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Measurement of intracellular cytokines by flow cytometry in normal and immunodeprived subjects Wu, Vivian Fung Fei 2001

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M E A S U R E M E N T OF INTRACELLULAR CYTOKINES B Y FLOW C Y T O M E T R Y IN N O R M A L AND IMMUNODEPRIVED SUBJECTS by VIVIAN FUNG FEI WU B.Sc. The University of British Columbia, 1972 R.T. Canadian Society of Laboratory Technologists, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Medicine, Experimental Medicine Programme) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 2000 ©Vivian Fung Fei Wu, 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study.-1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of rny department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of A/fOj/jyd^O^L^ T h e Univers i ty of Dritiah C o l u m b i a Vancouver, Canada Date DE-6 (2/88) Z00L1 SN0I103TI03 IVIOadS Z.8S6 ZZ8 l>09 XVd 8T:0T 00 /SZ/60 Abstract Cytokines are polypeptide protein hormones which are transiently produced by cells of the immune system and act as both autocrine and paracrine mediators to modulate the growth, activation, differentiation and proliferation of target cells. They play an important immuno-regulatory role in normal health, and their production is substantially disturbed in disorders such as transplantation and acquired immunodeficiency. This study has examined the use of flow cytometry as a rapid, sensitive and clinically relevant method to measure the intracellular production of IL-2 and IFNy, key cytokines in the human immune response. PBMC were activated with PMA and ionomycin in the presence of monensin. Fluorochrome conjugated monoclonal antibodies were used to define the intracellular cytokine and surface phenotype. In normal controls IL-2 and IFNy were expressed in 34% and 26% of T cells respectively after 5 hours of stimulation. IL-2 and IFNy expression did not differ between patients on hemo- and peritoneal dialysis (IL-2: 40% and 26%, IFNy: 22% and 29%>) nor with normal controls. Expression of IL-2 was significantly reduced in stable renal transplant patients receiving immunosuppressive pharmacological agents ( pO.OOl). The expression of IFNy was similar to normal controls after 5 hours of stimulation, the fluorescence of IFNy was reduced (p<0.05). The proportion of IL-2 and IFNy in CD3+ T cells was found to vary with each individual immuno compromised renal transplant patient in an elapsed period of 6 months . In HIV positive patients, IFNy expression was similar to normal controls but the fluorescence of IFNy was significantly reduced (pO.OOl). The expression of IL-2 was also significantly reduced (p<0.001); although differential expression in CD4+ and CD8+ T cells parallelled that in normal controls. The measurement of intracellular IL-2 and IFNy by flow cytometry is a novel, adaptable in vitro method for quantification of the immune competence of cells in a heterogeneous population. n Table of Contents Abstract ii Table of Contents iii List of Tables v List of Figures vii Acknowledgment ix Chapter One Introduction End stage renal failure 1 Renal transplantation 2 Clinical suppression 3 Measurement of lymphocyte function 5 Chapter Two Molecular and Cellular Events in Graft Rejection The sequence of graft rejection 7 Transplantation antigens 8 Antigen recognition 9 Signal transduction ' 11 Cytokine synthesis 13 Interleukin-2 14 Interferony 15 Chapter Three Immune Monitoring Using Cytokine Measurement In vitro measurement of cytokine production 17 Application of flow cytometry for cytokine measurement 19 Chapter Four Materials and Methods Objectives 22 Normal controls and study subjects 23 Cell culture and staining 23 Flow cytometry 26 Statistical analysis 30 Chapter Five Results Description of study subjects 36 Development of intracellular cytokine measurement assay 37 Comparison of intracellular cytokine expression in normal subjects and patients with chronic disease 52 iii Chapter Six Discussion Development of the intracellular cytokine measurement assay 74 Comparison of intracellular cytokine expression in normal subjects and patients with chronic disease 84 Potential customization of the assay to detect cytokines 91 References 94 iv List of Tables Table 1 Antibody combinations in the test panel 25 Table 2 Serial dilutions of antibodies to surface antigens 36 Table 3 Serial dilutions of antibodies to intracellular cytokine proteins 38 Table 4 Comparison of different stimuli in the expression of intracellular IL-2 and IFNy40 Table 5 Kinetics of CD69, intracellular IL-2 and IFNy Expression 41 Table 6 Comparison between permeabilized and non permeabilized T cells for intracellular IL-2 and IFNy expression 42 Table 7 Comparison of differences in the response between different anticoagulants and sample age 48 Table 8 Positivity and fluorescence of CD69, intracellular IL-2 and IFNy in unstimulated and stimulated T cells in normal controls 51 Table 9 Positivity and fluorescence of CD69, intracellular IL-2 and IFNy in unstimulated and stimulated T cells in PD patients 52 Table 10 Positivity and fluorescence of CD69, intracellular IL-2 and IFNy in unstimulated and stimulated T cells in HD patients 53 Table 11 Positivity of CD69, intracellular IL-2 and IFNy in unstimulated and stimulated T cells in PT patients 54 Table 12 Fluorescence of CD69, intracellular IL-2 and IFNy in unstimulated and stimulated T cells in PT patients 55 Table 13 Positivity and fluorescence of CD69, intracellular IL-2 and IFNy in unstimulated and stimulated T cells in HIV patients 56 Table 14 Positivity of intracellular IL-2 in T cells for all study groups 58 Table 15 Fluorescence of intracellular IL-2 by T cell for all study groups 60 Table 16 Positivity of intracellular IFNy for all study groups 62 Table 17 Fluorescence of intracellular IFNy by T cells for all study groups 64 Table 18 Differential expression of CD69 on T cell subsets between normal controls and PT and HIV patients 66 Table 19 Differential expression of intracellular IL-2 in T cell subsets between normal controls and PT and HIV patients 69 Table 20 Expression of intracellular IL-2 and IFNy by CD3+ T cell at the initial and 6 months assessments for the PT patients 71 vi List of Figures Figure 1 Delineation of leukocytes and lymphocyte gate 31 Figure 2 Gating of CD3 positive T cells 31 Figure 3 Gating of CD4+CD3+ and CD8+CD3+ cells 32 Figure 4 Histogram of an isotype control 33 Figure 5 Overlay of the intracellular IL-2 and the isotypic control histograms 34 Figure 6 Overlay of the intracellular IFNy and the isotypic control histograms 34 Figure 7 Overlay of the non permeabilized and the permeabilized T cells histograms, for IL-2 expression 43 Figure 8 Overlay of the non permeabilized and the permeabilized T cells histograms for IFNy expression 44 Figure 9 Comparison of unblocked and blocked intracellular IL-2 expressions 46 Figure 10 Comparison, of unblocked and blocked intracellular IFNy expressions 47 Figure 11 Comparison of different anticoagulants and specimen age 49 Figure 12 Summary of positive CD69 expressions between unstimulated and stimulated T for all study groups 57 Figure 13 Box and whisker plot of T cell intracellular IL-2 expression for all study populations 59 Figure 14 Box and whisker plot of T cell intracellular IL-2 fluorescence for all study populations 60 Figure 15 Box and whisker plot of T cell intracellular IFNy expression for all study populations 63 Figure 16 Box and whisker plot of T cell intracellular IFNy fluorescence for all study populations 64 Figure 17 Comparison of intracellular IL-2 expression of PT and HIV patients to normal controls in T helper cells and T suppressor cells. 68 vii Figure 18 Relationship of intracellular IL-2 expression by T helper cells to T cells in of HIV Pos patients 69 Figure 19 Relationship of intracellular IL-2 expression by T helper cells to T cells in of PT patients 69 Figure 20 Relationship of intracellular IL-2 expression by T helper cells to T cells in of normal controls 69 Figure 21 Relationship of intracellular IL-2 expression by T suppressor cells to T cells in of normal controls 70 Figure 22 Relationship of intracellular IL-2 expression by T suppressor cells to T cells in of PT patients 70 Figure 23 Relationship of intracellular IL-2 expression by T suppressor cells to T cells in of HIV positive patients 70 Figure 24 Bar chart of the L M C F of intracellular IL-2 expression by T cells at the initial and 6 months assessments 72 Figure 25 Bar chart of the L M C F of intracellular IFNy expression by T cells at the initial and 6 months assessments 73 Vl l l Acknowledgements I would like to express my sincere gratitude to Dr. Paul A. Keown for giving me the opportunity to obtain my Master of Science degree and for his tutorship as my supervisor. Special thanks to members of my committee, Dr. Jean LeRiche and Dr. David Landsberg, for their valuable time and helpful comments. I am indebted to the Immunology Laboratory at Vancouver General Hospital and especially to Janet Fitzpatrick. She gave me endless support, advice and encouragement. Above all, I am very grateful to my husband and children who have demonstrated great tolerance, patience, and understanding over the past years as I juggle my professional career and graduate studies with my duties as wife and mother. ix Chapter 1 Introduction End Stage Renal Failure The kidney is a vital organ whose primary function is the maintenance of homeostasis in blood by the regulation of fluid and electrolyte exchange. The kidney also functions as an endocrine organ controlling the production of erythrocytes, skeletal mineralization and vascular tone through the production of erythropoietin and other hormones. The kidney may be damaged through a wide variety of congenital or acquired disorders, such as glomerulonephritis, pyelonephritis, autoimmune nephropathy, chronic hypertension, cystic diseases, nephropathies caused by drugs or chemicals, or by diseases such as diabetes and amyloidosis resulting in the progressive loss of renal function and acute or chronic renal failure1. Patients with end stage renal disease (ESRD) are often treated by hemodialysis or peritoneal dialysis. In the former, they are connected to an external dialysis membrane through a vascular access system for approximately 12 hours per week, while in the latter, dialysis fluid is introduced into the peritoneal cavity several times daily, in order to maintain normal homeostasis. In 1997 there were 12,090 Canadian patients on renal dialysis of which 29.6% were on peritoneal dialysis and 70.4% were on hemodialysis2. In 1997, British Columbia has 1164 renal patients on dialysis of which 32.7% were on peritoneal dialysis and 67.3% were on hemodialysis3. Dialysis is able to regulate fluid and electrolyte exchange and remove some of the uremic toxins while erythropoietin and vitamin D can be taken orally to replace the endocrine function of the 1 kidneys. Nonetheless, this treatment is imperfect and does not restore normal metabolic functions. The disease remains debilitating and the patient's quality of life is adversely affected by the stress of dialysis and the accompanying fatigue, anorexia, anemia, renal osteodystrophy, and neuropathy. In addition, although advances in dialysis technology can prolong the patient's life, both forms of dialysis repetitively expose the depressed immune system of the ESRD patient to an increased risk of infection. Infectious complications contribute to the significant morbidity and mortality of the ESRD patients4. Renal Transplantation Kidney transplantation is the therapy of choice for most patients with ESRD. With successful transplantation, fluid regulation is restored and glomerular filtration returns towards normal levels during the post operative period. As renal function improves, cardiovascular function improves, anemia and neuropathy are corrected, osteodystrophy is reversed, and calcium and phosphorus metabolism reverts to normal5. Even ESRD induced infertility is restored to normal6. The patient returns to a relatively normal quality of life. In Canada, 2778 patients were waiting for a kidney transplant and 969 renal transplantations were performed in 1997. In British Columbia there were509 patients on the renal transplant waiting list and 139 renal transplants were performed in 1999, comprising 72 living donor and 67 cadaveric donor transplantation7. The kidney for renal transplantation may come from a cadaver, from a living blood relative, or even from a genetically unrelated living donor. Prior to transplantation, the patient and the potential donor are typed for A B O and H L A compatibility, and the recipient serum is investigated for the presence of anti-HLA antibodies to his/her donor. Matching of the M H C between the donor and the recipient is used to improve the outcome of the transplantation. However, the advance of molecular genetic techniques has revealed a tremendous degree of polymorphism within the M H C class I and class II loci8. This polymorphism reduces the possibility of an exact M H C match between donor and recipient in allograft transplantation. Because of this disparity, renal transplantation is not universally successful. Acute rejection of the graft occurs in 30-50% of patients, and is the leading cause of graft loss in the first year. Acute rejection may also increase the risk of chronic rejection, which results in graft failure months or years after transplantation. By 1997 the 1,3 and 5 year graft survivals in Canada were 92%, 88% and 81%o for living donor transplantations and 83%, 75%, and 67% for cadaveric grafts. The half life of the cadaveric grafts was 8.7 years9. By 1999 in British Columbia the 1, 3 and 5 year graft survivals were 94.1%, 92% and 90% for living donor transplants and 87.3%, 80.2%, and 71.7% for cadaveric grafts10. Clinical Immunosuppression Pharmaceutical or biological agents are used in all except identical twin transplants to reduce the immune response of the recipient and permit engraftment of the transplanted organ. The earliest of these included azathioprine, cyclophosphamide and corticosteroids1Azathioprine, a purine analog, and predisone, a corticosteroid, are among the most common agents used for immunosuppression in clinical transplantation. Neither is highly potent, however, and prednisone had many undesirable long-term consequences. Polyclonal antilymphocyte sera (ALS) and monoclonal antibody therapy was developed to remove or inactivate circulating T lymphocytes. The monoclonal antibody OKT3 is still one of the most effective clinical tool used for reversing renal allograft rejection12. 3 In 1976 Borel 1 3 first described the immunosuppressive effects of cyclosporine (CsA). CsA and tacrolimus are cyclic peptides which bind to cytosolic receptor proteins and function as potent inhibitors of calcineurin activity and cytokine gene transcription. Both are widely used in all forms of transplantation, although the microemulsion formulation Neoral is currently accepted as the baseline therapy for de novo transplantation in most centres14. Over the past decade the repertoire of immunosuppressive agents have expanded to include mycophenolate mofetil, sirolimus and brequinar sodium. Sirolimus binds to similar cytoplasmic receptor proteins but act at discrete molecular sites, blocking the downstream effect of the IL-2 receptor signal15. Preliminary studies show that sirolimus acts synergistically with CsA to produce highly effective clinical immunosuppression. In order to ensure effective immunosuppression, these agents are normally administered alone or in combination at the highest tolerable dose for all patients, even though many patients might remain clinically and immunologically quiescent at much lower drug dosage. Many of these drugs have important side effects, including marrow suppression, kidney failure, liver toxicity and hyperlipidemia, which may be mitigated to an extent by combining agents to reduce the individual dose. However, these drugs often produce additive or synergistic suppression of the immune response, resulting in an increase in the risks of infections and malignancy. For these reasons, the use of these agents requires careful and continuous monitoring to optimize the therapeutic risk/benefit ratio. Therefore there is a need for a reliable system to measure the competence of the immune response so that episodes of rejection can be predicted, and excessive suppression can be avoided. The most widely used method for monitoring immunosuppressive drug therapy at present is the pharmacokinetic measurement of drug trough levels, which is performed at routine intervals throughout the transplant course. Recent data has shown that this approach does not necessarily reflect the in-vivo biological action of the drug, however, a problem which may be more complex when several of these agents are used in combination. Direct monitoring of the host immune status is therefore preferable, but difficult to achieve with simplicity and accuracy. Although a number of preliminary approaches have been proposed, including measurement of calcineurin activity or IL-2 secretion, no reproducible method has as yet been devised which is suitable for this purpose. Measurement of Lymphocyte Function Currently, few methods exist for measurement of T cell immunity. Limiting dilution assays (LDA) have been used to estimate the frequency of T cells from the kidney recipient capable of differentiating into cytotoxic specific for the M H C antigens of the donor. The cells of the recipient and donor cells are plated in an arithmetic series and cultured for three to twelve days. After the incubation period, the cultures are tested for cytolytic activity in a standard 5 1 Cr release assay.51 Cr labeled target cells, such as the NK sensitive K562 cell lines or 5 1 Cr labeled Con A blast cells are added to the wells and after further incubation, analyzed for y emission from the oxidized 5 1 C r 2 + released by the dead cells'6. L D A can also use graded numbers of donor cells cultured with irradiated E B V transformed lymphoblastoid cell lines. The supernatant from each well is transferred and re-cultured with C T L L line to determine the IL-2 bioactivity of the supernatant, while the viability of the donor cells is determined by colorimetric measurements of the M T T assay using an ELISA reader system17. M T T [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] is a biochemical dye that is reduced by N A D H dehydrogenase of actively growing cells to an insoluble 5 formasan product which can be visualized spectrophotometrically18. Overall lymphocytic response to donor antigens can be measured in a mixed lymphocyte culture (MLC) with irradiated stimulator cells for three days. Tritiated thymidine [3H] added to the culture, is incorporated into the DNA of proliferating cells, and counted by a beta counter19. These assays quantitating T cells activity by limiting dilution, direct measurement of cytotoxic T cell activity and [ H] thymidine uptake are highly labour intensive with a slow turn-around, and thus have little practical relevance as routine monitoring tools. 