice how an increased antigenic response to the T-dependent antigen N P - K L H . Titres of antibodies generated against NP were determined by ELISA for 7 mice per genotype. Statistically significant IgM (a) and IgG (b) responses to N P - K L H were detected. ***p <0.05 over all genotypes, **p <0.005 over wildtype and SHIP+/-, but not significant over Pten+/-. * p<0.05 over wildtype and SHIP+S-. Red bars represent the average titre in each group. wt= wildtype, SH= SHIP+/-, PH= Pten ^-, +//  +//  +  DH=Pten+/SHlP+/--  108  1.8  3.2  1.2  0.9 anti-Pten  9.4  4.4  10.9  5.8 anti-SHIP  */+  S H I P + / - Pten+/- Pten+/-  SHIP+/-  Figure 15. Reduced protein levels of Pten and SHIP in Pten+I-SHIP+I- T cells. Whole cell lysates from expanded T cells were examined for levels of both Pten and SHIP by western blot analysis. Numbers represent densitometric values for the bands revealing reduced levels of Pten in Pten+I- and Pten+I-SHIP+I- lysates and reduced levels of SHIP protein in SHIP+I- and Pten+I-SHIP+I- lysates. These results are representative of lysates from two independent sets of littermate control mice.  109  Homogeneous Time Resolved F R E T  Figure 16. Schematic of FRET technique for PIP analysis. In this assay PIP3 is conjugated to an A P C molecule via streptavidin/biotin interaction. The P H domain from Grpl which is specific for PIP3 is conjugated to europium chelate. In the system, if the europium chelate labelled P H domain interacts with the PIP3 bound to the A P C molecule, light at a wavelength of 340nm will excite the europium chelate which will in turn excite the A P C molecule and cause it to emit light at a wavelength of 665nm. Unlabelled PIP3 introduced into this system, competes the labelled PH domain away from the A P C molecule, and decreases the fluoresence emission at 665nm. A standard curve with known amounts of unlabelled PIP3 can be generated to allow for quantitation of sample levels. Figure courtesy of Dr. Alexander Gray and this technique has been described in Gray, 2003.  110  • wildtype  • SHIP+/• Pten+/3.5  • Pten+/-SHIP+/-  3 2.5 2  b  •jr •ri—i  1.5  o  ! 0.5 1  2  5  10  30  minutes of stimulation with anti-CD3  Figure 17. Pten+I-SHIP+I- T cells do not show significant increases in PIP3 levels. levels were measured by a FRET technique before and after stimulation with anti-CD3 antibody. The graph represents the pooled results of three independent experiments performed on expanded T cells from littermate controls expressed as fold increase over wildtype levels. Error bars represent the S E M from the three experiments. PIP3  Ill  a)  wildtype 0  2 5 10 20 0  SHIP+/-  Pten+/-  2 5 10 20 0  2 5 10 20 0  ...  Pten+/-SHIP+/2 5 10 20 mm-mm9  minutes P-Akt Akt  +/+  SHIP+/-  Pten+/-  Pten+/-SHIP+/-  Figure 18. Pten+I-SHIP+I- T cells show modest increases in Akt phosphorylation after stimulation with anti-CD3. Expanded T cells from each genotype were stimulated with anti-CD3 for the indicated time periodsand 70-80 ug of protein lysate was immunoblotted with P-Akt antibody. Blots were stripped and re-probed with and-Akt antibody for loading control, (b) Combined densitometric analysis of the ratio of P-Akt/Akt shows a trend towards increased phosphorylation of Akt in Pten+1-SHIP+I- cells after 20 minutes of stimulation. The graph represents the mean fold increase over wildtype from 4 independent western blots and the error bars represent the standard error.  