6 Chapter 2 Molecular and Cellular Events in Graft Rejection The Sequence of Graft Rejection The immune system functions to protect the host from invasive pathogens by its exquisite ability to distinguish between self and non self. This surveillance function is achieved by the complex interaction of immunocompetent cells that circulate within the blood and lymphatic systems, and are resident in most organs. The introduction of an allograft into the host induces an immune response initiated by the presentation of foreign peptides. This may occur either directly on the graft cells or via donor antigen presenting cells (APC) which have migrated out of the graft to local recipient lymph nodes, or indirectly by the presentation of the donor peptides engulfed by self antigen-presenting cells which are then expressed in the context of the host's own major histocompatibility complex (MHC). The consequent appearance of donor-specific antibodies and cytotoxic T-cells in the peripheral blood, and a rise in concentration of inflammatory cytokines or their receptors in the serum and graft effluent, coincide with acute graft rejection. Alloactivated T lymphocytes and alloantibodies infiltrate the graft, where they produce direct lysis of target cells bearing relevant M H C molecules, and trigger subsequent events in the inflammatory cascade20'21 . Secondary macrophage infiltration and activation results in the liberation of proteolytic enzymes, excited oxygen radicals, macrophage-derived procoagulant activity, inflammatory and chemotactic mediators. These induce vasoconstriction, promote platelet aggregation, trigger microvascular coagulation and fibrin deposition, and increase vascular permeability, intensifying cellular recruitment into the graft.2 2'2 3 7 Messenger RNA levels of the cytokines IL-2 and IL-6 have been shown to increase up to 20 fold with graft rejection; this is not specific, however, and the same response may be observed during episodes of sepsis.24'25 The mRNA of other cytokines have been detected within the rejecting graft by in situ hybridization, and are reported to vary with the organ evaluated: IL-2, IL-4 and TNF-B mRNA were observed during cardiac graft rejection whereas IL-6 and TNF-a were reported in kidney graft rejection.26'27 Transplantation Antigens The principal transplantation antigens in humans are the human leukocyte antigen (HLA), coded by the major histocompatibility complex (MHC) on the short arm of chromosome 6. Products of these genes are highly expressed on lymphocytes and other mammalian cells, and are divided into two classes by structure and function. Class I H L A molecules function to present predominantly intracellular peptides including viral gene products, and class II present predominantly extracellular peptides, to the T cell receptor (TcR) complex. The H L A system is the most polymorphic gene region yet discovered. Principal polymorphisms exist in the variable regions of the and a2 domains of the class I molecules and mainly in the p chains of the class II molecules clustering around the antigen binding sites where the M H C class I and class II molecules contact endogenous and exogenous peptides and present them to the T lymphocytes. The T receptor can then recognize simultaneously the foreign peptide and the polymorphic determinants of the H L A molecule, enabling the immune system to distinguish self from non self and to initiate the appropriate response28. 8 Class I molecules, encoded by HLA-A, -B and -C genes, are composed of 45kDa transmembrane glycoproteins and are found on all nucleated cells and consist of 3 extracellular domains, a , a 2 and a 3 which are non covalently associated with a 12 kDa p 2 microglobulin polypeptide. The region between a j and a2 forms the antigen binding site involved in the presentation of endogenous peptides of 8-10 amino acids in length. Class II molecules are encoded by HLA-DP, -DQ, and -DR genes and are constitutively expressed on antigen presenting cells such as macrophages, monocytes, dendritic cells, B lymphocytes and on activated T cells by cytokines such as IFNy. The class II molecules consists of 2 non covalently associated polypeptides, the 30-34 kDa a and 26-29 kDa P chains. Each chain consists of 2 domains, ax and a 2 or P, and P 2 . The region between a! and Pj form the antigen binding site involved in the presentation of exogenous peptides that are over 15 amino acids in length, extending out of the binding site29. Antigen Recognition The T cell receptor (TcR), which recognizes foreign peptides in association with H L A molecules, consists of an extracellular heterodimer with transmembrane peptides and short cytoplasmic tails. On >90% of T cells the heterodimer is composed of a and P subunits, while on the remainder it consists of y and 6 subunits. Each chain is encoded by individual genes, designated A, B, G, and D. Each gene consists of V, D, J and C segments that are capable of rearrangement conferring great diversity to the variable regions of the TcR. The variable region is responsible for the recognition of foreign peptides presented in the context of the MHC class I and class II molecules30. The CD3 complex is functionally associated with the TcR and is composed of 5 polypeptides, designated y, 6, e, C and r| and is present on the cell surface of all mature T lymphocytes. The cytoplasmic domains of the polypeptides y, 8, e, £ and n contains antigen recognition activation motifs (ARAM) 9 or tyrosine activation motifs (TAM) which are phosphorylated after TcR engagement. Interaction between the TcR and MHC normally occurs with low affinity and is facilitated by a number of associative recognition molecules that may have co-stimulatory functions. CD8 serves this function for cytotoxic T cells which recognize foreign peptides in association with M H C class I molecules while CD4 serves the same role for helper T cells which recognize foreign peptides in association with M H C class II molecules. Both molecules are expressed on individual subsets of T lymphocytes, and promote adhesion between the foreign peptide, the TcR and the M H C molecule. Binding to the TcR is essential, but not sufficient for T cell activation, and a second signal is required under normal circumstances. This is provided by co-stimulatory molecules including CD2, CD28, CTLA4, CD40 and CD40L. CD2 is expressed on the T cell surface, and binds to its ligand CD58 (known as leukocyte functional antigen (LFA)-3) on antigen presenting cells, thereby providing adhesion between the APC and the T cell. CD2 serves as an adhesion molecule and functions in cytoplasmic signal transduction during activation as the proline rich sequences triggers p56 / c / c tyrosine kinase activity that is independent of TCR-CD3 complex 3 1 , 3 2. Cytotoxic T lymphocyte associated antigen (CTLA-4) and CD28, are encoded by two genes co-localized on chromosome 2. They can be expressed in monomeric or homodimeric forms. The ligand for both CD28 and CTLA-4 is the B7 molecule on the APC. B7 is a member of the immunoglobulin (Ig) gene superfamily encoded by a gene in chromosomal region 3ql3.3-3q21 on chromosome 1233. 10 Resting T cells express CD28, but the expression is up regulated several hundred fold upon activation. CTLA-4 expression is restricted to activated CD4+ and CD8+ T cells and is approximately 1/3Oth - l/50th the levels of CD28. Though present with relatively lower expression on activated T cells, CTLA-4 binds the B7 ligand with high avidity while the CD28 molecule has a lower avidity for B7 3 4 . The binding between the B7 on the APC and the CTLA-4 and CD28 molecules on the T cells functions in stabilizing the adhesion of the activated T cell to the APC while CD28 also functions in signal transduction and enhances the translation of the IL-2 gene. CD40 is a 45-50 kDa glycoprotein that is expressed on B cells, monocytes, and dendritic cells. The ligand for CD40 is CD40L, which is rapidly induced on CD4+ T cell upon activation. Engagement of CD40 to CD40L also enhances the expression of B7 molecules on the surface of dendritic cells and the production of IL-12 by these cells. While B7 and CD28 interaction amplifies the T cell response, IL-12 secretion promotes the CD4+ T cells into Thl effector cells and induces the production of IFNy 3 5. Signal Transduction Engagement of the H L A and the TcR/CD3/CD4 or CD8 complex on the T cell surface leads to lymphocyte activation through a series of intracellular signal transduction events. The cross linking of CD4 or CD8 with CD45, a cell surface glycoprotein, initiates tyrosine phosphatase activity in the cytoplasmic tail of CD45 which activates the Src family of kinases, p56fc/£ and p59^" within the CD4 or CD8 cytoplasmic tails. The binding of CD4 and/or CD8 to the antigen-MHC complex brings p56 / c / c and p59^yn into close proximity to the CD3 ( domains enabling the phosphorylation of the tyrosine residues within the CD3 A R A M The phosphorylated tyrosine A R A M then recruits ZAP-70 11 kinase36. A protein SLP-76, in turn, is phosphorylated by ZAP-70 generating docking sites for Src homology 2 domain (SH2) binding proteins, required for the phosphoinositide and ras pathways37. The activated protein tyrosine kinases also activate phospholipase C y l (PLCyl) which in turn hydrolyses phosphatidyl inositol 4,5 biphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 binds to receptors of C a 2 + storage vesicles in the cytosol, triggering their release and raising the level of intracellular C a 2 + . Experimentally this rise in C a 2 + is mimicked by the presence of ionomycin. The increases in C a 2 + activates calcineurin, which in turn dephosphorylates the ubiquitous cytostolic NFATc and its homologue the pre-existing cytoplasmic component NF ATp, members of the NFAT transcription complex. This results in their translocation to the nucleus38. Meanwhile D A G activates protein kinase C (PKC) which has downstream effects on the ras pathway. Activation of ras regulates MAP kinase cascade. MAP kinase, known as extracellular signal regulated kinase (ERK1), translocates to the nucleus and induces the expression of fas, myc and jun to form the AP-1 transcription factor complex39. The signal provided by the peptides bound to the M H C class II molecules and presented to the TcR/CD3 complex is amplified by the co-stimulatory binding of CD28 on the T cells to the B7 molecules on the APC. This results in the phosphorylation of the tyrosine residues in the cytoplasmic domains of CD28. PTK Phosphoinositide 3-kinase also binds to this site and activates P L C y l 4 0 . N F A T proteins play an important role in the regulation of cytokine gene expression41. N F A T sites may be flanked by AP-1 in the 5' promoter region as in the IL-2 gene, IL-4 and tumor necrosis factor (TNF)a genes or adjacent to binding sites of other transcription factors42. IFNy does not to have 12 N F A T binding sites, but it has two P sequences, Pl and P2, which behave like the N F A T in the IL-2 gene. The binding of NFAT-like proteins in the cytoplasm of activated T cells undergo nuclear translocation and binds to the promoter sequence of the IFNy gene after T cell stimulation43. The activation of PKC can be imitated experimentally by phorbol esters such as phorbol 12-myristate 13-acetate (PMA). Direct stimulation by intracellular signalling molecules PMA and ionomycin can bypass surface TcR stimulation and induce the activation of CD4+ T cells44. These signalling events lead to transcriptional activation of cytokine genes, transiently initiating the synthesis of mRNA, resulting in increased cytokine synthesis and secretion. Cytokine Synthesis Human T cells consist of heterogeneous populations whose subsets respond to activation signals by secreting different cytokines. Once secreted, the cytokines diffuse through intercellular space to their target cells where they interact with their cell surface receptors to regulate the activation, differentiation, proliferation and mobility of the target cells. CD4+ T cells may be separated into two defined subpopulations based on their function, known individually as type 1 helper T cells (Thl) and type 2 helper T cells (Th2). Thl cells preferentially secrete IL-2 and IFNy. IL-2 can feed back in an autocrine manner and further activate cytokine genes to produce IFNy, IL-4, IL-5, IL-7, IL-10 and colony stimulating factors (CSF). IFNy and granulocyte-macrophage colony stimulating factors (GM-CSF) in turn activate granulocytes to produce pro-inflammatory cytokines IL-1, IL-6 and TNFa, as well as IL-10 and IL-12. Th2 cells preferentially secrete IL-4, IL-6, IL-10 and IL-13 which are growth and differentiation factors for B cells. Th2 also secrete IL-5 which causes eosinophil differentiation and activation. Precursors to Thl/Th2 cells, known as ThO secrete all the 13 cytokines. Thl and Th2 cells cross regulate each other's function by reciprocal antagonism. Th2 cells inhibit Thl cell function through the secretion of IL-10, whereas Thl cells inhibits the function of Th2 cells by antagonizing IL-4 with the production of IFNy. Clonal expansion of Thl cells preferentially promotes cell mediated responses, while antagonizing the effect of Th2 cells in promoting the humoral response. Patients who suffer from chronic illnesses such as delayed type hypersensitivity (DTH) 4 5 demonstrate such polarization toward a Thl response. Interleukin-2 Interleukin-2 (IL-2), first described as T cell growth factor (TCGF), is a 153 amino acid (a.a.), 15.5 kDa glycoprotein, encoded by a gene containing four exons and three introns located on long arm of chromosome 4 (4q26-28)46,47. Analysis of the three-dimensional structure of IL-2 shows that it is 3A in size, and is composed mainly of a-helical segments which are interconnected by a disulphide bond between cystine (Cys) 58 and Cys 105 to form a globular structure. At position 3 there is O-linked glycosylation of the threonine residue. Transcription of the gene is controlled by the 5' enhancer region proximal to the nuclear factors NFAT-1, AP-1 and NFIL-2A 4 8 . IL-2 production is contingent upon activation of the T cells, which requires two independent co-stimulatory signals. One is induced by foreign peptides presented in the context of either major histocompatibility complex (MHC) class II in CD4+ T cells or M H C class I in CD8+ T cells. The other co-stimulatory signal for CD4+ T cells may be provided by several cell surface molecules, of which the principal molecule is CD28 whose ligand B7 is found on B cells, monocytes, dendritic cells and macrophages. Cross linking of CD28 and B7 initiates the transduction signals mobilizing the nuclear factor, CD28 response complex (CD28RC) which binds to the 5'of the AP-1 response element, CD28RE, stabilizing the mRNA 4 9 and augmenting IL-2 production50. IL-2 acts as an 14 autocrine signal which induces the clonal expansion of activated cells, promoting progression from G l to S phase in the cell cycle. It also functions as a paracrine signal to promote B cell differentiation, enhance antibody production, and activate lymphokine activated killer cells (LAK). Interferon-y Interferon-gamma (IFNy) is a type II interferon, also known as immune interferon. It is a 143 a.a. peptide with a molecular mass of 34-50 kDa, which is encoded by the gene containing four exons and three introns located on chromosome 12 (12q24.1)51. The regulatory elements of the IFNy gene have been mapped to a region 700 bp immediately 5' to the transcription start sites of N F A T like, AP-1 binding sites52. It appears that IL-2 from activated T cells is essential for promoting the early transcription of the IFNy gene and this induction is dose independent53. Young et al in 1996 postulated the existence of a complex in the AP-1 region known as ying-yang-1 (YY-1) which has both silencer and promoter functions. YY-1 blocks the transcription of the IFNy gene in resting cells. When the cell is activated, other DNA proteins bind to AP-1 and displace YY-1 permitting transcription of the IFNy gene54. The IFNy polypeptides forms a biologically active, compact and globular homodimer 35A in size55. It has a helical structure, whose tertiary shape is maintained by noncovalent forces. The molecule is therefore labile to heat (56°C), acidity (pH<4.0) and alkalinity (pH>9.0)56. Each individual polypeptide chain contains two N-linked glycosylation sites at asparagine (Asn) 25 and 97 at position 100 which may be differentially glycosylated into two 20 K and 25 K weight forms. Although the biological activity is not affected, the two weight forms affect the proteolytic degradation, stability and solubility and thus the circulatory life of the molecule57. 15 IFNy is produced by CD8+ T lymphocytes, NK cells and activated CD4+ T (Thl) lymphocytes, and is induced by foreign peptides in the context of either M H C class I (in CD8+ T cells) or class II (in CD4+ T cells). It up-regulates the expression of MHC class I antigens, and induces the synthesis of class II antigens on cells that do not constitutively express these markers. In addition, IFNy increases the expression of IL-2 receptors on T cells, regulates immunoglobulin isotype switching on B cells, up regulates the expression of cell surface receptors (FcR) for IgG, increasing antibody-mediated cellular cytotoxicity (ADCC) and induces the secretion of biologically active proteins such as C2 of complement pathways58. 16 Chapter 3 Immune Monitoring Using Cytokine Measurements Production of the cytokines IL-2 and IFNy is central to the development of the immune response when foreign peptides of the graft are presented to the host T-cells either directly, or indirectly by self A P C in the context of autologous M H C class I or class II molecules. For this reason, measurement of these cytokines has been used to monitor immune activation following transplantation. Cytokine production may be measured in vivo in the venous drainage from the graft, or in urine in the setting of renal transplantation. However the rapid dilution which occurs in both of these situations makes this approach unreliable. Cytokine production may also be triggered in vitro, in which case peripheral blood mononuclear cells (PBMC) are usually activated in cell cultures by direct stimulation of the T cell receptor/CD3 complex, with mitogens such as P H A or conavalin A (conA), or biochemically with P M A and ionomycin. The supernatant is removed after a few days of culture, and a variety of techniques are used to measure the net outcome of secreted cytokines over the incubation period. In vitro Measurement of Cytokine Production Biological assays depend upon the ability of the reference cytokine to support the proliferation of specific cytokine-dependent cell lines in vitro. The presence of IL-2 may be assessed using the IL-2 dependent cell line C T L L , measuring the proliferation by [3H]-thymidine incorporation5 9 into newly formed D N A strands during the S phase of the cell cycle. The presence of IFNy can be confirmed by the expression of M H C class II on W E H I 3 cells 6 0. Fluorochrome conjugated anti-MHC class II antibody is used to label the cells after exposure to the IFNy and analysed by flow cytometry. 17 Immunoassay techniques may be used to quantitate cytokines directly in the supernatant using, for example, a specific E L I S A sandwich methodology in which anti-cytokine antibodies are immobilized on micro titer plates. Dilutions of cytokine secreted from cultured cells are incubated with the immobilized anti-cytokine antibody. A substrate is added to bound antibody complexes and the enzymatic reaction is demonstrated by colour change analysed by the E L I S A reader. The E L I S A reader can also be used to analyze the reduction of M T T tetrazolium salt (3-(4, 5-dimethylthiazol-2-ys)-2, 5-diphenyl tetrazolium bromide) to formazan salt crystals by mitochondria of viable cytokine dependent cell lines in the culture wells 6 1 . Resting macrophages have high mannosyl receptor ( M M R ) activity which is down regulated by IFNy. This down regulation can be assayed by trace labelling the ligand to M M R , mannosylated-BSA withNa[ 1 2 5 I] iodide and analyzing the degradation o f [ 1 2 5 I] mannosylated-BSA by a gamma counter6 2. A variety of other mechanisms also exist for cytokine quantitation. The E L I S P O T analyses cytokine production at the single cell level. Plates coated with mouse anti-human cytokine monoclonal antibodies are incubated with cells at different dilutions. After incubation, the plates are incubated with biotinylated anti-cytokine monoclonal antibodies and visualized with alkaline phosphatase conjugated streptavidin as spots6 3. Molecular methods to detect m R N A have used in situ hybridization, and polymerase chain reaction (PCR) methodology. In the former, cells are hybridized with antisense R N A probe for the cytokine gene. The c D N A sample is then treated with RNase, dehydrated and coated with nuclear track emulsion, developed and counter stained for analysis by microscopy 6 4 . P C R uses 5' and 3' oligonucleotide primers specific for the cytokine m R N A were reverse transcribed and amplified. The amplified D N A fragments of nucleotides were separated by electrophoresis, visualized by autoradiography and analysed by densitometry 6 5 ' 6 6 ' 6 7. 