112  1  2  3  4  IL-4  IL-13  IFN-y  L32  GAPDH  Figure 19. Increased cytokine R N A by Pten+/-SHIP+/- T cells. Expanded T cells were stimulated with anti-CD3 and R N A levels were analysed by RNAse protection assay. Pten+/-SHIP+/- samples showed increases in IL-4, IL-13, and IFN-y R N A after 8 hours of stimulation. These results are from one experiment and are representative of at least three experiments on T cells isolated from seperate mice. l=wildtype, 2= SHIP -, 3=Pten -, 4=Pten SHIP -. +/  +/  +/  +/  113  wildtype  SHIP+/-  Pten+/-  Pten+/-SH1P+/-  wildtype  SHIP+/-  Pten+/-  Pten+/SHIP+/-  wildtype  SHIP+/-  Pten+/-  Pten+/-SHIP+/-  Figure 20. Increased cytokine R N A by Pten+I-SHIP+I- T cells. Expanded T cells were stimulated and R N A levels were analysed by RNAse protection assay. Samples were quantitated by densitometric analysis using Quantity One software and were normalized for loading using the internal G A P D H control. Pten+I-SHIP+I- T cells show increases in a) IL-13, b) IL-4, and c) IFN-y R N A after 8 hours of stimulation. These results are representative of three experiments on T cells isolated from independent mice. 114  a) 20000 18000 16000 14000 _, 12000 |10000 8000 6000 4000 2000 -I  • wildtype • SHIP+/• Pten+/• Pten+/-SH1P+/-  a  12 hours  b)  • wildtype 6000  • SHIP+/-  5000  • Pten+/-  4000 |  • Pten+/-SHIP+/-  3000  ~ 2000 1000  12 hours  C)  • wildtype 140000 i 120000  • SHIP+/• Pten+/• Pten+/-SHIP+A  100000 •g HOOOO S  60000 40000 20000  12 hou  Figure 21. Increased cytokine protein by Pten+I-SHIP+I- T cells. Expanded T cells were stimulated with 10 ug/ml soluble anti-CD3 and cytokine protein levels were measured by ELISA. Pten+I-SHIP+I- T cells produce significantly increased amounts of a) IL-13, b) IL-4 after 12 hours of stimulation. Levels of IFN-y protein (c) were significantly increased over wildtype and Pten+/~ supernatants. ***p<0.001 over all genotypes, **p<0.05 over Pten+I- and wildtype. These are the combined results from 4 to 5 experiments from independent mice. Error bars represent the S E M from all experiments.  115  10000•  1 1.35  10000 -f 1.26  41.S  1  .21000  -glOOO •5  o "i  U 100  52.7.  b)  -  'j  a 100  -i  X 10  -l|'''l'"i"Vl'lMU 10  i ' 12.4  " . • 8.17  1  iij • I -i -i-ivrft] r- i"f | •••nf • 10 100 1000 FL1-H: Annexin V  1000 100 FL1-H: Annexin V  11000  48.4'  C)  10000  innng -i 1.24 d) KIOOOH  .§1000  •5  o  1 1.o 100 £ I  I CN  CN  ^  :-*:*ss3!te&7  . 100  10  i  10  '•'!."•='.""•  •• •  •  13 3  1 1imVj""l"t \ | iVii| - -I 1 || I III/ 10000 100 1000 10 FL1-H: Annexin V  i 10.9  ""I i i 11 I I I I | — i 100 1000 FL1-H: Annexin V r  i i I'li'i' 10000  Figure 22. Increased cytokine production cannot be attributed to increased survival by Pten*' SHIP*' cells without stimulation. T cells were left un-stimulated for 12 hours and cell viability was assessed by FACS for AnnexinV/PI. The percentage of viable cells did not differ significantly between genotypes, a) wildtype, b) SHIP*', c) Pten* ', d) Pten* ' SHIP*'. Results are representative of at least three independent experiments. 