18 None of these tests are ideal for purposes of clinical monitoring. The techniques are time consuming and labour intensive; accurate calibration of laboratory standards is required; the toxic dyes and radioactive methods employed can be hazardous; inhibitors or substances other than cytokines may be present in culture fluids leading to false negative and false positive results; contamination can be a problem with P C R methods and the presence of cytokine m R N A does not guarantees its translation. In addition, the described methodologies are unable to provide simultaneous information about the production of different cytokines from individual cells. Application of Flow Cytometry for Cytokine Measurement Flow cytometry is the measurement of physical and/or chemical properties of biological cells in a fluidic medium as they flow in a single file past a measuring source. The earliest reports of cytometry were confined to the enumeration of cells passing through a capillary tube which were counted using a photoelectric sensor6 8. This was subsequently extended in 1947 by the incorporation of forward scattered light measurements and the use of an intense light source and photomultiplier tube as detector which permitted more sophisticated analyses including the counting of bacterial cells in aerosols 6 9. The use of synthetic dyes in flow cytometry proved an important advance. In 1941 Papanicolaour and Traut reported the use of fluorescent staining techniques to distinguish malignant cells from normal cells in vaginal carcinoma smears7 0. In 1950, Friedman using berberine sulfate dye found that nuclei of malignant cells stained more intensely than those of normal cells 7 1 . Coons and his collaborators later used isocyanate fluorescein which emitted a yellow-green fluorescence to discriminate cells from autofluorescence72. Immunofluorescent staining has since become a widely used technique. In 1956, Wallace Coulter suspended cells in saline, passed them through a small orifice and detected a change in impedance by which he was able to accurately count and size those 19 cells73. The flow cytometers today in clinical and research laboratories still use the Coulter principle. The use of flow cytometry to investigate intracellular antigens was initially described in 1969 by Van Dilla. This technique was first used to study DNA content, which was later followed in 1980 by studies in enzymatic activity such as peroxidase activity of leukocytes, acid phosphatase, esterases and oxidative enzymes and other intracellular functional assays including intracellular C a 2 + concentrations, intracellular pH and membrane and cytoplasmic membrane potentials74. In 1990 Andersson et al reported the use of flow cytometry to investigate the production of intracellular cytokines. To gain access to the interior of the cell, the cell membranes were permeabilized with saponin allowing the penetration of anti-cytokine monoclonal antibodies75. This technique was further refined by Sander et al in 1991 who reported the simultaneous detection of surface antigens and intracellular cytokines by two or three colour staining using fixation with paraformaldehyde and permeabilization by saponin76. In 1993, Jung et al used monensin to disrupt intracellular protein transport by the Golgi apparatus. The staining of the accumulated protein enhanced fluorescent signals allowing simultaneous analysis of intracellular cytokines and cell surface markers of single cells within a heterogeneous population by flow cytometry77. The advent of computers, lasers, novel statistical methods and an increasingly improved array of synthetic dyes and fluorochrome conjugation methodologies have further advance this field. The increased flexibility and functional capability of the modern flow cytometer have enabled the technology to become a sophisticated tool for the analysis of cell biology. The potential advantages of flow cytometry for measurement of intracellular cytokine production include 1) simultaneous detection of two or more cytokines in a single cell, enabling Thl/Th2 T cell functional subset 20 discrimination78,2) the analysis of simultaneous cytokine expression among different cell types, 3) detection of cytokines in rare or specific cell populations, 4) a short stimulation period which reduces the risk of induced apoptosis and 5) the speed and reproducibility of the method makes the technology easily applicable to clinical studies79. As with any clinical test, operational parameters of cytokine flow cytometry must be optimized, standardized and trial tested to determine the sensitivity and limitations before clinical studies can be applied. 21 Chapter 4 Materials and Methods 4.1 Objectives The intent of the current studies was to develop a simple, rapid and reproducible flow cytometric assay for intracellular measurement of IL-2 and IFNy which could be used to monitor immunologic status in renal transplantation. The specific objectives were as follows: 1. To establish the optimal parameters for the assay. (a) To compare the triggers for in vitro lymphocyte activation. (b) To determine the optimal incubation time for the expression of IL-2 and IFNy. (c) To evaluate the importance of cell membrane permeabilization. (d) To determine the specificity of the monoclonal antibodies to intracellular IL-2 and IFNy. (e) To assess the effects of different anticoagulants and specimen storage time on the expression of intracellular IL-2. 2. To determine the expression of CD69, IL-2 and IFNy in normal subjects. 3. To examine the production of intracellular cytokines in patients with chronic renal disease. 4. To evaluate the production of intracellular cytokines in patients with acquired immune deficiency disease. 5. To measure the production of intracellular cytokines in stable patients following renal transplantation and to determine the change over time. 22 4.2 Normal Controls and Study Subjects F i v e groups o f subjects were examined i n this study. Group 1 consisted o f no rma l disease-free controls w h o were not rece iv ing any medicat ion for immune-related disorders. They were selected f rom the laboratory personnel at the Vancouve r Genera l Hospi ta l . Groups 2 and 3 consisted o f stable adult patients w i t h end-stage renal disease who were rece iv ing therapy w i t h peri toneal d ia lys is or hemodialys is respectively and who were not on immunosuppressive medicat ion. B o t h were recruited f rom the renal disease program at the Vancouve r General Hosp i t a l . Group 4 consisted o f patients w i t h a funct ioning renal transplant who were at least one year post-transplantation. A l l were rece iv ing immunosuppress ion wi th cyclosporine, azathioprine and steroids, and were recruited f rom the renal transplant c l i n i c at the Vancouver General Hosp i t a l . Group 5 consisted o f patients w h o were pos i t ive to the H I V virus , and were being tested i n the Immuno logy Labora tory o f the V a n c o u v e r Genera l Hosp i t a l . 4.3 Cell Culture and Staining M o n o n u c l e a r C e l l Separation Per ipheral b l o o d (14 ml ) was obtained by venipuncture into sod ium hepar in anticoagulated vacutainers at r o o m temperature. Unde r sterile condit ions inside a laminar f l o w hood, the b l o o d was di lu ted 1:1 w i t h phosphate buffered saline ( P B S ) (Gibco B R L , Gaithersburg, M D ) and layered over f i c o l l hypaque (Pharmacia Bio tech , Uppsa la , Sweden) density separation gradient, and centrifuged at 400g at r o o m temperature for 30 minutes. The mononuclear ce l l layer was removed and washed twice i n P B S , once at 150g and again at 400g at r o o m temperature for 10 minutes. The washed cel ls were resuspended i n 1-1.5 m l o f R P M I 1640 m e d i u m (Gibco) , supplemented w i t h 2 % v / v o f L -glutamine, 2 % v / v penic i l l in-s t reptomycin . Whi t e b lood ce l l concentrat ion was determined by an electronic c e l l counter. 23 Monoclonal Antibody Titrations Monoclonal antibodies to human surface antigens CD3 (Beckon Dickinson (BD), San Jose, CA), CD4 (BD), CD8 (BD), and CD69 (BD), intracellular IL-2 (PharMingen, San Diego, CA), and IFNy (BD) and related isotypic controls were purchased from their manufacturers conjugated to fluorochromes fluorescein isothiocyanate (FITC), phycoerythrin (PE) or peridinin chlorophyll (PerCP). Each of these antibodies were titrated in a two fold dilution series. Unstimulated PBMC from healthy volunteers were used in the series to titrate anti-human CD3, CD4, CD8 and CD69 antibodies, while PBMC stimulated with PMA at 10 ng/ml and ionomycin at 1 /uM/ml with monensin at 2 //M/ml for 5 hours were added to the IL-2, and IFNy titration series. Lymphocyte Activation PBMC at a concentration of l-2xl0 6 cells/ml were suspended in a total volume of 1 ml of RPMI 1640 supplemented with 15%v/v heat inactivated fetal calf serum (FCS) (Hyclone Laboratory Inc., Logan, UT) in 17x100 mm polypropylene tubes (BD). Lymphocyte activation was triggered by the addition of (a) PMA (Sigma Chemical Co., St. Louis, MO) at 10 ng/ml and ionomycin (Sigma) at 1 AiM/ml, (b) 5%v/v PHA (Gibco), or (c) a combination of OKT3 (Ortho Biotech, North York, Ont) at 1 /ug/ml, 2 /ug/ml, 4 /ug/ml, 10 /ug/ml, 20 /ug/ml and 40 /ug/ml and CD28 (Serotec Inc., Raleigh, NC) at 0.25 /ug/ml, 0.5 /ug/ml, 1 /ug/ml, 2.5 /ug/ml, 5 /ug/ml and 10 /ug/ml. The tubes were loosely capped and incubated in 5% C 0 2 humidified incubator at 37°C for 1-24 hours. Physiological Blockade of the Golgi Apparatus Physiological blockade of cytokine export from the Golgi apparatus was achieved by the addition of monensin (Sigma) at 2 /^M/ml into the stimulated cell cultures. Monensin was added at the same time to unstimulated control cultures. 24 Cel l Surface Labelling with Monoclonal Antibodies After incubation, the cell cultures were washed with 2 ml of wash solution consisting of P B S , l % v / v heat inactivated F C S (Gibco) and 0 .1% v / v sodium azide (NaN 3 ) (Sigma), at p H 7.4 - 7.6, and centrifuged at 400g for 10 minutes. The supernatant was discarded and the remaining cell pellet was resuspended in T C 199 medium (Gibco), supplemented with 2 % v / v F C S into the original volume. Cel l suspension at 2 - 4 xlO"5 concentrations were aliquoted into a test panel of six polypropylene 12x75 mm tubes containing monoclonal antibodies (Mab) directed against cell surface markers described in table 1. Tube a PerCP (Surface) FITC (If used) P E 1 C D 3 C D 4 / C D 8 (Surface) I g G l (Surface) 2 C D 3 C D 4 / C D 8 (Surface) C D 69 (Surface) 3 C D 3 C D 4 / C D 8 (Surface) IgG2a (Intracellular) 4 C D 3 C D 4 / C D 8 (Surface) IL-2 (Intracellular) 5 C D 3 IgG2a (Intracellular) 6 C D 3 IFNy. (Intracellular) Table 1 Test panel composition of monoclonal antibody combinations for cell surface and intracellular staining. The panel of tubes was incubated at 4 ° C for 30 minutes. After incubation, each tube was washed with 1 ml of wash solution, centrifuged at 400g for 10 minutes. The supernatant was aspirated to remove any unbound Mab. Cells in tubes #1 and #2 (table 1) stained with Mab to surface antigens only were fixed with l % v / v paraformaldehyde (PFA) (J .S .EM Services Inc., Pointe Claire-Dorval, Que) and stored at 4 ° C for analysis by flow cytometry. 25 Cell Fixation and Permeabilization Cold 4%v/v PFA (0.5 ml) was added to each of the remaining tubes (tubes #3-#6), which were vortexed immediately and incubated at 4 °C for 10 minutes. At the end of PFA incubation, the tubes were centrifuged at 700g for 10 minutes and the supernatant was aspirated. Each tube was lightly vortexed to loosen the cell pellet and washed once with 2 ml of permeabilization buffer at pH 7.4 -7.6 containing PBS, l % v / v heat inactivated FCS, 0.1%v/v NaN 3 and 0.1%v/v saponin (Sigma). The tubes were centrifuged at 700g for 10 minutes and the supernatant was again aspirated. Intracellular Cytokine Labelling The tubes were vortexed gently to loosen the cell pellet and 25 iA of permeabilization buffer was added to each tube. Optimal concentrations of PE conjugated anti-IL-2 and its isotypic control IgG2a (PharMingen, San Diego, CA), and of FITC conjugated IFNy FITC and its isotypic control, IgG2a FITC, (BD) were added to the tubes as shown in table 1. The anti-cytokine Mab was diluted in saponin-PBS (PBS, 10%v/v heat inactivated FCS and 0.1%v/v saponin). After the second antibody incubation, the tubes were washed once with 1 ml permeabilization buffer, and centrifuged at 700g for 10 minutes. After aspiration of the supernatant, the tubes were vortexed gently to loosen the cell pellet and resuspended in 0.3 ml of wash solution and analysed within 24 hours. 4.4 Flow Cytometry The flow cytometer used in this study was an Epics Profile I flow cytometer (Coulter Corp., Miami, FL). The fluidic, optical and data analysis systems are described briefly below: Fluidic System Sheath solution, an electrolyte buffered cell free medium (Biosure Controls, Grass Valley, CA), was delivered under pressure of 6.5 psi into the flow chamber also known as flow cell. Sheath flowed 26 down the sides of the flow chamber to form a laminar flow. The cell sample was injected from the sample insertion tube at a controlled rate of 25 yul/min. in single file into the laminar flow inside the flow chamber. Hydrodynamic focusing was achieved with the cells positioned by the laminar flow in the central region of the stream, stabilizing the cellular flow as it passed through the interrogation point where the cells were illuminated by the laser beam. The used sheath solution and sample were returned to the waste tank for disposal. Optical System The light source of the flow cytometer was an argon laser, with an emission wavelength of 488 nm. A s the intensity of a laser beam demonstrated Gaussian distribution, the beam was shaped through a series of lenses into an elliptical shape. This resulted in a larger area of constant illumination within the beam width in which the cells travel. This wavelength of 488 nm was used to excite the conjugated fluorochromes FITC, PerCP and PE . Photons from the laser beam were either scattered or absorbed by the cells as they passed through the interrogation point. Forward scatter (FS), also known as forward angle light scatter ( F A L S ) , measured the size of the cell. A t the same time, 488 nm argon light scattered by the cytoplasmic granules known as side scatter (SS) or 90° light scatter (90LS), measured the granularity of the cell distinguishing granulated from non-granulated cells. The fluorescent dyes conjugated to the Mab absorbed photons at 488 nm raising the fluorochromes to an excited state, from which they relapsed to the ground state with the emission of light energy. F ITC emits in the green spectral band with peak emission at 525 nm, P E emits in orange spectral band with peak emission at 575 nm and PerCP emits in red spectral band with peak emission at 680 nm. The analog signals of different wavelengths of emitted light were spectrally separated and the fluorescence from each fluorochrome followed an optical path that was controlled by optical filters and mirrors. Dichroic filters placed at 45° to the 27 light source enables light of transmissible wavelength to pass through the filter, while other wavelengths were reflected at 90° to a second set of photomultiplier (PMT) detectors. Each separated wavelength passed through a band pass filter such that each PMT only detected a specific narrow spectrum of light. The emitted light impact upon its internal dynode plates, accelerating the electrons until the analog signal exits the PMT as an electrical current80. The green wavelength passed through 520 - 530 nm, orange wavelength was transmitted through 570 - 580 nm and red passed through 655-695 nm band pass filters. Despite the use of filters, there remained a small percentage of spectral overlap which was compensated (eliminated) electronically. Data Analysis System The analog electrical signal from each PMT was electronically converted to linear digital signals by an electronic analog-to-digital converter (ADC). The dynamic range from the dimmest to the brightest expression was increased by logarithmic amplifiers, and displayed by the onboard computer as scatter plots or histograms on four decade logarithmic scale. Fluorescence from each cell can be measured simultaneously at different wavelengths by using multiple detectors. White blood cells can be separated into subpopulation of lymphocytes, monocytes and granulocytes by the flow cytometer based on their properties of size and granularity as determined by forward and side scatter (figure 1), and/or by fluorescence analysis of differential unique expression of surface antigens (figure 2). A gate or bitmap can be manually drawn around cell populations of interest. The acquisition of digital data from within these gates is displayed as a single parameter, two dimensional histograms or as two parameter dot plots. Single parameter histograms display relative fluorescence intensity of the conjugated fiuorochrome of interest on the X axis, which consists of a four decade logarithmic fluorescence scale, and the number of cells labelled by the fiuorochrome that is counted by the flow cytometer along the Y axis. Analysis 28 cursors are drawn across the distribution of interest and the statistical software from the cytometer's onboard computer provides frequency distribution which includes percentage, mean, standard deviation and coefficient of variance of cell fluorescence. The instrument's conversion chart converts the mean logarithmic fluorescence intensity along the X axis to linear mean channel fluorescence (LMCF). The reported percentage (% positive) is the relative proportion of positive cells within the bitmap that is displaying the antigen of interest. Flow Cytometric Data Analysis The 15 mW output laser emitting at 488 nm wavelength was aligned daily with DNA-Check (Coulter) polystyrene beads. Fluorescence in each of the fluorescence channel was standardized to the target channel daily using Flow-Set (Coulter) polystyrene beads. Fluorescence compensation was electronically set for optimal separation of FITC, PE and PerCP immunofluorescence. A double anchor gating strategy with two different types of gates was used in the analysis of each tube. A bitmap was manually drawn around the lymphocyte population as illustrated in figure 6 and 10,000 events from within the bitmap were counted by the cytometer. A second fluorescence gated bitmap targeting total T cells, as determined by fluorescence of anti-CD3 Mab and lymphocyte SS characteristics (figure 2), or individual subpopulations of T cells, T helper cells (CD4+CD3+) or suppressor T cells (CD8+CD3+) (figure 3) was manually drawn around the population of interest. Expressions of CD69 and intracellular cytokine expressions of IL-2, and IFNy as well as their respective isotype controls from the gated cells were displayed to single parameter, two dimensional fluorescence histograms. Isotype controls were used to label cells with an irrelevant antibody of the same isotype class and fluorescence as the test reagent. The analysis cursor was placed at the base of the negative event 29 distribution, extending across the X-axis as illustrated in figure 4. The same cursor setting was applied to the test. This has been depicted in figures 5 and 6, where the expressions of intracellular IL-2 and IFNy were overlaid onto histograms displaying the isotype controls. If the events within the isotypic control cursor were < 2% positive, the percentage of events occurring within the test was recorded. When the isotypic events were > 2%, this frequency was subtracted from the test results. Occasionally it was necessary to draw a second analytical cursor when there was a slight shift in the negative events. 4.5 Statistical Analysis Expressions of IL-2 and IFNy within CD3+ lymphocytes and the two principal T cell subpopulations (CD4+CD3+ and CD8+CD3+ T cells) from each of the five study groups were analysed using one way analysis of variance (ANOVA). Statistical significance for all analysis was set at a = 0.05, testing the sample means with the null hypothesis HQ •' M (normal) = f2 (HIV Pos) = / " (PD) = ^ (HD ) = / " (PT) The analysis data was expressed as the percentage of positive staining cells or as L M C F . When the null hypothesis was rejected, Newman-Keul's post hoc multiple comparisons method was applied. 30 Figure 1 Diagram to depict the delineation of leukocytes into lymphocytes, monocytes and granulocytes by the flow cytometer according to their forward (FS) and side scatter (SS) properties. Bitmap (A) has been drawn around the lymphocyte population, CO Q U SS Figure 2 A gate or bitmap (B) has been drawn around CD3 positive T cells which was determined by reactivity to anti-human CD3 monoclonal antibodies and low side scatter properties. 31 LFL 3 (CD3) Figure 3 Two parameter dot plot of CD4 (or CD8).(Y axis) vs CD3 (X axis). Gate or bitmap (B) has been drawn around CD4+CD3+ T helper (or CD8+CD3+ T suppressor) cells in quadrant 2. 32 T 1 1 I I I I 11 1000 LFL 2 (Isotype Control] Figure 4 Single parameter histogram of an isotype control. An analysis cursor was set at the base of the negative population, extending across the 4 decade log fluorescence scale on the X-axis and cell count is on the Y-axis. 33 1000 LFL 2 (IL-2) F i g u r e 5 Histogram of 16% of cells showing the intracellular IL-2 expression overlaid onto the isotypic control histogram. LFL 2 (IFNy) F i g u r e 6 Histogram of 33.9% positive cells showing the intracellular IFNy expression overlaid onto the isotypic control histogram. 34 Chapter 5 Results 5.1 Description of Study Subjects Group 1 (Normals) consisted of 11 laboratory personnel (8 females and 3 males) with a mean age of 34 years (range: 25 - 45 years) with no physical signs of illnesses. Group 2 (PD) was composed of 10 clinically stable peritoneal dialysis patients (3 females and 7 males) with a mean age of 55 years (range: 44 - 73 years) who have been on peritoneal dialysis for a mean of 17 months (range: 1-39 months). Group 3 (HD) consisted of 8 patients (3 females and 5 males) with a mean age of 60 years (range: 26-81 years) who have been clinically stable on hemodialysis for a mean of 14 months (range: 2 - 5 1 months). None of these patients were receiving immunosuppressive medication or other drugs known to influence the immune system. Group 4 (PT) consisted of 10 patients (4 females and 6 males) with a mean age of 47 years (range: 35 - 56 years) who have had their renal grafts for a mean of 5.9 years (range: 2-15 years). All were receiving cyclosporine immunosuppression therapy on a twice daily basis, along with azathioprine and predisone. Group 5 (HIV) was composed of 12 patients (3 females and 9 males) who were positive to p24 antigen of human immunodeficiency virus (HIV). Their ages ranged from 26 - 47 years. 35 5. 2 Development of Intracellular Cytokine Measurement Assay 5.2.1 Titration of monoclonal antibodies to cell surface molecules The results of titration of the fluorochromes PerCP, FITC and PE conjugated anti-human antibodies to surface antigens CD3, CD4, CD8 and CD69 using unstimulated PBMC from healthy volunteers are shown in table 2. CD3 PerCP Concentration (/zg/ml) llllllllli 0.03 llllilillllll llllilillllll O.OS 0.04 % Positive 82.3% 81.9% 81.8% 80.1% 78.7% 75.8% L M C F 134 133 127 120 98 73 CD4 FITC Concentration (,ug/ml) 0.3 0.15 0.08 0.04 0.02 0.01 % Positive 43.3% 43.0% 42.6% 39.6% 40.2% 39.0% L M C F 129 130 128 124 110 98 CD8 FITC Concentration (/ug/ml) Ijjllljlll ().()3 0.16 0.08 0.04 % Positive 14.2% 13.1% 12.9% 12.0% 11.7% 11.4% L M C F 128 124 122 116 114 111 CD69 PE Concentration (,ug/ml) 2.5 1.25 lllllllllll 0.3 1 0.16 0.08 % Positive 91.1% 83.9% 61.4% 49.5% 39.9% 31.1% L M C F 112 111 99 94 92 90 Table 2 Results of two fold serial dilutions of CD3 PerCP, CD4 FITC, CD8 FITC and CD69 PE expressed as % positive and L M C F for each concentration of antibody. 36 There was a one channel decrease in L M C F in CD3 PerCP titration between the concentrations 1.25 ug/ml and 0.63 ug/ml, and larger decreases in fluorescence with subsequent dilutions. The percentage of positive staining cells stayed relatively stable between concentrations 1.25 - 0.63 ug/ml. A concentration of 0.63 ug/ml PerCP conjugated anti-CD3 antibody was selected for use. Anti-CD4 FITC showed a one channel increase in L M C F from 129 to 130 as the concentration decreased from 0.3 to 0.15 ug/ml while the percentage of positive cells remained relatively stable between 0.3 to 0.08 pg/ml. The concentration of 0.15 ug/ml was therefore selected for FITC conjugated anti-CD4 antibody. There was 1.1% decrease in the percentage of positive cells and a decrease of 4 linear channels between the manufacturer's recommended concentration of 1.25 ug/ml and the dilution at 0.63 ug/ml. The % positive cells and L M C F continued to decrease throughout the range of the titration series. 0.63 ug/ml was selected as the concentration for use in labeling surface CD8 antigens. There was a decrease of 1 linear channel between manufacturer's recommended concentration of 2.5 ug/ml and 1.25 ug/ml and a steady decrease thereafter. The percentage of positive cells decreased from 91.1% to 83.9% between 2.5 ng/ml and 1.25 ug/ml with a larger decrease to 61.4% at 6.3 ug/ml and steadily decreased thereafter. The concentration of anti-CD69 was therefore selected as 1.25 ug/ml. 37 5.2.2. T i t ra t ion o f monoc lona l antibodies to intracellular cytokines Ti t ra t ion o f P E conjugated anti-human I L - 2 P E and F I T C conjugated ant i-human I F N y used performed us ing P B M C . The cells were stimulated by P M A at 10 n g / m l and i o n o m y c i n at 1 fuM/ml i n the presence o f monens in at 2 fjM/ml for 5 hours. Results are shown i n table 3. Concentra t ion (/-tg/ml) l l l l l l l l l 0.5 i i i i i i i i i i i i i i i l l l l l l l l l l l 0.063 0.031 % Pos i t ive 54.6% 37 .5% 35 .7% 32 .9% 3 0 . 5 % 2 7 . 5 % L M C F 155 138 137 129 113 99 I F N y F I T C Concent ra t ion (jug/ml) l l l l l l l l l 1.25 i i i i i i i i i i 0.3 1 0.16 0.08 % Pos i t ive 20 .3% 20 .8% 20 .7% 19.0% 17.7% 17.6% L M C F 182 166 146 134 124 114 Table 3 Resul ts o f two fo ld serial di lut ions o f I L - 2 P E and I F N y F I T C expressed as % posi t ive and L M C F for each concentration o f antibody. There was a decrease i n L M C F and i n % posi t ive cells f rom 1 ug /ml . L M C F remained constant f rom 0.5 - 0.25 p g / m l fo l l owed by a further steady decrease f rom 0.13 to 0.031 p g / m l . The percent posi t ive cel ls also remained constant f rom 0.5 - 0.25 ug /ml w i t h further sl ight decreases f rom 0.063 to 0.031 u g / m l . A concentration o f P E conjugated ant i - IL-2 o f 0.5 u g / m l was selected for use. There was a steady decrease i n fluorescence i n the titration series for I F N y The percentage o f pos i t ive cel ls remained relat ively stable between 2.5 to0.63 u g / m l and decreased steadily thereafter. A concentrat ion o f 1.25 ug /ml was selected as the concentration for F I T C conjugated anti- I F N y antibody. 38 5.2.3. Comparison of PHA. PMA and ionomycin and OKT3 and anti-CD28 stimulation on CD69 and intracellular IL-2 and IFNy expression Two additional stimuli, the plant lectin phytohaemagglutinin (PHA) and the monoclonal antibodies anti-CD28 and anti-CD3 (OKT3), were compared to PMA and ionomycin for their ability to trigger intracellular cytokine production. The results are shown in tables 4. Target Protein 4 Hours 24 Hours Unst imulated (with monensin) CD69 (% Pos) 2.6% 1.1% CD69 (LMCF) 81 102 IL-2 (% Pos) 0.3% 0.4% IL-2 (LMCF) 98 93 IFNy (% Pos) 0.4% 0.3% IFNy (LMCF) 80 77 St imula t ion by 5 % v / v P H A (with Monens in) CD69 (% Pos) 46.2% 78.0% CD69 (LMCF) 114 129 IL-2 (% Pos) 4.6% 2.2% IL-2 (LMCF) 103 100 IFNy (% Pos) 1.5% 1.0% IFNy (LMCF) 88 93 St imula t ion by P M A (10 ng/ml) and ionomyc in (1 yuM/ml) (with Monens in) CD69 (% Pos) 45.6% 76.1% CD69 (LMCF) 94 131 IL-2 (% Pos) 26.6% 20.1% IL-2 (LMCF) 139 111 IFNy (% Pos) 15.8% 28.5% IFNy (LMCF) 152 126 39 Stimulation by O K T 3 ( 4 0 Aig/rnl)and anti-CD28(10>g/ml) (with Monensin) CD69 (% Pos) 78.0% N T CD69 (LMCF) 120 -IL-2 (% Pos) 3.1% N T IL-2 (LMCF) 106 -IFNy (% Pos) 4.6% N T IFNy (LMCF) 118 -Table 4 Percentage (% positive) and L M C F of cells expressing intracellular IL-2 and IFNy in T cells after 4 and 24 hour incubation, stimulated by 5 % v / v P H A , O K T 3 and anti-CD28 and P M A and ionomycin. PHA, PMA and ionomycin Normal control P B M C were incubated in R P M I 1640 media with 15% v / v F C S and 2 u M / m l monensin for 4 hours and 24 hours, and were stimulated with 5% v / v P H A or a combination of P M A at 10 ng/ml and ionomycin at 1 u M / m l throughout the period of incubation. A s shown in table 4 lymphocytes were activated by both stimuli resulting in a significant increase in CD69 expression. However, less than 5% of cells stimulated with 5% v / v P H A produced intracellular IL-2 and IFNy production above control values after 4 and 24 hours, compared with over 20% of cells which were positive for IL-2 and IFNy after stimulation with P M A and ionomycin. Stimulation with OKT3 and anti-CD28 Normal control P B M C were stimulated with differing concentrations of O K T 3 and rat anti-human CD28 for 5 hours in the presence of monensin. Only 1.9% of unstimulated CD3+ T cells were positive for CD69 , while 2.7% of cells expressed intracellular IL-2 and 1.3% expressed IFNy . Following stimulation, as shown in table 4, lymphocytes were activated and 70% to 83.8% were positive for surface antigen CD69. However, the intracellular expression of IL-2 and IFNy remained at control levels. A ten fold increase in the concentration of O K T 3 and CD28 showed only marginal improvement on the overall expression of surface CD69 and intracellular IL-2 and IFNy . 40 5.2.4. Kinetics of cell surface CD69 and intracellular IL-2 and IFNy expression Normal control PBMC were stimulated with PMA at 10 ng/ml and ionomycin at 1/JVl/ml. The expression of surface CD69 and intracellular IL-2 and IFNy were measured hourly throughout the first 6 hours of incubation period. The results are shown in table 5. 1 hour 2 hour 3 hour 4 hour 5 hour 6 hour CD69 7.2% 15.5% 15.2% 12.4% 10.9% 9.2% Unst. LMCF 74 76 78 75 76 77 i i i i i i i l i i i i i 7.8% 53.2% 75.0% 82.8% 88.3% 88.7% Stim. LMCF 73 76 84 87 94 97 IL-2 i i i i i i i l i i i i i i i i 0.1% 0.2% 0.3% 0.1% 0.1% 0.2% Unst. LMCF 59 62 61 62 63 79 2.7% 24.1% 29.9% 32.0% 37.1% 32.4% Stim. LMCF 72 94 103 114 120 120 IFNy 0.2% 0.3% 0.3% 0.5% 0.5% 0.6% l l l l l l l l l l l l l l 93 96 96 90 96 96 l l l i l i l i l i i i i 11.9% 26.3% 29.3% 29.8% 29.4% 29.1% Stim. LMCF 111 148 173 179 183 183 Table 5 Results of the kinetic study of surface CD69, intracellular IL-2, and IFNy stimulated with P M A and ionomycin and unstimulated cells for 5 hours in the presence of monensin expressed as % positive and L M C F . CD69 expression increased steadily during first 3 hours and 82.8% to 88.7% of cells were positive for this marker from 4-6 hours. Intracellular IL-2 expression increased progressively during the first 3 hours and remained constant from 4 - 6 hours. Expression of intracellular IFNy followed a parallel course, and remained stable from 3 - 6 hours. A period of five hours was therefore selected 41 as the optimal incubation time to measure peak expression of CD69, intracellular IL-2, and IFNy. 5.2.5. Effect of cell membrane permeabilization Normal control PBMC were stimulated with PMA and ionomycin in the presence of monensin for 5 hours. The cultured cells were then stained for their surface CD3 antigens and either treated with saponin to induce membrane permeation or left untreated. Cells were labelled with antibodies to IL-2 and IFNy. The intracellular expression of IL-2 and IFNy is summarized in table 6 and illustrated in figures 7 and 8. Permeabilized cells exhibited both brighter fluorescence for IL-2 (LMCF: 149 vs 97) and IFNy (LMCF: 174 vs 99) and a higher percentage of positive staining cells (IL-2: 43.4% vs 18.5%o, IFNy: 23.6% vs 7%), than those in which the membrane was intact. Intracellular Cytokine Expression % Positivity Permeabilized Not Permeabilized IL-2 % Positive 43.4% 18.5% , L M C F 149 97 IFNy % Positive 23.6% 7.0% L M C F 174 99 Table 6 Results of comparison between membrane permeabilized and not permeabilized T cells for IL-2 and IFNy, expressed as % positivity and L M C F . 42 Effects of Permeabilization in Expression of Intracellular IL-2 in CD3+ T Cells Log Fluorescence (IL-2) Figure 7 Histogram of non permeabilized C D 3 + T cells expressing 18.5% intracellular IL -2 with L M C F of 97 was overlaid with histogram of permeabilized C D 3 + T cells expressing 43.4% intracellular IL-2 with L M C F of 149. 43 Effects of Permeabilization in Expression of Intracellular IFNy in CD3+ T Cells Q V E R L A Y : S I N < 3 L E P A R A M E T E R Log F l u o r e s c e n c e ( IFNg) Figure 8 Histogram of non permeabilized CD3+ T cells expressing 7% intracellular IFNy with L M C F of 99 was overlaid with a histogram of permeabilized CD3+ T cells expressing 23.6% intracellular IFNy with L M C F of 174 44 5.2.6. Specificity of intracellular anti-IL-2 and anti-IFNv labelling Blocking experiments were performed using recombinant human (rh) IL-2 and IFNy proteins (PharMingen) to confirm the specificity of antibody binding. Normal control PBMC were stimulated with PMA and ionomycin in the presence of monensin for 5 hours. The cultured cells were initially stained for their surface CD3 antigens. PE conjugated anti-human IL-2 and FITC conjugated anti-human IFNy antibodies were pre-incubated with the appropriate recombinant proteins at 2 to 5 fold antibody excess. An isotype control was used to preset the analysis cursor. The results of the analysis are illustrated in figures 9 and 10. In unblocked cultures, 36.8% of cells expressed intracellular IL-2 and 19.0% showed intracellular IFNy. Rh IL-2 protein at 3X anti-human IL-2 antibody concentration blocked most of the epitopes on the IL-2 protein, so that only 3.8% of cells remained positive for IL-2. Similarly, the addition of rh IFNy at 2-5X the anti-human IFNy antibody concentration markedly reduced the intracellular anti-IFNy antibody binding. Thus the use of recombinant human cytokine proteins demonstrated the specificity of the monoclonals R A H IL-2 and M A H IFNy antibodies in intracellular labeling. 45 IL-2 Log Fluorescence Figure 9 Comparison of intracellular IL-2 between unblocked and blocked expression by recombinant human IL-2 protein in excess of Mab concentrations. 46 I F N Y Log Fluorescence Figure 10 Comparison of unblocked and blocked expression of intracellular IFNy by recombinant human IFNy protein in excess of Mab concentrations. 4 7 5.2.7. Anticoagulant and age of blood sample comparisons Blood samples from normal controls were drawn into three different anticoagulants: acid citrate dextrose (ACD), heparin and ethylenediaminetetraacetic acid (EDTA), and were processed on the same day or stored at room temperature for 24 and 48 hours. The effects of these variables on stimulated intracellular IL-2 expression are shown in table 7 and illustrated in figure 11. Same Day 24 Hours 48 1 lours A C D % Positive 54.3% 42.2% 22.2% i i i i i i i i j i i i i i i i i 147 131 129 EDTA % Positive 51.4% 36.6% 18.9% I . M C I 146 134 129 Heparin % Positive 55.4% 44.1% 33.9% 148 136 135 Table 7 Comparisons of differences in the response of intracellular IL-2 expression to A C D , E D T A and heparin anticoagulants and between different chronological ages of samples. There was no significant difference in L M C F or the percent of cells staining positive for IL-2 between the three anticoagulants when the samples were processed on the day of venipuncture. L M C F decreased with increasing duration of storage in all three anticoagulants, falling to 12% of control in A C D , 12% in EDTA and 9% in heparin by 48 hours. The percent of cells staining positive for IL-2 decreased even more rapidly, falling to 59% of control in A C D , 63% in E D T A and 39% in heparin at the same time point. Therefore, although the use of heparin appears to mitigate the decline in both L M C F and percent of cells positive for intracellular IL-2 by comparison with A C D and EDTA, blood samples should be used within 24 hours of collection to ensure consistency of measurement. 48 Comparison of Anticoagulants and Specimen Age (IL-2 Expression) Same Day 24 Hours Specimen Age % Positive (YI) ACD(Y1) EDTA (Y1) 48 Hours LMCF (Y2) Heparin (Y1) Figure 11 When specimens were processed the same day of the venipuncture, there was comparable positive expression of intracellular IL-2 (51.4% - 55.4%) and fluorescence intensity of this expression (LMCF 146 - 148) among the three anticoagulants (ACD, E D T A and heparin). After 24 hours there was decrease in expression and fluorescence intensity of intracellular IL-2 with further decreases after 48 hours. 49 5.2.8. Standardization of the assay Based on the results described, peripheral blood samples were drawn into sodium heparin anticoagulated tubes and processed within 24 hours. White blood cells harvested by ficoll hypaque density gradient and cultured in RPMI 1640 media supplemented with 15%v/v FCS, 2%v/v L -glutamine, and 2%v/v penicillin-streptomycin were left unstimulated or stimulated with PMA at 10 ng/ml and ionomycin at 1 uM/ml in presence of 2 uM/ml of monensin for 5 hours. The cultured cells were washed and labeled with predetermined concentrations of antibodies to surface antigens CD3, CD4, CD8 and CD69. This was followed by fixation with paraformaldehyde and permeabilization by saponin. The permeabilized cells were subsequently intracellularly labelled with antibodies to IL-2 and IFNy and analysed by flow cytometry. Bitmaps were drawn around lymphocytic population and T cells of interest. The results from the bitmaps were displayed in single fluorescence histograms, expressed as percentage of positive events and as L M C F , within the single parameter analysis cursor which had been preset by the isotype control. 50 5.3. Comparisons of CD69 and Intracellular Cytokine Expression in Normal Subjects and Patients with Chronic Disease 5.3.1. Comparison of intracellular IL-2 and IFNy expression within populations 5.3.1.1. Normal Subjects CD69 1|;!|:;;1|1|||||I | i | | | : | l n s | iH i | | | | i Slim. Unstim. Slim. • p ^ i i ^ ^ i i i i i i Slim. %Pos LMCF %Pos LMCF %Pos LMCF %Pos LMCF %Pos LMCF %Pos LMCF NI 2.9 86 79.4 103 0.8 60 19.3 132 0.3 56 19.4 155 N2 6.7 82 75.0 97 0.7 124 36.9 161 0.3 75 22 167 N3 6.9 92 78.2 98 0.6 124 28.9 142 0.2 81 37.7 164 N4 4.4 94 73.6 91 1.8 113 44.9 156 0.3 76 25.2 182 N5 6.4 81 75.8 94 0.3 78 51.9 134 0.5 54 25.3 159 N6 3.8 85 76.8 96 0.5 81 34.3 149 0.2 79 42.2 158 N7 5.0 82 64.2 96 1.0 86 45.7 140 0.3 78 17.1 164 N8 7.1 79 74.7 94 4.3 118 40.5 148 1.8 68 20.5 164 N9 2.3 82 75.5 98 3.5 136 42.0 152 0.2 76 22.7 178 N10 18.6 88 93.5 115 0.5 111 38.6 146 0.3 79 26.8 165 N i l 13.0 86 84.8 105 0.6 96 26.9 141 0.1 71 28.1 165 X 7.0 85 77.4 99 1.3 102 37.3 146 0.4 72 26.1 166 sd 4.8 5 7.3 7 1.3 24 9.4 9 0.5 9 7.7 8 Table 8 Positive (%) and L M C F of CD69, cytokines IL-2, and IFNy in unstimulated, and stimulated control CD3+ T cells of normal, healthy volunteers, x = mean Table 8 shows that the percentage of cells expressing CD69 (PO.0001), intracellular IL-2 (PO.0001) and IFNy (PO.0001) was significantly higher in stimulated cultures by comparison with unstimulated controls. The L M C F of CD69, intracellular IL-2 and IFNy expressed by stimulated cultures were also significantly higher than the unstimulated cultures (PO.001). 