1  1  116  1  10  100 1000 FL1-H: Annexm V  10000  1  10  100 1000 FL'l-H: Annexm V  10000  Figure 23. Increased cytokine production cannot be attributed to increased survival by Pten+/-SHIP /~ cells after stimulation. T cells were stimulated for 12 hours with anti-CD3 and cell viability was assessed by FACS for Annexin/PI. The percentage of viable cells did not differ between genotypes, a) wildtype, b) SHIP+I-, c) Pten+I-, d) Pten+I-SHIP+I-. Results are respresentative of at least three independent experiments. +  117  100D0  TT56  1.45  10000-< 2,14  1.55  1000-  100-  104 B8.3f';»  I ' I ""I 100 1000 IFNgFITC 1  1  1  1  8.69. 1  I ''^'|  10000  10  T  1—I J I I I I |  ~l  1—I | I I I I ]  100 1000 IFNg FITC  r 10000  Figure 24. Pten+/-SHIP+/- cells appear to make more IL-4 cytokine on a per cell basis. Intracellular staining for IL-4 and IFNg was performed on CD4+ T cells stimulated with anti-CD3. While the percentage of detectable cells making IL-4 in all genotypes is quite low, (a, wildtype, b) SHIP+/-, c) Pten+I- d) Pten+I-SHIP+I-), the percentages do not differ significantly. Graphs are respresentative of 5 independent experiments.  118  wt  SH  PH  DH  P-STAT6  STAT6  5  wildtype  SHIP+/-  Pten+/-  Pten+/-SHIP+/-  Figure 25. Pten+I-SHIP+I- cells do not show significantly increased levels of phosphorylated STAT6. a) T cell lysates were analysed by western blotting for levels of phosphorylated STAT6. Blot were stripped and reprobed with antibody against total cellular STAT6. b) The graph represents the pooled results of three independent experiments of P-STAT6/STAT6 loading and expressed as fold increase over wildtype levels. Error bars represent the standard error between experiments. wt=wildtype, SH= SHIP+I-, PH=Pten+l-, DH= Pten+I SHIP+I-  119  wildtype 0  b)  2.5  2  10  Pten+ -SHIP+ /  15  30  0  2  10  /  15  30  i  • wildtype • Pten+/-SHIP+/-  1.5  1H  0.5  2  5  10  30  Time after anti-CD3 stimulation  Figure 26. Pten+/-SHIP+/- cells do not show increases in phosphorylated p42/44 M A P K before or after stimulation. a) T cell lysates were assessed for levels of phosphorylated p42/44 M A P K by western blotting and blots were stripped and reprobed to establish levels of total p42/44 M A P K . b) Graph represents the pooled densitometric values from three independent stimulations normalizing P-p42/44 M A P K to total p42/44 M A P K and expressed as fold increase over wildtype. Error bars represent the standard error between experiments.  120  wildtype 0  a )  2  5  10  Pten+ISHIP+I20  0  2  5  10  20 P-p38 p38  b) 3.5 j  3  &  I  O Wildtype • Pten+/-SHIP+/-  ,  |  2.5 -I 2  S  1.5  S  I  . 0.5  2  5  10  20  minutes after anti-CD3 stimulation  Figure 27. Pten+I-SHIP+I- cells do not show increases in phosphorylated p38 M A P K before or after stimulation. a) T cell lysates were assessed for levels of phosphorylated p38 M A P K by western blotting and blots were stripped and reprobed to establish levels of total p38 M A P K . b) Graph represents the pooled densitometric values from three independent stimulations normalizing P-p38 M A P K to total p38 M A P K and expressed as fold increase over wildtype. Error bars represent the standard error between experiments.  