51 5.3.1.2. Stable Peritoneal Dialysis Patients (PD) The results presented in table 9 shows the surface expression of CD69 was seven fold greater on PMA and ionomycin stimulated cells compared with unstimulated cells. The amplitude of the responses in PMA and ionomycin stimulated cells measured by the % positivity and L M C F for CD69, intracellular IL-2 and IFNy, were similar to those seen in the normal subjects (table 8). !l||fi | |n^ IFNy %Pos LMCF %Pos LMCF %Pos LMCF %Pos L M C F PD-l 10.6 89 75.4 100 32.9 125 17.0 153 PD-2 9.1 85 89.1 117 36.4 146 18.4 169 PD-3 4.4 82 70.3 94 44.2 152 25.7 157 P D - l 8.4 92 87.5 107 32.3 135 20.0 156 I'D-* 1.0 92 75.2 97.0 40.9 136 14.7 148 PD-(> 2.3 78 65.4 106 35.6 145 23.2 167 PD-7 2.9 84 87.0 106 44.4 143 20.3 149 PD-8 10.3 86 80.8 102 51.0 153 27.8 162 PD-9 4.2 85 62.4 97 44.3 142 31.7 168 I'D-lo- 16.9 82 94.4 97 46.8 140 11.2 103 rn ean 7.0 86 78.8 102 40.9 142 22.2 153 sd 4.9 4 10.8 7 6.3 8 5.2 19 Table 9 Positive (%) expression and L M C F of CD69 in unstimulated, and CD69, cytokines IL-2, and IFNy in stimulated, CD3+ T cells of stable peritoneal dialysis patients. 52 5.3.1.3. Stable Haemodialysis Patients (HD) The results presented in table 9 show the surface expression of CD69 was eight fold greater on PMA and ionomycin stimulated cells compared with unstimulated cells. The amplitude of the responses in PMA and ionomycin stimulated cells compared to normal subjects were the same as the PD group in 5.3.1.2. | | | | | | | n s t i i w ^ llllllllllli CD69 CIW) l l l l M IFNy %Pos L M C F %Pos L M C F %Pos L M C F %Pos L M C F HD - l 7.9 87 73.4 98 33.7 141 19.0 164 HD-2 7.5 84 78.7 100 36.0 149 24.5 164 HD-3 16.2 86 92.7 111 42.4 140 35.1 169 HD-4 6.8 85 84.2 102 42.2 136 33.2 168 HD-5 5.9 93 82.5 104 49.7 149 33.0 160 HD-6 4.5 94 79.3 102 61.0 152 23.2 164 HD-7 6.0 93 84.1 110 36.3 131 23.6 149 HD-8 3.6 87 75.3 106 22.2 128 41.3 156 mean 7.3 89 81.3 104 40.4 141 29.1 162 sd 3.9 4 6.1 5 11.5 9 7.6 7 Table 1 0 Positive (%) expression and L M C F of CD69 in unstimulated, CD69, cytokines IL-2, IFNy in stimulated, CD3+ T cells of stable haemodialysis patients. 53 5.3.1.4. Stable Post Renal Transplant Patients (PT) The percentage of cells expressing the surface antigen CD69 and intracellular IL-2 and IFNy at the initial and 6 months assessments are shown in table 11 and their L M C F are shown in table 12. 111 ; ;|fnM Stimulated Ill|;llir|>6ll|illlllll l l l l l l i l l l l l Initial 6 mos Initial 6 mos Initial 6 mos Initial 6 mos PT-1 5.3 11.8 50.7 34.4 10.5 3.6 32.8 9.3 PT-2 5.8 5.0 63.0 70.8 33.6 29.4 32.8 34.2 PT-3 4.6 3.1 68.4 85.9 23.9 17.3 25.4 25.8 PT-4 9.9 4.1 79.8 42.8 5.3 32.6 9.4 15.2 PT-5 2.5 2.4 88.5 87 3.6 2.0 6.8 4.2 PT-6 9.5 11.9 76.1 82.9 21.1 22.3 42.6 55.0 PT-7 8.6 11.0 65.4 64.4 21.8 1.3 28.1 32.3 PT-8 4.2 5.1 42.2 59.5 3.9 30.9 45.7 32.2 PT-9 6.5 16.4 41.8 58.2 3.7 1.7 62.7 49.6 PT-10 1.6 5.4 23.5 60.4 5.1 33.2 5.3 30.7 Mean 6.3 7.6 64.0 64.6 14.2 17.5 31.8 28.9 11^111111111 2.5 4.7 16.4 17.6 11.2 14.1 17.5 16.2 Table 11 Positive (%) expression of CD69 in unstimulated, CD69, cytokines IL-2, and IFNy in stimulated, CD3+ T cells of stable post renal transplant patients at initial and 6 months analysis, deviation CD69 expression was six fold greater on PMA and ionomycin stimulated cells compared with unstimulated cells at both assessments. The amplitude of the responses in PMA and ionomycin stimulated cells compared to normal subjects were also the same as the PD group in 5.3.1.2. There was no significant difference in the expression of intracellular IL-2 between the initial and the 6 month assessments (P>0.05). However the expression of intracellular IL-2 was significantly 54 suppressed when compared to those expressed by the normal subjects (P<0.001). There was no significant difference in the expression of IFNy between the two time points or when compared to normal subjects (P>0.05). Unstimulated Stimulated CD69 LMCF CD69 LMCF IFNy LMCF Initial 6 mos Initial 6 mos Initial 6 mos Initial 6 mos I 'M 89 85 95 106 129 124 144 138 PT-2 88 92 95 92 126 142 149 151 PT-3 87 90 93 99 128 129 157 145 89 95 110 98 119 140 138 145 PI-5 89 102 111 104 122 111 135 128 PT-6 87 92 98 108 138 142 167 159 IT-7 86 99 97 84 145 114 172 138 PT-8 82 93 88 91 109 142 128 162 PT-9 79 89 99 89 91 109 125 145 PT-10 93 87 87 101 118 147 99 134 mean 87 92 97 97 123 130 141 145 .sd 4 5 8 8 15 15 22 11 Table 12 L M C F of CD69 in unstimulated, CD69, cytokines IL-2, and IFNy in stimulated, CD3+ T cells of stable post renal transplant patients at initial and 6 months analysis. The results presented in table 12 shows the that there was no significant difference in the fluorescence of the intracellular IL-2 and IFNy between the initial and the 6 month assessments (P>0.05). However the fluorescence of intracellular IL-2 was significantly less when compared to those expressed by the normal subjects (P<0.001). There was also significant difference in the fluorescence of IFNy of the two assessments and those expressed by the normal subjects (P<0.01). 55 5.3.1.5. HIV Positive Patients (HIV Pos) Stimulated CD69 CD69 IFNy %Pos L M C F %Pos L M C F %Pos L M C F %Pos L M C F 1IIV-I 10.4 80 56.0 112 0.6 109 2.4 101 l l l \ -2 NSQ N A 67.0 122 2.1 91 3.3 115 HIV-3 4.6 96 54.4 94 29.7 139 26.6 155 IIIV-4 13.9 84 34.5 102 14.3 125 6.3 102 II1V-5 5.8 86 78.7 112 21.8 130 26.0 101 IllV-ft 7.0 85 61.4 109 7.8 124 18.3 99 IIIV-7 14.8 88 36.6 111 14.1 133 18.7 116 HIV-8 2.5 72 71.3 110 26.5 128 9.4 101 HIV-9 13.3 87 57.7 102 12.7 149 37.5 147 HIV-10 7.1 84 67.8 96 11.9 124 25.5 147 IIIV-I 1 6.0 84 59.6 98 24.5 146 35.3 155 IM\- I2 9.3 79 56.5 99 . 5.8 107 41.0 143 niL'ini 8.6 84 58.5 106 14.3 125 20.8 124 sd 4.1 6 12.8 8 9.6 17 13.4 24 Table 13 Positive (%) expression of CD69 in unstimulated, CD69, cytokines IL-2, and IFNy and IL-4 in stimulated, CD3+ T cells of HIV positive patients. "Nsq" is "not sufficient quantity" of cells for analysis. CD69 was five fold greater on PMA and ionomycin stimulated cells compared with unstimulated cells, at both assessment intervals. The amplitude of the responses in PMA and ionomycin stimulated cells compared to normal subjects were also the same as the PD group in 5.3.1.2. Compared to the normal patients, the expression and fluorescence of intracellular IL-2 of the HIV patients was significantly reduced (PO.OOl). Although there was no significant difference in the expression of intracellular IFNy between the HIV patients and the normal patients (P>0.05), the 56 fluorescence of intracellular IFNy was significantly higher in the HIV positive group (PO.OOl). 5.3.2.1 Comparison of CD69 between the Study Populations The expression of CD69 in unstimulated CD3+ T cells after 5 hours of incubation was similar for all the study populations. The expression of CD69 in stimulated CD3+ T cells showed a seven to eight fold increase above the unstimulated cells with no significant difference between the HD, PD, HIV positive and the normal groups. There was a six fold increase in the expression of CD69 in stimulated CD3+ T cells for the PT groups at both their initial and 6 moths assessments (PO.05) and this was significantly lower than the normal, HD, PD and HIV positive groups (figure 12). 1 0 0 8 0 4) > "</) g_ 6 0 (A (A |_ 4 0 x UJ cn to Q O 2 0 l i t N o r m a l s p=ns p=ns K M p=ns p<0.05 p<0.05 P D H D PT( in i ) S t u d y P o p u l a t i o n s P T ( 6 m o s ) H iv P o s U n s t i m C D 6 9 S t i m C D 6 9 Figure 12 Summary of positive CD69 expressions between unstimulated and stimulated T cells for normal controls, PD patients, HD patients and HIV patients. 57 5.3.2.2. Comparison of IL-2 expression between patient populations The expression of intracellular IL-2 in normal controls was compared to that in the four patient categories of: (a) peritoneal dialysis patients, (b) haemodialysis patients, (c) post renal transplant patients and (d) patients with HIV. The percentage of T cells expressing intracellular IL-2 is shown in table 14 and depicted in figure 13. The fluorescence intensity of these intracellular IL-2 expressions is shown on table 15 and illustrated in figure 14. Sample # Normals !S!!!!ii!!lll! HD PT (ini) PT (6 mos) HIV Pos 1 19.3% 32.9% 33.7% 10.5% 3.6% 0.6% l l l l l l l l l 36.9% 36.4% 36.0% 33.6% 29.4% 2.1% i i i i i l i i i l i : i i i 28.9% 44.2% 42.4% 23.9% 17.3% 29.7% 4 44.9% 32.3% 42.2% 5.3% 32.6% 14.3% iiiiiiiiiiiii 33.6% 40.9% 49.7% 3.6% 2.0% 21.8% !!!!!ii!!lll! 34.3% 35.6% 61.0% 21.1% 22.3% 7.8% 22.6% 44.4% 36.3% 21.8% 1.3% 14.1% 8 40.5% 51.0% 22.2% 3.9% 30.9% 26.5% 42.0% 44.3% 3.7% 1.7% 12.7% 10 38.6% 46.8% 5.1% 33.2% 11.9% 11 26.9% 24.5% 12 5.8% mean 33.5% 40.9% 40.4% 13.3% 17.5% 14.3% sd 8.2 6.3 11.5 10.9 14.1 9.6 Table 14 The percentage of CD3+ T cells expressing intracellular IL-2 in normal controls and study patient populations. 58 Comparison of Intracellular IL2 Expressed by T Cells 70 - i 6 0 -> -4—1 CO 5 0 -o CL 4 0 -£= O 3 0 -LO CO <D L _ 2 0 -Q . X LU 1 0 -0 -p=ns Normals p=m p<0.001 p<o.001 p<0.001 P D HD PT(ini) PT(6mo) H I V P o s Study Populations Figure 13 Box and whisker plot showing the distribution, mean and p value intracellular IL-2 expressed by T cells among the study populations. 59 Sample # Normals HD PT (ini) PT (6 mos) HIV Pos 1 132 125 141 129 124 109 l l l l i l i l l l l l l 161 146 149 126 142 91 l l l ^ l l l l l i l l l l l 142 152 140 128 129 139 4 156 135 136 119 140 125 i l l l l l ! l ! ! ! ! ! l 134 136 149 122 111 130 6 149 145 152 138 142 124 140 143 131 145 114 133 8 148 153 128 109 142 128 9 152 142 91 109 149 10 146 140 118 147 124 11 141 146 12 107 mean 146 142 141 123 130 125 sd 9 8 9 15 15 17 Table 15 The fluorescence intensity of intracellular IL-2 in CD3+ T cells is expressed as linear mean channels for normal controls and study patient populations. Comparison of Linear Mean Channel Fluorescence for Intracellular IL2 in T cells 175 150-125-P=ns p=ns p<a_oi P<_£P5 P<_^°1 100 4, Normals PD HD PT(ini) PT(6rno) HIV Pos Study Populations Figure 14 Box and whisker plot depicting the distribution, mean and p value of the fluorescence intensity of intracellular IL-2 expressed by T cells among the study populations 60 Statistical comparisons of the data showing using A N O V A indicated that there was a significant difference in the production of intracellular IL-2 per CD3+ T cell between the population means. Results from Newman-Keul's post hoc multiple comparisons tests for the proportion of CD3+ T cells producing intracellular IL-2 and their fluorescence are depicted by box and whisker plots in figures 18 and 19. These analyses indicated that the proportion of cells expressing intracellular IL-2 and their fluorescence were significantly reduced in patients with HIV and in patients following renal transplantation (P<0.001). 61 5.3.2.3 Comparison of IFNy expression between patient populations The expression of intracellular IFNy in normal controls was compared to that in the four patient categories of: (a) peritoneal dialysis patients, (b) haemodialysis patients, (c) post renal transplant patients and (d) patients with HIV. The percentage of T cells expressing intracellular IFNy is shown in table 16 and depicted in figure 15. The fluorescence intensity of these intracellular IFNy expressions is shown on table 17 and illustrated in figure 16. Sample # Normals i i i i i i i i i i i i i HD PT (ini) PT (6 raos) HIV Pos 1 19.4% 17.0% 19.0% 32.8% 9.3% 2.4% ! ! ! ! ! ! ! ! 22.0% 18.4% 24.5% 32.8% 34.2% 3.3% 37.7% 25.7% 35.1% 25.4% 25.8% 26.6% 4 25.2% 20.0% 33.2% 9.4% 15.2% 6.3% 5 25.3% 14.7% 33.0% 6.8% 4.2% 26.0% 6 42.2% 23.2% 23.2% 42.6% 55.0% 18.3% 7 11.0% 20.3% 23.6% 28.1% 32.3% 18.7% 8 20.5% 27.8% 41.3% 45.7% 32.2% 27.9% 9 22.7% 31.7% 62.7% 49.6% 37.5% 10 26.8% 22.9% 5.3% 30.7% 25.5% 11 28.1% 35.3% 12 41.0% mean 25.54% 22.17% 29.11% 29.16% 28.9% 22.4% sd 8.5 5.2 7.6 18.5 16.2 13.0 Table 16 The percentage of CD3+ T cells expressing intracellular IFNy in normal controls and study patient populations. 62 Comparison of Intracellular IFNj Expressed by T cells p=ns p=ns p=ns p=ns p=ns Normal; PD HD PT(ini) PT(6rno) HIV Pos Study Populations Figure 15 Box and whisker plot of the distribution, mean and p value of intracellular IFNy expressed by T cells among the study populations. 63 Sample # Normals l l l l l i l l l l l : l l l l l i l l l l l l l : PT (ini) PT (6 mos) HIV Pos 1 155 153 164 144 138 101 2 167 169 164 149 151 115 164 157 169 157 145 155 182 156 168 138 145 102 5 159 148 160 135 128 101 6 158 167 164 167 159 99 l l l l l l l l l l l s l l 164 149 149 172 138 116 !!!§!!!!!!!§;: 164 162 156 128 162 101 l l i ^ l l l l l l l l l l l 178 168 125 145 147 l l ; l ! l l l l ! l l l i : 165 103 99 134 147 165 155 12 143 mean 166 153 162 141 145 124 sd 8 19 7 22 11 24 Table 17 The fluorescence intensity of intracellular IFNy in CD3+ T cells is expressed as linear mean channels for normal controls and study patient populations. 200 175 H LL 150 u 125 100 75 Comparison of Linear Mean Channel Fluorescence for Intracellular IFNy in T cells p=ns p=ns p<0.0 p<0.05 p<0.001 Normals PD HD PT(ini) PT(6rno) HIV Pos Study Populations Figure 16 Box and whisker plot depicting the distribution, mean and p value of the fluorescence intensity of intracellular IFNy expressed by T cells among the study populations 64 Statistical comparisons of the data showing using A N O V A indicated that there was no significant difference in the number of CD3+ T cells producing intracellular IFNy between the population means. Results from Newman-Keul's post hoc multiple comparisons tests indicated that the fluorescence of intracellular IFNy was slightly lower in patients following renal transplantation (P<0.05) and significantly reduced in patients with HIV (P<0.001). 65 5.3.3.1. Expression of CD69 in T cell subsets Table 18 shows the differential expressions of CD69 by stimulated cells examined in CD4+CD3+ (Th) and CD8+CD3+ (Ts) subsets of T cells in 4 normal samples, 10 PT and 8 HIV positive samples. Th CD69 l l l l l l l l l l l i i i i i i i i i i Th CD69 | | | i i o s i | | Ts CD69 (%Pos) Th CD69 (%Pos) Ts CD69 i i i l i i i i NS 87.5 35.6 I'T-I 32.9 16.2 IIIV-5 80.4 67.4 N9 81.7 37.3 l l l l f l l l l l 25.6 14.9 IIIV-6 63.0 41.0 Nil) 81.1 42.1 lllBllllll 22.5 7.4 HIV-~ 71.7 36.5 N i l 81.2 29.9 I'T-I 50.6 13.2 IIIV-S 73.6 58.3 l'T-5 81.2 26.6 IIIV-9 82.9 33.8 I'l-() 53.1 47.0 IIIV-10 78.5 37.9 PT-7 N T NT 111V-1 1 67.4 36.7 IT-S 59.3 20.0 IIIV-12 68.1 56.6 i i i i i i i i i i NT NT PT-10 61.0 42.1 82.9 36.2 48.3 23.4 73.2 46 sd 3.1 5 l l l l l^l l l l l l i l i 20 14.2 sci 7 12.8 Table 18 Differential expression of CD69 expressed as % positive stimulated Th and Ts cells in normal controls, PT and HIV positive patient populations. "NT" indicates sample was not tested for the expression of this marker. The expression of CD69 on CD4+CD3+ T cells was twice that expressed on the CD8+CD3+ T cells. There was a significant difference in the expression of CD69 by the Th subset between the normal samples and the PT study groups (PO.OOl), but there was no difference between the normal samples and the HIV patients (P>0.05). The expression of CD69 by the Ts subset was not different when compared between the normal samples, and the PT and the HIV patients, although there was a sight difference in its expression when the PT patients were compared to the HIV patients (PO.01). 66 5.3.3.2. Expression of IL-2 in T cell subsets The differential expression of intracellular IL-2 by stimulated Th and Ts cells are shown in table 19. Th iiiiiii s : i i : i s l l : l : l l l l l i l i l l l i JjpPolfK Hi llllllllll l l l l l s l ? ' -11111-2111 j|j|Bi!ifs;s lliHIllll lillllllllll ; iiiiiiii T s IL-2 (%Pos) N8 25.1 2.5 PT-1 6.4 1.2 i i i i l l l l 36.4 9.9 N9 25.1 6.7 l l l l l l l l 38.2 10.4 l l l l l l l l l 29.3 7.0 N10 46.3 25.1 21.3 5.8 :-pii||| 35.7 8.6 N i l 40.0 10.6 38.5 7.5 FIIV-8 28.1 27.9 1.4 0.8 l l l l l l l 35.2 6.4 l l l l l l l l l 31.4 8.1 l l l l l l l l 19.7 4.0 l l l l l l l l 2.0 0.5 l l l l l l l l 41.9 11.9 llllllllllll 35.5 14.3 l l l l l l l l l 20.2 5.7 ! l l | | | | | | | 4.3 1.0 : | : l l l | | | | 31.4 26.2 mean 34.1 11.2 mean 21.0 7.6 llmllthl::: 30.8 10.2 sd 10.7 9.8 lllllllllllll 15.9 8.1 l l l l l l l l 8.0 7.6 Table 19 Differential expression of intracellular IL-2 expressed as % positive, produced by stimulated Th and Ts cells in normal controls, PT and HIV positive patient populations. The expression of intracellular IL-2 by CD4+3+ T helper cells and CD8+CD3+ T suppressor cells of PT and HIV positive study populations demonstrated no statistical difference from normal controls (P>0.05) as depicted in figure 17. There was also no difference in the intracellular IL-2 fluorescence in T cell subsets between the Normal controls, PT and HIV study groups (data not shown). 67 Proportion of IL-2 Expressed in T Helper (Th) &T Suppressor (Ts) Populations p=ns p=ns p=ns p=ns " Normals(Th) P T (Th) HIV(Th) Normals(Ts) P T (Ts) HIV (Ts) Study Populations Figure 17 Comparison of intracellular IL-2 expression of PT and HIV patients to normal controls in CD4+CD3+ T helper cells (Th) and CD8+CD3+ T suppressor cells (Ts). 68 5.3.4 Relationship Between T Cell Subsets and the IL-2 Secreting CD3+ T Cells Regression analysis was used to investigate the relationship between the T cell subsets and the number of IL-2 secreting CD3+ T cells. Normal controls and HIV positive patients demonstrated a positive linear relationship between the percentage of CD4+CD3+ T cells and the proportion of IL-2 producing T cells (coefficient of determination (R = 0.92) in normal controls (figure 20) and R 2 = 0.81 in HIV positive patients (figures 18). No relationship was observed between CD4+CD3+ T cells and the proportion of IL-2 producing T cells in PT patients (R = 0.05) (figure 19). B. A. I o.o4— 1 1 1 1 1 1 °- 0 10 20 30 4 0 50 60 CD4+3+ C e l l s (%) Figure 18 HIV Pos patients 69 Normal controls demonstrated a negative linear relationship between the percentage of CD8+CD3+ T cells and the proportion of IL-2 producing T cells (R 2 = 0.95) (figure 21), while PT (figure 22) and HIV positive (figure 23) patients demonstrated no relationship (R 2 = 0.49 and R2=0.11 respectively). CD8+3+ Cells (%) CD8+3+ Ce l l s (%) Figure 21 Normal controls Figure 22 PT patients 0.3-+ CT 8 0 . 2 -0.1 £o.o R2=0.11 1 1 1 1 1 20 30 40 50 60 70 CD8+3+ Cells (%) Figure 23 HIV positive patients 7 0 5.4. Expression of intracellular IL-2 and IFNy among PT patients over time The PT patients were assessed initially for production of intracellular IL-2 and IFNy and reassessed after six months are summarized in table 20. IL-2 (initial) IL-2 (6 mos) IFNy (initial) IFNy (6 mos) PT-1 10.5% 3.6% 32.8% 9.3% PT-2 33.6% 29.4% 32.8% 34.2% P f-3 23.9% 17.3% 25.4% 25.8% PT-4 5.3% 32.6% 9.4% 15.2% PT-5 3.6% 2.0% 6.8% 4.2% P 1-6 21.1% 22.3% 42.6% 55.0% PT-7 21.8% 1.3% 28.1% 32.3% PT-8 3.9% 30.9% 45.7% 32.2% PT-9 3.7% 1.7% 62.7% 49.6% Pi-10 5.1% 33.2% 5.3% 30.7% mean 14.2% 17.5% 31.8% 28.9% sd 11.2 14.1 17.5 16.2 Table 20 Comparison of intracellular IL-2 and IFNy expression by proportion of CD3+ T cells assessed initially and after six months between normal controls and the PT study population. Although there was no difference in the mean expression of intracellular IL-2 and IFNy by the proportion of CD3+ T cells at both assessments, PT-4, PT-8 and PT-10 demonstrated apparent differences in the proportion of T cells expressing intracellular IL-2 between the initial and the 6 month assessments. PT-10 also showed differences in the proportion of T cells expressing intracellular IFNy between the initial and the 6 month assessments. There was also no apparent differences in the fluorescence of intracellular IL-2 and IFNy between the initial and the 6 month assessments (figures 24 and 25). 71 PT-2(5yr) I PT-4(2yr) I PT-6(14yr) I PT-8(15yr) I PT-10(5yr) PT-1(3yr) PT-3(4yr) PT-5(3yr) PT-7(4yr) PT-9(4yr) Stable T R a n s p l a n t Patients (PT) & Graft Age (Yr) 2 IL-2(ini) Q IL -2 (6mos) Figure 24 Bar graph to show individual L M C F of intracellular IL-2 expressed by stimulated CD3+ T cells in the PT study group at the initial and at 6 moths assessments. 72 2 0 0 1 5 0 o ^ 1 0 0 5 0 1 7 2 1 4 4 7\ 1 3 8 i y 151 1 5 7 V 1 4 5 1~45 138^ 1 5 9 1 6 2 1 3 8 1 2 8 • -I-45-1 2 5 1 3 4 9 9 *5i P T - 2 ( 5 y r ) I P T - 4 ( 2 y r ) I P T - 6 ( 1 4 y r ) I P T - 8 ( 1 5 y r ) I P T - 1 0 ( 5 y i P T - 1 ( 3 y r ) P T - 3 ( 4 y r ) P T - 5 ( 3 y r ) P T - 7 ( 4 y r ) P T - 9 ( 4 y r ) Stab le T r a n s p l a n t Pat ients (PT) & Graft A g e (Yr) IFNg(ini) IFNg(6 mos) Figure 25 Bar graph to show individual LMCF of intracellular IFNy expressed by stimulated C D 3 + T cells in the PT study group at the initial and at 6 moths assessments. 