121  a)  b)  c)  140000 120000 • -a loooooI  80000  V, 60000 • 40000 • 20000  wildtype  SHIP+/-  Pten+/-  Pten+/-SHIP+/-  Figure 28. Young Pten+/-SHIP+/- mice demonstrate significant increases in circulating IgGl, and IgE but not IgG2a. Serum from 8 week old mice was examined by ELISA for levels of circulating a) IgGl, b) IgE and c) IgG2a. ***p<0.001 over all genotypes, **p<0.025 over wildtype and SHIP+I- only. Data was derived from serum samples from at least 7 mice per genotype assessed in duplicate readings. Error bars represent the SEM of the multiple readings. 122  1-6 IL-3^""  Ptitripoterrl StemCell  1-1 iL-6  IL-6 / L tl-1.11-2 IL-7/ S mCell yL-6.IL-7  M y x o i d Stem Cell GM-CSF It. 3  [Thymus]  Pre B Cell  CFUGEMM j GM-CSF Jl-3 x  *' / GM-CSF 1-3  GM-CSF IL-3^'  BFU-E  CFU-Meg  GM-CSF t-3  GM-CSF  IL-3  • • GM-CSF IL-3  CFU-C  GM-CSF G-CSF  GM-CSF 1-3|  CFU-M  i GM-CSF  IM-CSF CFU-E  Meijakat yooiasi GM-CSF 1113  Myeloblast I GM-CSF ! G-CSF  Monoblast  JBPGM-CSF M-CSF  IL-1 IL-2 IL-4 IL 5 IL -S  CFU-Bas  CFU-GM  n %  GM-CSF  IL-3/IL-4  CFU-Eo  GM-CSF il-5  w  Myeloblast!  GM-CSF R.-5  'IP  Myeloblast  •  B Lyrophoblast  anvan Megakaryocyte Thromc-opoieir  EPO  c ... £r yt nrocyfce  NeutropNiie Myelocyte I GM-CSF i G-CSF  GM-CSF M-CSF  «  w  Thrombocytes  Promonocyte  Polymorph©-  nucleated Neutrophil  Monocyte | GM-CSF | M-CSF  Macrophage  Eosinophilic Myelocyte j GM-CSF  m  T LyrnphoWast  IL-11L-4 antigen  Proerythroblast  Prothymocyte  amige-t  Onver  Basophilic Myelocyte  L-ait-4  9 Eosinophil  Basophil IL-3.11-4  m  Masl Cell  Plasma Cell  Figure 29. Overview of hematopoiesis and the role of cytokines in lineage differentiation. From Harmening,2002.  123  Figure 30. Pten /-SHIP-/- mice have comparable body weights to SHIP-/- mice. The average body weights of mice between the ages of 4 and 5 weeks are presented. The data represents weights from 4-7 animals per genotype. Error bars represent the SEM.**p<0.001 over wildtype and Pten+/- only. +  Table 4. Pten+'SHIP' mice have significant peripheral blood abnormalities.  Genotype wildtype Pteri'' SHIP'' Pten'-SHIF ' 1  RBCs (10 /L) 8.11 ± 0 . 1 9 8.01 ± 0 . 1 5 6.95 ± 0.54 4.88 ± 0.29* 12  Peripheral CBC values Hgb (g/L) 137.66 ±3.51 137.66 ±2.74 115.50 ± 9.51 83.37 ± 5.03*  Hematocrit (UL) 0.41 ±0.01 0.42 ±0.01 0.35 ± 0.03 0.26 ± 0.02*  WBCs (10 /L) 3.65 ± 0.30 3.83 ±0.77 5.77 ± 1.34 18.76 ±3.00* 9  PLTs (10 /L) 662.20 ± 110.01 745.00± 75.63 492.00± 37.72 106.25 ± 26.96* 9  Blood samples were collected and analyzed on a Coulter Gen-S counter. Data represent analysis of 4 to 8 mice per genotype ± SEM. *p s 0.005 compared to all genotypes, A N O V A , Tukey test for multiple comparisons.  Table 5. Increased W B C in Pten* SHIP' mice is attributable to increased neutrophils. WBC differentials  Genotype wildtype Pteri ' SHIP'' Pten'-SHIP'1  Neutrophils  Lymphocytes  Monocytes  Eosinophils  Basophils  8.15 ± 1.00 8.96 ± 1.23 9.08 ± 2.92 26.81 ± 8.5F  82.60 ± 6.38 78.03 ± 7.03 75.86 ± 5.63 48.21 ±6.04*  8.48 ±5.61 12.33 ± 5.46 14.36 ± 5.84 16.20 ±4.71  0.16 ±0.16 0.83 ± 0.54 1.00 ±0.51 0.45 ±0.17  ND 0.16 ± 0.16 0.25 ± 0.22 8.03 ± 7.89  Blood smears were stained with Wright-Giemsa and percentages were determined out of 100 W B C scored. Data represent differentials from 6 to 8 mice per genotype ± S E M . p=0.06 compared to all genotypes, *p=s 0.025 compared to all genotypes.  