73 Chapter 6 Discussion 6.1 Development of the Intracellular Cytokine Measurement Assay IL-2 and IFNy are among the primary cytokines produced by activated CD4+ T cells during a Thl cell-mediated immune response. They play a central role in the regulatory and inflammatory response, and activate B cells, macrophages and trigger other cytokines secreted by activated T cell in the immune system. The studies reported here have produced a simple, rapid and reproducible clinical tool for the intracellular measurement of IL-2 and IFNy. The assay was standardized by determining the optimal monoclonal antibody concentrations for use in cell surface and intracellular labelling and the optimal incubation period needed for the expressions of these intracellular cytokines. Biological, mitogenic and pharmacological stimuli were compared to establish the most efficient method for lymphocyte activation to induce cytokine expressions. The requirements for permeabilization of cell membranes and the specificity of intracellular labelling were ascertained. Finally the optimal anticoagulant and maximum duration of blood sample storage were determined. In these studies, CD69 was used to monitor the level of cellular activation. CD69, known also as activation inducer molecule (AIM), early activation antigen (EA-1), Leu 23 and MLR-3 antigen, is a cell surface glycoprotein found on hematopoietic cells, but is undetectable or detected at very low levels on unstimulated, or resting lymphocytes. AIM is expressed on the cell surface within 30 minutes after lymphocyte activation8'. The antigen is a phosphorylated, homodimer, consisting of 28 kDa and 32 kDa chains that is disulphide linked. The extracellular portion is approximately 138 74 a.a. in length with a single transmembrane domain. The molecule is encoded in the short arm of chromosome 12, in the region of 12p 12.3-p 13.282. CD69 expression is up-regulated by the activation of Ras kinase Raf-1 of the Map kinase cascade. Although its expression was reported by Taylor-Fishwick et al 8 3 to be induced by PMA and ionomycin which result in RNA transcription and protein synthesis of the CD69 molecule, we also found its up-regulation in PHA and OKT3 and anti-CD28 stimulation experiments. Since CD69 is an easily detectable surface marker that is expressed quickly following stimulation and is involved in the activation process, AIM is, therefore, a valuable rapid and sensitive marker to assess the presence of lymphocyte activation and to discriminate between unstimulated and stimulated cell populations. Cytokines are synthesized into the endoplasmic reticulum (ER) by the ER membrane ribosomal translation of the mRNA, and are transported to the Golgi apparatus for final processing before being discharged from the cell into intercellular space. The Golgi apparatus consists of stacks of flattened cis, medial and trans cisternae which are chemically, structurally and functionally different from each other84. As the cytokine proteins move through the cisternae, they undergo glycosylation with the phosphorylation of N-linked, O-linked oligosaccharides, removal of mannose and other sugars and the addition of N-actylglucosamine, galactose and sialic acid85. Once these products are sequentially matured in the trans region, they are encapsulated in secretory vesicles which bud off from the trans region and eventually fuses with the plasma membrane and discharge their contents from the cell. Transport of protein to the cell surface is effectively blocked at the trans cisternae by the action of monensin, which has little or no effects on protein synthesis or ATP levels86 and is widely used as 7 5 a biochemical and biological investigative tool to study Golgi apparatus transport function and vesicular traffic pathways. Monensin, a metabolite derived from Streptomyces cinnamonensis, is an open chain molecule with a ten fold affinity for sodium (Na+) over potassium (K +) and little to no affinity for calcium (Ca+). When monensin complexes Na + , it becomes cyclic, stabilized by hydrogen bonds between carboxyl and hydroxyl groups. This monovalent ionophore is then capable of collapsing intracellular Na + and hydrogen (H+) gradients. The internal lumen of the trans region is normally acidic due by the presence of H + ATPase hydrolysing enzymes. Monensin complexes with N a + in the cytoplasm, diffuses across the membrane into the trans region of the Golgi apparatus and releases the ion. It then picks up a proton, re-crosses the membrane to the outside and releases the proton in favour of complexing with a Na + . In this manner, monensin induces a 1:1 N a + / H + exchange leading to net uptake of Na + and Cl" resulting in the entry of water. Consequently the trans cisternae of the Golgi apparatus swells. The minimum concentration of monensin needed to cause this effect is about 10"7 M . The swollen cisternae appear to be devoid of contents and thus monensin effectively blocks the intracellular transport of protein to the plasma membrane without affecting protein synthesis87' 8 8 ' 8 9 . Monensin was used in the studies reported here to accumulate the intracellular cytokines in sufficient quantities to permit their measurement by flow cytometry. In order to label the accumulated intracellular cytokines, fluorochrome conjugated anti-cytokine Mab must gain access into the cytosolic component of the cell to label their target cytokine. Cellular membranes are permeabilized by the action of saponin, a plant glycoside from Quillaja bark, which has a high affinity for cholesterol in the cell membrane. This intercalates in the membrane and complexes the phospholipids and the cholesterol into a lattice of hexagonal rings with centre to centre spacing of 140 - 150 A (14 - 15 nm) and a central pore of about 70 - 95 A 9 0 (or 7 - 9.5 nm) 76 in both the cytoplasmic and nuclear membranes, while retaining the integrity of the morphology and the expression of most membrane antigens. The use of saponin has enabled the simultaneous analysis of cytoplasmic, nuclear and/or surface antigens91 without resulting in a rise of autofluorescence or cell aggregation. Saponin permeabilization enables both the anti-IL-2 IgG antibodies of molecular weight 150 kDa, and their conjugated PE molecule of 240 kDa molecular mass (diameter 0.2 - 0.3 nm 9 2 , 9 3 , 9 4 ) and the anti-IFNy antibodies whose molecular weight of 0.39 kDa were conjugated with FITC molecules to gain access into the cytosol. The cells were sequentially interrogated by the laser, and the results displayed as frequency distribution histograms from which the mean fluorescence intensity was determined. 43.4% of permeabilized cells demonstrated brighter PE signal from conjugated IL-2 antibodies bound to intracellular IL-2 protein, expressing a L M C F of 149 channels, while 18.5% of non-permeabilized cells expressed dimmer L M C F of 97 channels. The dim fluorescent intensity and the relatively lower expression (18.5%) may be due to autocrine action of residual IL-2 on the surface of the T cells. Permeabilized cells demonstrated 23.6% positivity in intracellular IFNy expression with a FITC linear fluorescent intensity of 174 channels, whereas non-permeabilized cells showed only 8% positivity and L M C F of 99 channels may also be from residual IFNy in the medium. The anomaly of IL2 with 43.4% positivity expressing a fluorescence intensity of 149 channels contrasted to the expression of IFNy with 23.6% positivity and a fluorescence intensity of 174 channels may be due to a difference in number of fluorochrome molecules conjugated to protein (F/P) ratio between the two fluorochromes. PE molecule has molecular weight of 240 kDa and crystal structure of 0.2 - 0.3 nm in diameter, while FITC molecule has molecular mass of 0.39 kDa 9 5. The molecular weight of IL-2 is 15 kDa and IFNy is 20-25 kDa. The F/P ratio of PE molecules to 77 anti-IL2 antibody molecule was 1:1 whereas the F/P ratio of FITC to anti-IFNy antibody molecule was 4 - 9 FITC molecules to each antibody molecule. Due to the proprietary nature of FITC conjugation the exact F/P ratio was unknown. Monoclonal antibodies to cell surface antigens CD3, CD4, CD8, CD69 and intracellular cytokines IL-2, and IFNy used for this study were mainly of immunoglobulins class G (IgG) and of IgGl and IgG2 subclass, containing both heavy (H) and light (L) chains. The difference between the 2 subclasses is defined by the number and arrangement of interchain disulphide bonds96. Each Mab was titrated to determine the level for saturation concentration needed to achieve the optimal discrimination between the positive signals expressed by the marker of interest and the negative signals which were derived from machine noise, autofluorescence and other cells within the bitmap. Both the linear mean fluorescence intensity (LMCF) and the proportion of cells within the positive signal were recorded. With each dilution of the antibody, the positive cell population became correspondingly less intense in fluorescence. After the concentration of the Mab were selected, the accompanying isotype controls were used at the same concentration. Isotype controls, also known as "irrelevant" antibodies, are polyclonal antibodies which will not bind to the target epitopes but may react with other proteins intracellularly or on the cell surface. The isotype controls were of the same IgG subclass and fluorescence as the test reagent and were used to estimate non-specific binding of the monoclonal conjugated antibody. And when < 2% of cells were stained by isotype controls, it demonstrated little or no non specific binding present. Consequently there was no mathematical adjustments made to the proportion of cells labelled by the antibody of interest. 78 Selection of 5 hours as the optimal incubation time for measurement of peak expression of CD69, intracellular IL-2 and IFNy was achieved by hourly assessment of their expression in a lymphocyte cultures activated by P M A and ionomycin. Expression of CD69, intracellular IL-2 and IFNy increased progressively throughout the activation process, becoming stable between 4 - 6 hours of culture. The induction of cytokine synthesis is a primary consequence of T cell activation, and is initiated by stimulation of TcR/CD3 complex in conjunction with other co-stimulatory cell surface receptors. Binding of these cell surface molecules triggers a cascade of intracellular signal transduction events resulting in the transcription of the cytokine genes. Although pharmacologic agents P M A and ionomycin were powerful and effective stimuli, other in vitro stimuli such as the mitogen P H A , and the biological agents O K T 3 and anti-CD28 were investigated as alternative potential stimuli for the clinical test. P H A is a lectin from Phaseolus vulgaris (red kidney bean) that is commonly used to stimulate and activate lymphoid cells 9 7. The lectin recognizes complex oligosaccharides structures on cell surfaces98 targeting galactose residues triggering a polyclonal response. It interacts with T cells through a number of sites, including a direct interaction with a 20 kDa molecule of the CD3 complex 9 9, binding or cross linking to T C R 1 0 0 as well as binding CD2. O'Flynn 1 0 1 et al found simultaneous binding of CD2 cell surface molecules resulted in Ca mobilization and generation of diacylglycerol within 2-3 min. of exposure to P H A . The binding or cross linking of P H A to CD3 triggers the transduction cascade, activation of P K C and increase of cytoplasmic Ca levels leading to the activation of the IL-2 gene. Meager reported in 1991 that P H A activated cells increase in size 79 at about 12 hours post-activation and progresses from G, to the S phase of the cell cycle between 24 to 48 hours'02 with detectable levels of IL-2 in culture supernatants after 18-24 hours of PHA stimulation103. Stimulation experiments by using either 5% v / v PHA and P M A and ionomycin showed that CD69 expression increased four fold after 4 hours of stimulation and seven fold after 24 hours of stimulation. However, these PHA activated cells produced very low levels of intracellular IL-2 and IFNy exceeding by only 2-5 times, with slight increases in fluorescence intensity. By comparison, stimulation by P M A and ionomycin resulted in productions of IL-2 and IFNy which was 15-20 times control values with a subsequent significant increase in fluorescence. Increasing the concentration of PHA beyond the range reported produced excessive agglutination of cells which could cause serious malfunction of the flow cell in the flow cytometer. The polyclonal activation of T cells by PHA alone for 4 or 24 hours was therefore insufficient to induce significant levels of intracellular IL-2 production. Activation of T cells and cytokine production requires two signals. The first is engagement of the TcR/CD3 and M H C class Il-peptide complexes. The second signal is the ligation of CD28, a 44 kDa T cell transmembrane glycoprotein and B7-1 and B7-2. B7-1 is induced on activated B lymphocytes and monocytes and B7-2 is expressed on activated T and B cells and constitutively expressed on monocytes'04. When CD28 initially binds to B7-1, CTLA-4 in T cells is up-regulated105 and binds preferentially to B7-1. B7-1, B7-2 and CTLA-4 expression peaks between 18 and 42 hours of stimulation106. The signal transduction of CD28 molecule, RelA, c-Rel andNficBl, are activated by Raf-1 kinase in the Ras signalling pathway and results in the binding of the CD28 response complex 80 (CD28RC) to the IL-2 promoter CD28 response element (CD28RE) 1 0 7 in the enhancer region of the IL-2 gene augmenting the production of IL-2. IL-2 mRNA levels increases more than 6 hours after T cell stimulation and the autocrine function of IL-2 inducing clonal expansion occurs in 2-3 days and functions to amplify the CD28-B7-1 response108'109. The binding of CD28 also increases the expression of IFNy" 0. In the studies reported here, the two signals required for T cell activation were simulated by ant-human antibodies cross linking CD3 and CD28. This resulted in the activation of lymphocytes as indicated by the expressions of CD69 (70% to 86% positivity). However, 5 hours of stimulation with various combinations and concentrations of anti-CD3 and anti-CD28 antibodies was insufficient to induce significant expressions of intracellular IL-2 and IFNy compared to the unstimulated normal controls. This type of stimulation combinations probably requires 48 - 72 hours with exogenous IL-2 present in the medium''' to observe its effect and to produce a measurable increase of cytokine levels. In contrast to PHA, OKT3 and CD28 stimulation, PMA and ionomycin stimulation for 4 hours and 24 hours resulted in 10 to 30 fold increase in IL-2 expression. PMA and ionomycin are powerful biochemical stimuli that bypass the binding of TCR/CD3 complex and CD3 and CD28 co-stimulation. PMA is phorbol 12-myristate 13-acetate ( C 3 6 H 5 6 0 8 , molecular weight 616.84), an analogue of diacylglycerol, activates protein kinase C; while the mobilization of Ca 2 + i s enhanced by the ionophoric polyether antibiotic produced from Streptomyces conglobatus, ionomycin calcium salt ( C 4 1 H 7 0 O 9 C a , molecular weight 747.1) which has a high affinity for Ca4"1". PKC activates the cytoplasmic components of N F K B and OCT translocating them into the nucleus, as well as activating 81 the ras pathway which leads to the activation of the cytoplasmic components of cFos and cJun and their translocation into the nucleus. These translocated components ultimately bind to and activate the promoter sites leading to the transcription of the cytokine genes and synthesis of IL-2 and IFNy . The levels of synthesized IL-2 and IFNy were significantly higher than when stimulated by P H A or O K T 3 and CD28 stimuli for the same time. To confirm the specificity of the assay, human recombinant IL-2 and IFNy proteins were pre-incubated with anti-human IL-2 and IFNy antibodies at excess concentrations. The recombinant human IL-2 and IFNy proteins were constructed commercially by inserting double stranded D N A sequence of the human IL-2 and IFNy genes into E. Coli D N A expression vectors containing highly active promoter sequence to the IL-2 and IFNy genes leading to the production of human recombinant IL-2 and IFNy proteins" 2. The antibodies were commercially produced by the immunization of laboratory animals to induce an immune response causing their B cells to produce clones of immunoglobulins of IgG class which recognizes the epitopes of specific antigens with which they were originally immunized. When the anti-human IL-2 antibodies were preincubated with excess antigen, 10.3% of cells were positive for IL-2 expression at 5 fold antigen excess and only 3.8% at three fold excess, compared to controls in which 36.8% of cells were positive for IL-2 expression. The binding between antibody and the recombinant antigen is dependent on the concentration of both antibody and antigen. This data suggests that this equilibrium was achieved at three fold antigen excess. A t greater antigen excess, steric hindrances may prevent binding to the antibodies resulting in unstable antigen-antibody complexes. When the unstable mixture was introduced into the permeabilized cell 82 suspension, some of the antibodies in the unstable antigen-antibody complexes were able to bind to the intracellular cytokine proteins, resulting in a degree of false positivity. Approximately 19.0% of control cells expressed intracellular IFNy which was blocked with 2 - 5 fold excess of recombinant IFNy protein. Therefore the use of monoclonal anti-human IL-2 and IFNy antibodies appeared to confirm the specificity of the assay. In preparation for the test to be used clinically, the effects of different anticoagulants and sample storage duration had to be established. Three common clinical anticoagulants were examined: A C D , heparin and E D T A . When blood samples were processed the same day as the venipuncture, there were minimal differences in the expression of intracellular IL-2 expression among the different anticoagulants. But when blood remained in the anticoagulant for 24 hours, there was a differential decrease in %positivity between heparinized, A C D and E D T A anticoagulated samples (44.1%, 42.2% and 36.6% respectively) with similar decreases in fluorescence intensity. There was a further decrease both in the number of positive staining cells and fluorescence intensity after 48 hours to 20%, 22% and 29% respectively. The difference between the three anticoagulants may be due to the diffusion of the anticoagulant across the cytoplasmic membrane through its membrane channels to affect the availability of cytoplasmic calcium. Citrate in A C D binds plasma calcium, however its action is reversible with the addition of extracellular calcium, and dextrose in A C D serves as a preservative and nutrient for the cel ls" 3 . E D T A chelates calcium and prevents calcium from ioniz ing" 4 . Heparin prevents the clotting process by binding antithrombin III which inactivates thrombin and prevents the formation of fibrin from fibrinogen" 5 . The induction of cytokine 9-1-production requires the activation of protein kinase C and an increase in cytoplasmic C a , leading to the transcription of the cytokine genes and the synthesis of cytokines. Since the action of heparin 83 does not i nvo lve ca l c ium, and the action o f A C D may be reversed by the addi t ion o f i o n o m y c i n , the expression o f intracellular I L - 2 f rom hepar inized b lood and A C D were the highest o f the three anticoagulants after 24 hours. Therefore heparinized samples were preferred w i t h i n 24 hours to op t imize the induc t ion o f intracellular I L - 2 and I F N y expression. 6.2 Comparison of Intracellular Cytokine Expression in Normal Subjects and Patients with Chronic Disease The potential o f the assay developed i n this study to moni tor the i m m u n o l o g i c a l status o f patients was compared i n several groups o f subjects. Volunteers w i t h no phys ica l signs o f i l lnesses were recruited as normal controls. Patients w i th end stage renal disease were examined to determine the effects o f u remia on the product ion o f intracellular I L - 2 and I F N y , and compared w i t h post renal transplant patients w h o retained functioning grafts for more than 1 year (range 2 - 1 5 years). A l l had good renal function, w i t h serum creatinine levels be low 200 / ^ m o l / L , no rma l ly considered the upper l i m i t o f the acceptable reference range for patients w i t h a single functional k idney . These patients were rece iv ing a standard immunosuppressive regimen consis t ing o f c y c l o s p o r i n A ( C s A ) , azathioprine and predisone. C s A is the most c o m m o n immunosuppress ive drug used i n transplantation. It is a c y c l i c nonapeptide from the fungus Tolypocladium inflatum Gams consis t ing o f 11 a .a ." 6 w h i c h binds to cytoplasmic transporter cy toph i l in , and inhibi ts the serine-threonine phosphatase enzymat ic act ivi ty o f the s ignal l ing peptide calcineurin . B y this mechanism, it prevents the m o b i l i z a t i o n and translocation o f cytoplasmic N F A T into the nucleus and b ind ing to the response element o f the cytokine genes, and subsequently inhibits the secretion o f I L - 2 and I F N y i n activated T c e l l s 1 1 7 ' 1 1 8 ' 1 1 9 . F i n a l l y , a group o f subjects w i t h H I V infect ion were selected as a pos i t ive control 84 group. CD4+ T cells are the prime cell source for IL-2 and IFNy, and these cells decrease with progression of HIV infection. There was no significant difference in the mean expression of CD69 on unstimulated CD3+ T cells between any of the five populations examined in this study, suggesting that the basal level of T cell activation was indifferent between the groups. For technical reasons, only non stimulated CD3+ T cells from normal control were examined for the expressions of IL-2 and IFNy. It is therefore not possible from the results available to determine whether the expression of these cytokines will be similar in the other study groups. Except for three individuals, one from normal control, PD and HD groups, the non-stimulated cells from all subjects were expressing CD69 that were within 2 sd of their group mean. The cells from the three individuals may already be activated in vivo and therefore were showed an elevated CD69 response. Following stimulation in vitro, >90% of cells in these three subjects were positive for CD69, the highest expression for this marker in these studies. Stimulated CD3+ T cells from adult patients with end stage renal disease who were maintained on peritoneal dialysis or haemodialysis exhibited a mean level of CD69 expression that was not different from normal controls (p = ns). However, CD69 expression in PT patients was statistically different from normal control cells (p<0.05), as was the expression of this marker on CD4+ T cells (p<0.01), while CD69 expression in the CD8+ T subset was not different from normal control cells (p=ns). This decreased expression of CD69 did not appear to be correlated with the daily cumulative dose of CsA (R2 = 0.07 and 0.13 respectively). 85 Previous studies reported differing IL-2 and IFNy gene expression and their subsequent protein production between hemodialysis patients, peritoneal dialysis patients and normal control. Gerez et al 1 2 0 showed a complete loss of the inducibility of the IL-2 gene and decreased expression of IFNy m-RNA in hemodialysis patients while the IL-2 and IFNy m-RNA were similar between the peritoneal dialysis and their normal controls. Morita et al 1 2 1 found stimulated PBMC from hemodialysis patients produced similar amounts of IL-2 and IFNy as normal controls. Using PHA stimulated PBMC and analyzing the IL-2 production by C T L L cell line proliferation technique, Weinstein et al 1 2 2 documented high levels of extracellular IL-2 in hemodialysis patients with no difference between their peritoneal dialysis patients and uremic ESRD patients while Ha et al 1 2 3 found no significant difference in the production of extracellular IL-2 between their normal controls, hemodialysis and peritoneal dialysis patients. In the studies reported here, there was also no statistical difference in the expressions of IL-2 (p = ns) and IFNy (p = ns) between normal controls and patients on peritoneal and hemodialysis patients respectively. The CD3+ T cells from the PT patients expressed significantly lower mean IL-2 (p<0.001). The consistency of production of intracellular IL-2 and IFNy in PT patients were assessed again after 6 months. At both initial and 6 months assessments, there was a significant difference in the proportion of CD3+ T cells expressing intracellular IL-2 between normal control cells and PT cells (p<0.001) and a similar reduction in the production by CD4+ T cells (p<0.05) while no difference was exhibited by the CD8+ subsets of T cells of the PT study population compared to normal controls. However, no correlation was found between the proportion of CD4+3+, CD8+3+ cells and the proportion of T cells expressing intracellular IL-2 (R = 0.05 and 0.49 respectively). 86 The PT patients demonstrated varying levels of intracellular IL-2 and IFNy expressions by CD3+ T cells between their initial and subsequent analyses.. Of the 10 PT patients, only PT #5 (graft age of 3 years) was below 1 sd of the group mean in IL-2 and IFNy synthesis, while PT#9 (graft age of 4 years) was below 1 sd of the group mean for IL-2 production and above 1 sd of the group mean for IFNy synthesis at both analyses 6 months apart. The other eight PT study patients expressed intracellular IL-2 and IFNy within lsd of the group mean. Manca et al 1 2 4 in 1986 found individual variations in sensitivity to CsA and described the presence of CsA resistant clones in vitro. In 1988 Kumagal et al' 2 5 suggested that CsA was able to abrogate the induction of T cell immunocompetence, but was unable to prevent IL-2 production and mitosis once immunocompetence has been achieved. Batiuk and Halloran, in 1997, showed that even at peak CsA levels, calcineurin activity was not completely inhibited126'127. Although there is evidence that CD69 generated intracellular signals by the induction of c-fos gene expression to form AP-1 and N F A T binding complexes and the subsequent transcription of IL-2 gene 1 2 8' 1 2 9' 1 3 0' 1 3 1, this has been shown in vitro to be CsA sensitive132' 1 3 3 . However, June et al 1 3 4 demonstrated that the activation of lymphocytes by PMA and phorbol ester in vitro results in IL-2 gene expression that is completely resistant to the action of CsA, suggesting that the CD28 pathway may be resistant to immunosuppressants in vivo. There was no significant difference (p=ns) in the production of intracellular IFNy by the CD3+ T cells between stable transplant patients and normal controls. This may be due to the presence of PMA/PHA responsive elements that have been mapped to the promoter region of the IFNy gene135. Rafig et al in 1998136 suggested that CsA enhances IFNy production by blocking the production of IL-4 and IL-10. Although IL-4 and IL-10 were not investigated in this study, the mean proportion of CD3+ T cells expressing intracellular IFNy in the PT group who were on twice daily doses of CsA approximated the IFNy expression of the control 87 group, the amount of intracellular IFNy, that constitute the fluorescence, produced by these cells was lower in the PT study group (P<0.05). Graft rejection in organ and bone marrow transplantation is a major obstacle in the success of solid and bone marrow transplantation. Cytokines play critical roles in the development of this rejection response, via the activation, differentiation and proliferation of alloreactive T cells, natural killer cells and macrophages that infiltrate into the allograft and ultimately causes graft rejection. The direct investigation of these intracellular cytokines coupled with their cellular source within the allograft may help to elucidate the mechanism of the orchestration of graft rejection. Immunosuppressive agents suppressing the proliferation of T cells are a key element of the pharmacological control of rejection. Measurement of the selective inhibitory effects of immunosuppressive pharmacological agents on the expression of intracellular cytokines may be beneficial to the clinician in customizing immunosuppressive therapy to fit the patient. Longitudinal studies of transplanted patients may also provide some insights into the intricate balance of intracellular cytokines in selected populations of immunoregulatory cells and the role they play in engraftment in transplantation. The studies reported here measured the expressions of intracellular IL-2 and IFNy in patients who were on standard regimen of immunosuppressive therapy with an engrafted kidney ranging from 2 to 15 years. Although numbers are small, the results of the PT patients examined indicate a substantial individualized response in the production of IL-2 and IFNy. This differential response may play a role in the susceptibility to acute rejection which occurs in 30 - 50% of transplanted patients. To achieve these goals a larger cohort needs to be followed using cytokine flow cytometric 88 techniques and probably employing a larger panel of cytokines such as IL-4, IL-6, IL-10 and/or IL-12. Intracellular cytokine flow cytometric technique could also be used to monitor the intracellular levels of IL-2 production in specific lymphoid subpopulations of PBMC or directly from renal biopsies or aspirates as the patients undergo rejection and respond to immunosuppression therapy. Finally, it will be important to analyze the correlation of intracellular IL-2 and levels of CsA or other immunosuppressive drugs. Perhaps as new immunosuppressive pharmacological agents becomes available, this versatile technique can be an adjunct to the monitoring tools that will be used to assess these agents. The human immunodeficiency virus (HIV) binds with high affinity to human CD4 molecules on Th cells, replicating itself at a high rate and disseminating widely throughout the host. As the disease progresses, the number of CD4+T cells becomes depleted as a consequence of HIV mediated direct cytopathic and syncytia formation, cytotoxicity, natural killer cells and autoimmune antibodies to its own M H C class II molecules which are homologous to the gpl20 and gp40 proteins of the HIV, and by apoptosis137. The ratio of Th cells to Ts cells becomes reversed compared to normal controls. Since CD4+ T cells play a central role in the immune response, the disease should greatly impact on the primary source of IL-2 and IFNy cytokines. Fan et al 1 3 8 suggested there was elevated serum IFNy and decreased levels of IL-2 gene expression with HIV infection, while Maggie et al 1 3 9 suggests HIV infected individuals secrete normal amounts of IFNy. In the studies reported here, the number of cells expressing intracellular IFNy expression was not significantly different (p = ns) from the normal controls, the fluorescence of intracellular IFNy produced by these cells was significantly lower in the HIV positive patients (P<0.001). Mean IL-2 expression in CD3+ T cells (P<0.001) and the fluorescence (P<0.01) from HIV patients was significantly lower than in normal 89 controls. The expression of intracellular IL-2 from the CD4+ T and CD8+ T subsets of the HIV patients were found to approximate that expressed by the normal control cells. As the PE fluorochromes have been commercially conjugated to the IL-2 antibody at a 1:1 F/P ratio, the relative fluorescence of the bound antibody-antigen complexes thus enables the antigen density to be estimated. IL-2 producing T cells demonstrated a positive correlation with CD4+CD3+ % positivity for both normal control cells (R2 = 0.92) and HIV positive samples (R2 = 0.81). This could suggest that the HIV positive patients whose CD4+ T cells were comparatively decreased may overcompensate in the production of IL-2 by the activated CD4+CD3+ T cells. There was a suggestion by Hagiwara et al in 1996140 that given the loss of CD4+ cells, steady state cytokine production may be a homeostatic attempt by the fraction of T cells that have increased and have become activated. In stimulated normal control cells, CD8+CD3+ T cells produced IL-2 at one third the capacity of CD4+CD3+ T cells. Despite an increase in CD8+CD3+ cell number responding to the depletion of CD4+ T cells, IL-2 production in HIV patients also approximated that produced by the normal controls. Although there was a near normal production of IL-2 by CD8+CD3+ T cells from the HIV patients, the relationship between IL-2 producing T cells and CD8+CD3+ % positivity cannot be established (Rz = 0.11). The reason for this is not clear at this time. This would be an area of interest to study in HIV patients as clonal expansion of CD8+CD3+ T cells may be beneficial to some HIV infected patients as these cells have inherent cytolytic functions. It would be interesting to study the differential expression of IFNy in CD4+ and CD8+ T cells and the relationship between intracellular IFNy and IL-2 of CD8+ T cells in HIV patients. It would be 90 important to determine whether the cytotoxic activity of the CD 8+ cells in immunocompromised patients is associated with the degree of expression of intracellular IL2 and IFNy. AIDS is associated with a profound dysregulation of cytokine expression, and the depletion of CD4+ T cells may also affect the function of cytotoxic T cells, and inflammatory responses. In 1995 during the First International Symposium on HIV and Cytokines, Reims, France, there was documentation of a shift in the cytokine pattern from Thl to Th2 during the course of HIV infection. Other researchers found antibodies to cytokines IL-4 and IL-10 or the addition of IL-12 can prevent apoptosis of T cells. Fauci indicated that IL-2 enhances the suppressive effects of CD8 and may help to boost CD4 cell numbers. Baiter reported from the symposium that cytokine therapy may be effective only in asymptomatic individuals and may be difficult to assess its efficacy in HIV infected individuals141. 6.3 Potential Customization of the Assay to Detect Cytokines The technique described here for measuring intracellular cytokine production by flow cytometry is versatile. Although not yet confirmed, it is possible that this technique can be combined with cell culture and customized by selecting individual stimuli to permit the production of several different cytokine. IL-1, a major inflammatory mediator, is produced by B lymphocytes and monocytes in PBMC can be induced by PMA and ionomycin, bacterial endotoxin, lipopolysaccharide (LPS), from gram negative bacterial cell walls and IFNy in the culture medium142. IL-4, a B cell growth factor, suppresses monocytic cytokine production of IL-1, IL-6, IL-10, IL-12, IFNp, TNFoc and M - and G-CSFs. IL-4 produced by Th2 cells can be induced by anti-CD3, anti-CD2 in combination with PMA, or PMA and ionomycin, or mitogens such as PHA, concanavalin A (Con A) or poke weed mitogen143. IL-6, a pleiotropic cytokine, is an essential factor in the maturation of B lymphocytes into plasma 91 cells, functions as a hematopoietic growth factor stimulating the proliferation and differentiation of progenitor cells, and regulates the production of acute phase protein productions by the liver. The production of IL-6 is also up-regulated in mesangial proliferative glomerulonephritis. HIV infection have been shown to increase IL-6 synthesis144. In vitro synthesis of IL-6 can be up-regulated in lymphocytes by PHA and PMA on T cells, LPS on monocytes, and Staphylococcus Aureus Cowan strain protein (SAC) on B cells145. IL-8 is mainly an inflammatory cytokine whose synthesis by leukocytes can be induced by PHA, Con A, LPS, IL-1, TNFa and phorbol esters146. IL-9 produced by T cells is particularly prevalent in T cell neoplasms and is inducible by PMA, PHA, anti-CD3, anti-CD28 antibodies in presence of IL-2 1 4 7. IL-10 is a regulatory cytokine that suppresses the activation of macrophages and dendritic cells and stimulates the proliferation and differentiation of T and B cells. IL-10 is produced by Thl , Th2 and B lymphocytes and monocytes. Anti-CD3 will induce T cells to produce IL-10, while LPS induces monocytic IL-10 1 4 8. IL-12 is a pleiotropic immunoregulatory cytokine that augments the lytic activities of NK, lymphokine activated killer cells and cytolytic T cells and promotes cell mediated immunity by enhancing the activation and differentiation of Thl cells. IL-12 is released by monocytes when cultured with LPS, and SAC can also induce B lymphocytes to produce IL-121 4 9. IL-13, produced by activated Thl and Th2 cells, inhibits the production of inflammatory cytokines, and induces expression of CD23 on B cells and promotes their differentiation and maturation. It also increases the M H C class II expression on monocytes. IL-13 can be stimulated in vitro by PMA, ionomycin, PHA, anti-CD2, anti-CD28 and anti-CD31 5 0. IL-15 exerts its influence by binding to the (3 and y subunits of the IL-2 receptor and synergizes with IL-12 to stimulate the proliferation of T cells. It is also an effector glycoprotein in B cell proliferation and differentiation, and activates NK cells through their IL-2R. Although IL-15 is primarily produced by monocytes after stimulation by LPS, it is also present in human tissue 92 including the kidney and pancreatic cells151. IL-17 is a proinflammatory cytokine which has an additive effect on IL-6 production is synthesized by PMA and ionomycin or immobilized CD3 and CD28 Mab stimulated PBMC or T cells'52. IL-18 augments the production of IFNy and proliferation of Thl clones is produced primarily by tissues including pancreas, kidney, liver and lung. The induction of IL-18 can be up-regulated by LPS' 5 3 . Superantigens Staphylococci enterotoxin A or B (SEA or SEB) binds to the TCR and the M H C class II molecules can also be used to elicit a strong primary response, activating the T cells to produce IL-2, IFNy and TNFa and TNFP' 5 4 although TNF can also be produced from monocytes by induction with LPS 1 5 5 . Different cell sources, including PBMC, tissue, bone marrow aspirates or biopsies, may be used to stimulate the production of the cytokine of interest. The incubation period of activation has to be optimized for the expression of each cytokine. Monensin can be added to a short incubation of less than 8 hours or added to the last 4-6 hours of a longer overnight incubation to accumulate the synthesized proteins. The specificity of fiuorochrome conjugated antibodies used in the flow cytometric technique allows analysis of cytokine production to be simultaneously characterized in rare cells such as activated CD38 positive CD8+ T cells or CD34 positive progenitors or specific cell populations such as subpopulations of lymphocytes or monocytes and this is only limited by the capabilities of the instrumentation. As the technology advances, the study of cytokines, their cell source, their interactions, and their role in pathology can be further elucidated. 93 Massry A.G. & Glassock R.J. (1995) Textbook of Nephrology, 3rd edn. p787. Williams & Wilkins, Baltimore. Annual Report (1996) Dialysis and Renal Transplantation, Canadian Organ Replacement Register, p. 1-1. Canadian Institute for Health Information, Ottawa, Ontario. Canadian Organ Replacement Register (CORR). (2000) Dialysis Statistics. Retrieved July 3, 2000 from the World Wide Web: http://www. cihi.ca/facts/corrdial.htm Keane W.F. & Maddy M.F. (1989) Host defense and infectious complications in maintenance hemodialysis patients. In: Replacement of Renal Functions by Dialysis (ed. J. F. Mahier), 3rd edn, p. 865. Kluwer Academic Publishers, Dordrecht, The Netherlands. Hamburger J., Crosnier J., Dormont J. & Bach J.