7  to  • wildtype • Pten+/• SHIP-/• Pten+/-SHIP-/-  granulocytes  lymphocytes  monocytes  Figure 31. Pten+I- SHIP-/- mice exhibit peripheral blood leukocytosis. Peripheral blood counts for each of the four genotypes. Data was derived from Tables 4 and 5 and error bars represent the S E M of pooled data. Granulocyte counts (x 109) include neutrophils, basophils and eosinophils. Significance: ***p<0.05 over all genotypes, ** p<0.025 over wildtype and Pten+I- alone. p=0.06 over SHIP-I-, *p<0.05 over wildtype alone.  126  Figure 32. Pten+I-SHIP-/- mice have splenomegaly and hepatomegaly at 4-5 weeks of age. (a) Organ weights are expressed as percentage of body weight. Values represent the average of wieghts from at least six animals per genotype and error bars represent the S E M of the wieghts. ***p<0.005 over all genotypes.  127  Figure 33. Extramedullary hematopoiesis in livers of 4-5 week old Pten+ISH1P-I- mice. Livers were fixed in 10% formalin, embedded in paraffin and sectioned at 6 microns. H & E staining reveals the presence of monocytic and granulocytic cells throughout the livers of Pten+I-SHIP-I- mice (a) that are absent in age matched SHIP-I- littermates (b) which were similar to Pten /- and wildtype livers (not shown). Magnification 40x. +  128  Table 6. FACS analysis of whole bone marrow reveals no differences between SHIP' and PterP'SHIP'- mice 'j  Marker B220 Ly6G CDllb CD14 CD41 Terll9 NK1.1 CD34 Marker B220 Ly6G CDllb CD14 CD41 Terll9 NK1.1 CD34 Average nucleated cellularity x 10  Percentage of bone marrow cells SHIP-/Pten+/Wild type*, '• 20.42 ± 0.93 23.82 ± 1.62 26.02 ± 1.22 50.92 ± 2 . 1 8 36.5 ±2.67 36.68 ±4.27 52.5 ±2.29 36.42 ± 3.05 36.06 ± 3.73 3.45± 0.85 2.8 ± 0.62 3.95 ± 1.56 6.81 ±0.98 10.93 ± 2.02 7.11 ±0.975 25.96 ± 5.16 19.83 ± 3 . 1 0 19.14 ±4.26 15.92 ± 2 . 0 1 8.38 ± 1.04 8.21 ±0.69 12.96 ± 1.34 7.88 ±0.54 7.68 ± 0.30 Absolute number in bone marrow x 10" SHIP-/Pten+/Wild type 6.31 ± 1.08 2.69 ± 0.43 7.42 ± 0.93 6.84 ± 1.29 9.33 ± 0.43 9.97 ±0.58 7.10 ± 1.41 9.31 ±0.59 9.86 ±0.55 0.71 ±0.14 0.51 ± 0 . 1 9 1.16 ±0.53 1.54 ±0.48 1.72 ±0.18 1.92 ±0.17 5.38 ± 1.42 3.61 ± 1.40 5.25 ± 1.03 2.08 ± 0.44 2.12 ± 0.19 2.35 ±0.38 1.98 ±0.14 1.83 ±0.42 2.19 * 0.16 1.32 ±0.23  2.60 ± 0.29  2.18 ±0.23  Pten+/-SHIP-/15.23 ± 3.22 50.55 ± 5.32* 49.9 ±3.18* 8.17 ±2.60 10.69 ± 2.34 28.23 ± 4.42 18.28 ±3.47 12.96 ± 1.34 Pten+/-SHIP-/2.27 ± 0.60* 6.05 ± 1.11* 6.01 ± 1.04* 0.81 ±0.18 1.24 ±0.31 3.17 ±0.52 2.15 ±0.54 1.49 ± 0 . 1 9 1.21 ± 0.19*  8  Whole bone marrow was blocked with anti-FcyRIIB antibody and then stained with FITC or PE conjugated antibodies followed by F A C S analysis. No significant differences between wild type and PterP ' marrow were observed. No significant differences between Pten 'SHIP' and SHIP' marrow were observed. Data represent the analysis of staining from 4-6 mice per genotype. * p<0.05 over wild type and PterP ' marrow but not over SHIP marrow. 1  +  1  1  129  Figure 34. Clonogenic assays for progenitor growth reveal differences between SHIP-Iand Pten+I- SHIP-I- in marrow, liver, and peripheral blood. a) Bone marrow cells were plated in duplicate in methylcellulose complete with growth factors and the number and type of colonies were scored 10-12 days later. Pten+I- SHIP-I- mice show decreases in G E M M , B F U - E and total overall progenitor numbers compared to littermate controls. b) Splenocyte CFA reveal increases in the granulocyte/macrophage lineages over wild-type and Pten+I- cells only, c) Hepatocytes show a significant increase in the frequency of C F U (predominantly granulocytic and macrophage) in livers from Pten+I- SHIP-I- mice. d) CFA performed on peripheral blood reveal a statistically significant increase in SHIP-I- C F U which is absent in Pten+I- SHIP-I- peripheral blood. ***p < 0.05 over all genotypes, **p<0.01 over wildtype and Pten+I- only. *p<0.025 over wild-type alone. Data is compiled from at least 3 mice per genotype ± S E M of the number of colonies.  130  •  0 001  0 01  0 1  (GM-CSF ngteU)  b)  • Pron'SHIpSIMP - Pten' wikliypc  liMimal giowlh  120 100 -  T  SO 60 • 40 •  20 •  le-  n u -  ft  0 05  01  05  1  Cll—3 ngfrnl)  c)  . pien'SllIP'SUM'Pten*'wildtype  120 100 S  SO -  —  IT  20 •  o 0  3  1  125  50  25  (SF ngtel)  Figure 35. Pten- -/- SHIP-/- mice show increased sensitivity to low doses of GM-CSF over SHIP-/- littermates. Bone marrow cells were plated in methylcellulose with decreasing amounts of the growth factors GM-CSF (a), IL-3 (b) and SF (c). Total number of colonies that grew after 10 days were scored and expressed as the percentage of colonies that grew in the maximal dose of each cytokine. Both Pten+/- SHIP-/- and SHIP-/- showed increased low dose sensitivity over Pten+/- and wildtype for GM-CSF, IL-3 and SF, and Pten /- SHIP-/- showed significantly increased response over all genotypes at 0.001 ng/ml GM-CSF. These are the combined results of data from 3-4 mice per group plated in duplicate.***p<0.05. Error bars represent the S E M . 1  +  131  Figure 36. Pten+Z-SHIP-/- mice show subtle decreases over SHIP-/- in the lymphoid compartmentof the bone marrow. Representative FACS analysis to evaluate small forward scatter 'lymphoid' cells and large forward scatter 'myeloid' cells reveals only subtle decreases in the lymphoid and increases in the myeloid compartments compared to SHIP-/- profiles a) wild type, b) Pten+/~, c) SHIP-/-, d) Pten /-SHIP-/-. These results are representative of analysis performed on 4-6 mice per genotype. +  132  Figure 37. Pten+I SHIP-I- mice show subtle decreases over SHIP-I- in the clonogenic potential of pre-B progenitors. Bone marrow cells were plated in methylcellulose with recombinant IL-7 and colony numbers were assessed 6 days later and were extrpolated to femur values from the marrow cellularity. **p<0.01 compared to wild type and Pten+I- only. The graph represents data pooled from at least 4 mice per genotype. Error bars represent the S E M of colony numbers.  133  a) 8  *<>  |  1  70 60  -I  wildtype  Pten-  SHIP-/-  Ptcn-/-SHIP-  ;  b) 1 40  4 5  =j  • wildtype 0 Pten+/• SHIP- -  t  30  I  "S  20  I  15  |  10  3  • Pten+ -SHIP-,-  25  CD43WB220+  lgM+/B22()+  Figure 38. Under-representation of mature B cells in the bone marrow of Pten+/-SHIP-/- mice. Cell surface staining reveals a statistically significant decrease in B220+ cells within the lymphoid compartment of Pten+I SHIP-1- marrow (a), as well as in the B220+/IgM+ subset of mature marrow B cells (b). ***p<0.001 over all genotypes. Graphs represent data pooled from at least four mice per genotype ± S E M .  134  Table 7. Absolute number and percentage of Lin S c a r c - k i t cells in the bone marrow. +  Genotype  Lin cells Absolute number Percentage of x 10 femur 5  Scal ckit +  Percentage of lincompartment  +  Absolute number x 10 2.18±0.45 3.45±1.21 3.58±0.97 1.65±0.37 4  11.58±1.62 2.01±0.50 0.48±0.12 wildtype 8.37±1.37 4.00±0.80 0.87±0.05 Pten+'2.48±0.53 14.46±3.12 0.91±0.24 SHIP' 1.30±0.34 13.35±1.17 0.58±0.15 Pten 'SHIP'Whole bone marrow was enumerated, lineage cell depleted, re-counted and then stained for cell surface Seal and c-kit expression. Values in the chart were extrapolated from femoral cell counts. Results represent pooled data from 3-4 mice per genotype ± S E M . +  135  Table 8. Recipients of Pten SHIP"' cells are anemic and show decreased donor reconstitution in the marrow Femur  Peripheral blood Recipients of cells from wildtype Pten'-SHIF ' 1  p value  (B) 30.53±0.86 26.50±0.78  WBC (x 10 /L) 9.85+2.12 5.87+1.38  RBC (x 10 /L) 9.51 ±0.66 3.88±0.48  cellularity (x 10 ) 27.80±3.77 15.68±3.62  5.2 (% of total) 80.60±1.44 46.00±5.86  5.2 /B220 (% of total) 13.52±1.69 3.13+0.68  5.2 /Macl (% of total) 54.30±2.09 23.7+4.13  0.004  0.143  <0.001  0.036  <0.001  <0.001  <0.001  Body weight  9  12  6  +  +  +  +  +  • wildtype • Pten+/• SHIP-/• Pten+/-SHIP-/-  G ,0  O  91  m 43  Ly5.2+  Ly5.2+B220+  Ly5.2+Macl +  Ly5.2+CD3+  Figure 39. Ability of Pten+I- SHIP-I- B M cells to repopulate irradiated recipients is compromised. Flow cytometry analysis of peripheral blood samples obtained from recipients of B M cells from the various donor genotypes is shown. Cells expressing Ly5.2+, Ly5.2+ and B220+, Ly5.2+ and Macl+, and Ly5.2+ and CD3+ are represented as a percentage of total peripheral blood leukocyte populations. The data was derived from two donors of each genotype; CD3 phenotyping was performed on 5-9 recipients/genotype, and B220 and M a c l analysis on 9-12 recipients/genotype ± S E M . ***p<0.05 over all other genotypes,**p<0.01 over wildtype and Pten+I-.  137  Figure 40. The proportion of donor derived B220+ and Macl+ cells does not differ between recipeints of SHIP-/- and Pten+ISHIP-1- cells. The proportion of Ly5.2 cells that were B220+, M a c l + or CD3+ was calculated to determine if there were discrepencies compared to SHIP-/- mice. The results revealed an increase in only the percentage of CD3+ cells in the donor derived compartment of recipients of SHIP-/- cells compared to recipients of Pten+I-SHIP-1- cells.***p<0.05 compared to all genotypes, **p<0.001 compared to wild type and Pten+I- alone. The data was derived from two donors of each genotype; C D 3 phenotyping was performed on 5-9 recipients/genotype, and B220 and M a c l analysis on 9-12 recipients/genotype ± the S E M .  138  

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