-F. (1972) Results of kidney transplantation in man. In: Renal Transplantation: Theory and Practice, p. 160. The Williams and Wilkins Company, Baltimore. Morris P.J. (1988) Results of renal transplantation. In: Renal Transplantation: Principles and Practice, p. 753. W.B. Saunders Company, Philadelphia. B.C. Transplant Society. (2000) Organ Transplantation Fact Sheet. Retrieved July 3, • 2000 from the World Wide Web: http://www.transplant.bc.ca/statistics.html Ball S.T. & M.J. D. (1995) Transplantation immunology. Current Opinion in Nephrology and Hypertension, 4, 465. Annual Report (1996) Dialysis and Renal Transplantation, Canadian Organ Replacement Register, p.4-1. Canadian Institute for Health Information, Ottawa, Ontario. B.C. Transplant Society. (2000) Patient and Graft Survival, B.C. and Canada Comparison. Retrieved July 3, 2000 from the World Wide Web: http://www.transplant.bc.ca/statistic.html Janeway Jr. C.A. & Travers P. (1994) Control and manipulation of the immune response. In: ImmunoBiology: The Immune System in Health and Disease, p. 12:2. Garland Publishing Inc., New York. Burdick J.F. (1992) Biology of immunosuppression mediated by antilymphocyte antibodies. In: Kidney Transplant rejection (ed. J. F. Burdick, L. C. Racusen, K. Solez & G. M . Williams), 2nd edn, p. 506. Marcel Dekker Inc., New York. Borel J.F. (1976) Comparative study of an in vitro and in vivo drug effects on cell mediated cytotoxicity. Immunology, 31, 631. 94 14. Keown P. A. (1998) Therapeutic strategies for optimal use of novel immunosuppressants. University of British C o l u m b i a , Canada. 15. Halloran, P.F. (1996) Molecular mechanism of new immunosuppressants. Clinical Transplantation. 10, 118, quoted in Keown, P.A. (1998) Therapeutic strategies for optimal use of novel immunosuppressants. University of British Columbia, Canada 16. Benvenuto R, Bachetoni A, Cinti P, Sallusto F, Franco A, Molajoni ER, Barnaba V, Balsano F, Cortesini R (1991) Enhanced production of interferon-gamma by T lymphocytes cloned from rejected kidney grafts. Transplantation 51 (4) 887 17. Zanker B, Jooss-Rudiger J, Franz HE, Wagner H, Kabelitz D (1993) Evidence that functional deletion of donot-reactive T lymphocytes in kidney allograft recipients can occur at the level of cytotoxic T cells, IL-2 producing T cells, or both. A limiting dilution study. Transplantation 56 (3) 628 18. Freshney Rl (1994) Measurement of viability and cytotoxicity In: Culture of Animal Cells 3rd edn p.296 Wiley-Liss New York 19. Alexander SI, Younes SB, Zurakowski D, Mira N M , Dubey DP, Drew MP, Harmon WE and Yunis EJ (1997) Cell mediated cytotoxicity: A predictor of chronic rejection in pediatric H L A haploidentical renal transplants Transplantation 27 (12) 1756 20. Granelli-Piperno A. (1993) Cellular mode of action of cyclosporin A. In: T Cell Directed Immunointervention. (Bach J-F ed.) p. 3 Blackwell Scientific 21. Thomson A.W. (1993) Cellular mode of action of cyclosporin A. In: T Cell Directed Immunointervention. (Bach J-F ed.) p. 26 Blackwell Scientific 22. Brady H.R., Spertini 0., Jimenez W., Brenner B.M., Marsden P.A., & Tedder T.F. (1992) Neutophils, monocytes, and lymphocytes bind to cytokine-activated kidney glomerular endothelial cells through L-selectin (LAM-1) in vitro. Journal of Immunology, 149, 2437. 23. Lowry R.P., Blais D., Marghesco D., & Powell W.S. (1987) Immune effector mechanisms in organ allograft rejection. VIII: Inflammatory mediators and cytotoxins in rejecting rat cardiac allografts. Transplantation Proceedings 19, 424. 24. Simpson M.A., Young-Fadok T.M. , Madras P.N., Freeman R.B., Dempsey R.A. Shaffer D., Lewis D., Jenkins R.L. & Monaco A.P. (1991) Sequential interleukin 2 and interleukin 2 receptor levels distinguish rejection from cyclosporine toxicity in liver allograft recipients. Archives of Surgery 126, 717. 25. YoshimuraN., Oka T., & Kalian B.D. (1991) Sequential determinations of serum interleukin 6 levels as an immunodiagnostic tool to differentiate rejection from 95 nephrotoxicity in renal allograft recipients. Transplantation. 51, 172. 26. Dallman M.J., Larsen C P . , & Morris P.J. (1991) Cytokine gene transcription in vascularised organ grafts: Analysis using semiquantitative polymerase chain reaction. Journal of Experimental Medicine. 174, 493. 27. Vanderbroecke C , Caillat-Zucman S., Legendre C , Noel L-H. , Kreis H., Woodrow D., Bach J-F, & Tovey M.G. (1991) Differential in situ expression of cytokines in renal allograft rejection. Transplantation. 51, 602. 28. Johnson H.K., Richie R.E. & Niblack G.D. (1983) The case for renal transplantation. In: End Stage Renal Disease: An Integrated Approach (ed. W. J. Stone & P. L. Rabin), p. 245. Academic Press, New York. 29. Lowry, R.P. & Takeuchi, T. (1992)Immunologic tolerance and its relationship to clinical transplantation. In: Kidney Transplant rejection (ed. J. F. Burdick, L. C. Racusen, K. Solez & G. M. Williams), 2nd edn, p. 93. Marcel Dekker Inc., New York. 30. Male D., Cooke A., Owen M. , Trowsdale J. & Champion B. (1996) Class I and II molecules of the MHC. In: Advanced Immunology, p. 4.1. Mosby, Italy. 31. Danielian S., Alcover A., Polissard L. , Stefanescu M. , Acuto O., Fischer S. & Fagard R. (1992) Both T cell receptor (TCR)-CD3 complex and CD2 increase the tyrosine kinase activity of p56/c/c. CD2 can mediate TCR-CD3-independent and CD45-dependent activation of p56/c/c. European Journal of Immunology, 22, 2915. 32. Bell G.M., Fargnoli J., Bolen J.B., Kish L. & Imboden J.B. (1996) The SH3 domain of p56 / c / l binds to proline rich sequences in the cytoplasmic domain of CD2. Journal of Experimental Medicine, 183, 169. 33. Linsley PS and Ledbetter JA (1993) The Role of the CD28 Receptor During T Cell Responses to Antigen Annual Review of Immunology 11, 191 34. Linsley P.S., Greene J.L., Tan P., Bradshaw J., Ledbetter J.A., Anasetti C. & Damle N.K. (1992) Coexpression and functional cooperation of CTLA-4 and CD28 on activated T lymphocytes. Journal of Experimental Medicine, 176, 1595. 35. Peng X, Kasran A, Warmerdam PAM, de Boer M and Ceuppens JL (1996) Accessory Signaling by CD40 for T Cell Activation: Induction of Thl and Th2 Cytokines and Synergy with Interleukin-12 for Interferon-y Production European Journal of Immunology26, 1621 36. TamuraT., Nakano H., Nagase H., Morokata T., Igarashi O., Oshimi Y., Miyazaki S. & Nariuchi H. (1995) Early activation signal transduction pathways of Thl and Th2 cell clones stimulated with anti-CD3. Journal of Immunology, 155, 4692. 96 37. Wardenburg J.B., Fu C , Jackman J.K., Flotow H., Wilkinson S.E., Williams D.H., Johnson R., Kong G., Chan A.C. & Findell P.R. (1996) Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function. Journal of Biological Chemistry, 271, 19641. 38. Northrop J.P., Ho S.N., Chen L. , Thomas D.J., Timmerman L.A. , Nolan G.P., Admon A. & Crabtree G.R. (1994) NF-AT components define a family of transcription factors targeted in T-cell activation. Nature, 369, 497. 39. Male D., Cooke A., Owen M. , Trowsdale J. & Champion B. (1996) T lymphocyte activation and maturation. In: Advanced Immunology. Mosby, Italy. 40. June CH, Bluestone JA, Nadler L M and Thompson CB (1994) The B7 and CD28 Receptor Families Immunology Today 7, 321 41. Luo C , Burgeon E., Carew J.A., McCaffrey P.G., Badalian T.M. , Lane W.S., Hogan P.G. & Rao A. (1996) Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes. Molecular & Cellular Biology, 16, 3955. 42. Park J., Takeuchi A. & Sharma S. (1996) Characterization of a new isoform of the N F A T (nuclear factor of activated T cells) gene family member NFATc. Journal of Biological Chemistry, 271, 20914. 43. Campbell P.M., Pimm J., Ramassar V. & Halloran P.F. (1996) Identification of a calcium-inducible, cyclosporine sensitive element in the IFN-gamma promoter that is a potential NFAT binding site. Transplantation, 61, 933. 44. Takahama Y. & Nakauchi H. (1996) Phorbol ester and calcium ionophore can replace TCR signals that induce positive selection of CD4+ cells. Journal of Immunology, 157, 1508. 45. Goldman M . & Druet P. (1995) The TH1/TH2 concept and its relevance to renal disorders and transplantation immunity. Nephrol Dial Transplant, 10, 1282. 46. Callard R. & Gearing A. (1994) The cytokines and their receptors. In: The Cytokine FactsBook, p. 39. Academic Press Ltd., San Diego. 47. Goldsmith M.A. & Greene W.C. (1994) Interleukin-2 and the interleukin-2 receptor. In: The Cytokine Handbook (ed. A. W. Thomson), 2nd edn. Academic Press Ltd., San Diego. 48. Meager A. (1991) Lymphocyte activation. In: Cytokines. P. 115. Prentice Hall, New Jersey. 97 49. Umlauf S.W., Beverly B., Kang S.M., Brorson K., Tran A.C. & Schwartz R.H. (1993) Molecular regulation of the IL-2 gene: rheostatic control of the immune system. Immunological Reviews, 1 3 3 , 177. 50. Schwartz RH (1992) Costimulation of T Lymphocytes: The Role of CD28, CTLA-4, and B7/BB1 in Interleukin-2 Production and Immunotherapy Cell 7 1 , 1065 51. Naylor S., Sakaguchi A., Shows T., Law M . , Goedel D. & Gray P. (1983) Human immune interferon gene is located on chromosome 12. Journal of Experimental Medicine, 157, 1020. 52. Ciccarone V., Chriva J., Hardy K. & Young H. (1990) Identification of enhancer-like elements in human IFN-gamma genomic DNA. Journal of Immunology, 1 4 4 , 725. 53. Arad G., Nussinovich R. & Kaempfer R. (1995) Interleukin-2 induces an early step in the activation of interferon-gamma gene expression. Immunology Letters, 4 4 , 213. 54. Young H.A. (1996) Regulation of interferon-gamma gene expression. Journal of Interferon & Cytokine Research, 16, 563. 55. Ealick S., Cook W., Vijay-Kumar S., Carson M . , Nagabhushan T., Trotta P. & Bugg C. (1991) Three dimensional structure of recombinant human interferon-gamma. Science, 2 5 2 , 698. 56. Mulkerrin M . & Wetzel R. (1989) pH dependence of the reversible and irreversible thermal denaturation of gamma interferons. Biochemistry, 2 8 , 6556. 57. Kelker H., Yip Y., Anderson P. & Vilcek J. (1983) Effects of glycosidase treatment on the physico-chemical properties and biological activity of human interferon-gamma. Journal of Biological Chemistry, 2 5 8 , 8010. 58. Meager A. (1991) Mediators of immune responses and inflammatory reactions. In: Cytokines, p. 144. Prentice Hall, New Jersey. 59. Schulick R.D., Weir M.B., Miller M.W., Cohen D.J., Bermas B.L. & Shearer G.M. (1993) Longitudinal study of in vitro CD4+ T helper cell function in recently transplanted renal allograft patients undergoing tapering of their immunosuppressive drugs. Transplantation, 5 6 , 590. 60. Hock H., Dorsch M . , Kunzendorf U., Qin Z., Diamantstein T. & Blankenstein T. (1993) Mechanisms of rejection induced by tumour cell-targeted gene transfer of interleukin 2, interleukin 4, interleukin 7, tumour necrosis factor, or interferony. Proceedings of the National Academy of Science of U.S.A., 90, 2774. 61. Wadhwa M . , Bird C , Page L. , Mire-Sluis A. & Thorpe R. (1995) Quantitative biological assays for individual cytokines. In: Cytokine: A Practical Approach (ed. F. R. Balkwill), 2d edn, p. 366. Oxford University Press, New York. 98 62. Doyle A., Stein M . , Keshav S. & Gordon S. (1995) Assays for macrophage activation by cytokines. In: Cytokines: A Practical Approach (ed. F. R. Balkwill), 2d edn, p. 273. Oxford University Press, New York. 63. Merville P., Pouteil-Noble C , Wijdenes J., Potaux L . , Touraine J. & Banchereau J. (1993) Detection of single cells secreting IFN-y, IL-6 and IL-10 in irreversibly rejected human kidney allografts, and their modulation by IL-2 and IL-4. Transplantation, 55, 639. 64. Tovey M . , Deglise-Favre A. & Schoevaert D. (1993) Differential in situ expression of cytokine gene in human renal transplantation. Kidney International, 43, 129. 65. Fan J., Bass H.Z. & Fahey J.L. (1993) Elevated IFN-gamma and decreased IL-2 gene expression are associated with HIV infection. Journal of Immunology, 151, 5031. 66. McLean A., Hughes D., Welsh K., Gray D., Roake J., Fuggle S., Morris P. & Dallman M . (1997) Patterns of graft infiltration and cytokine gene expression during the first 10 days of kidney transplantation. Transplantation, 63, 374. 67. Lewis A.E . (1991) Detecting cytokine production at the single cell level. Cytokine, 3, 184. 68. Moldavan A. (1934) Photoelectric technique for the counting of microscopic cells. Science, 80, 188. 69. Gucker F.J., O'Konshi C. & Pickard H. (1947) A photoelectric counter for colloidal particles. Journal of American Chemical Society, 69, 2422. 70. Papanicolaou G. & Traut H. (1941) The diagnosis value of vaginal smears in carcinoma of the uterus. American Journal of Obstetrics and Gynecology, 42, 193. 71. Friedman H.J., Jr. (1950) The use of ultraviolet light and fluorescent dyes in the detection of uterine cancer by vaginal smear. American Journal of Obstetrics and Gynecology, 59, 852. 72. Coons A., Creech H. & Jones R. (1941) Immunological properties of an antibody containing a fluorescent group. Proceedings of Society of Experimental Biology, 47, 200. 73. Coulter W. (1956) High speed automatic blood cell counter and cell size analyser. Proceedings of National Electronics Conference, 12, 1034. 74. Shapiro H. (1995) History. In: Practical Flow Cytometry, 3rd edn, p. 68. Wiley-Liss Inc., New York. 99 75. Andersson U., Andersson J., Lindfors A., Wagner K., Moller G. & Heusser C. (1990) Simultaneous production of interleukin 2, interleukin 4 and interferon-y by activated human blood lymphocytes. European Journal of Immunology, 2 0 , 1591. 76. Sander B., Andersson J. & Andersson U. (1991) Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunological Review, 119, 65. 77. Jung T., Schauer U., Heusser C., Neumann C. & Rieger C. (1993) Detection of intracellular cytokines by flow cytometry. Journal of Immunological Methods, 159, 197. 78. Prussin C. & Metcalfe D.D. (1995) Detection of intracellular cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. Journal of Immunological Methods, 188, 117. 79. Caiman P. (1996) Cytokine flow cytometry: Assessing cytokine production at the single cell level. Clinical Immunology Letter, 16, 85. 80. Shapiro H. (1995) How Flow Cytometers Work. In: Practical Flow Cytometry, p.128. 3rd edn. Wiley-Liss Inc., New York. 81. Mardiney III M . , Brown M.R. & Fleisher T.A. (1996) Measurement of T cell CD69 expression: A rapid and efficient means to assess mitogen or antigen induced proliferative capacity in normals. Cytometry, 26 , 305. 82. Testi R., D'Ambrosio D., De Maria R. & Santoni A. (1994) The CD69 receptor: a multipurpose cell surface trigger for hematopoietic cells. Immunology Today, 15 , 479. 83. Taylor-Fishwick D.A. & J.N. S. (1995) Raf-1 provides a dominant but not exclusive signal for the induction of CD69 expression on T cells. European Journal of Immunology, 25 , 3215. 84. Mollenhauer H.H. & Morre D.J. (1991) Perspectives on Golgi apparatus form and function. Journal of Electron Microscopy Technique, 17, 2. 85. Alberts B., Bray D., Lewis J., Raff M . , Roberts K. & Watson J. (1994) Vesicular traffic in the secretory and endocytic pathways. In: Molecular Biology of The Cell, 3rd edn. Garland Publishing Inc., New York. 86. Tartakoff A . M . (1983) Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell, 32 , 1026. 87. Morre D.J., Morre D.M., Mollenhauer H.H. & Reutter W. (1987) Golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices. European Journal of Cell Biology, 43 , 235. 100 88. Mollenhauer H.H., Morre D.J. & Rowe L.D. (1990) Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity. Biochimica et Biophysica Acta, 1031, 225. 89. Mollenhauer H.H., Morre D.J. & Minnifield N. (1992) Swelling response of Golgi apparatus cisternae in cells treated with monensin is reduced by cell injury. Cell Biology International Reports, 16, 217. 90. Glauert A . M . , Dingle J.T. & Lucy J.A. (1962) Action of saponin on biological cell membrane. Nature, 196, 952. 91. Jacob M.C. , Favre M . & Bens J.C. (1991) Membrane cell permeabilization with saponin and multiparametric analysis by flow cytometry. Cytometry, 12, 550. 92. Ficner R., Lobeck K., Schmidt G. & Huber R. (1992) Isolation, crystallization, crystal structure analysis and refinement of B-phycoerytlirin from the red alga Porphyredium sordidum at 2.2 A resolution. Journal of Molecular Biology, 228, 935. 93. Ficner R. & Huber R. (1993) Refined crystal structure of phycoerythrin from Porphyridium cruentum at 0.23 nm resolution and localization of the gamma subunit. Eurpean Journal of Biochemistry, 18, 103. 94. Chang W.R., Jiang T., Wan Z.L., Zhang J.P., Yang Z.X. & Liang D.C. (1996) Crystal structure of R-phycoerythrin (R-PE) from Polysiphonia urceolata at 2.8 A resolution. Journal of Molecular Biology, 262, 721. 95. Recktenwald D., Prezelin B., Chen C.H. & Kimura J. (1990) Biological pigments as fluorescent labels for cytometry. New Technologies in Cytometry and Molecular Biology, 1206, 106. 96. Coleman R.M., Lombard M.F. & Sicard R.E. (1992) Effectors of humoral immunity. In: Fundamental Immunology, 2nd edn. Wm. C. Brown, Dubuque. 97. Skinnider L.F. & McAskill J. (1980) A comparison of phorbol myristate acetate with phytohemgglutinin as a stimulator of in vitro colony growth of peripheral human leucocytes. Experimental Hematology, 8, 477. 98. Dupuis G. & Leclair B. (1982) Studies on Phaseolus vulgaris phytohemagglutinin. FEBS Letters, 144, 29. 99. Valentine M.A., Tsoukas C D . , Rhodes G., Vaughan J.H. & Carson D.A. (1985) Phytohemagglutinin binds to the 20-kDa molecule of the T3 complex. European Journal of Immunology, 15, 851. 100. Chilson O.P., Boylston A.W. & Crumpton M.J. (1984) Phaseolus vulgaris phytohaemagglutinin (PHA) binds to the human T lymphocyte antigen receptor. 101 EMBO Journal, 3, 3239. 101. O' Flynn K., Krensky A . M . , Beverley P.C., Burakoff S.J. & Linch D.C. (1985) Phytohaemagglutinin activation of T cells through the sheep red blood cell receptor. Nature, 313, 686. 102. Meager A. (1991) Lymphocyte activation. In: Cytokines, p. 120-122. Prentice Hall, New Jersey. 103. Bruserud O., Degre M . & Thorsby E. (1986) Interleukin 2- and interferon-production and expression of interleukin 2 receptors by human mononuclear cells stimulated with mumps virus or phytohaemagglutinin. Acta Pathologica, Microbiologica, et Immunologica Scandinavica - Section C, Immunology, 94, 51. 104. Van Gool S.W., Kasran A., G. W., De Boer M . & Ceuppens J.L. (1995) Accessory signalling by B7-1 for T cell activation induced by anti-CD2: Evidence for IL-2 independent C T L generation and CsA resistant cytokine production. Scandinavian Journal of Immunology, 41, 23. 105. Lenschow D.J., Walunas T.L. & Bluestone J.A. (1996) CD28/B7 system of T cell costimulation. Annual Review of Immunology, 14, 233. 106. Hathcock K.S., Laszlo G., Pucillo C., Linsley P. & Hodes R.J. (1994) Comparative analysis of B7-1 and B7-2 costimulatory ligands: Expression and function. Journal of Experimental Medicine, 180, 631. 107. Lai J., Horvath G., Subleski J., Bruder J., Ghosh P. & Tan T. (1995) RelA is a potent transcriptional activator of the CD28 response element within the interleukin-2 promoter. Molecular and Cellular Biology, 15, 4260. 108. June C.H., Bluestone J.A., Nadler L . M . & Thompson C.B. (1994) The B7 and CD28 receptor families. Immunology Today, 15, 321. 109. Koulova L. , Clark E.A., Shu G. & Dupont B. (1991) The CD28 ligand B7/BB1 provides costimulatory signal for alloactivation of CD4+ T cells. Journal of Experimental Medicine, 173, 759. 110. Thompson C.B., Lindsten T., Ledbetter J.A., Kunkel S.L., Young H.A., Emerson S.G., Leiden J.M. & June C H . (1989) CD28 activation pathway regulates the production of multiple cell derived lymphokines/cytokines. Proceedings of the National Academy of Sciences of U.S.A., 86, 1333. 111. Schwartz R. (1992) Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell, 71, 1065. 112. Alberts B., Bray D., Lewis J., Raff M . , Roberts K. & Watson J. (1994) Basic genetic mechanism. In: Molecular Biology of The Cell, 3rd edn. p. 321. Garland 102 Publishing Inc., New York. 113. Inwood M.J., Thomson M.S. & Bryant N.J. (1983) Immunohaematology and Haematology. In: Lynch's Medical Laboratory Technology (ed. S. S. Raphael), 4th edn. p. 585. W.B. Saunders Comp., Philadelphia. 114. Burtis C.A. & Ashwood E.R. (1996) In: Tietz Fundamentals of Clinical Chemistry, p. 29. W.B. Saunders Comp., Philadelphia. 115. Hoffbrand A.V. & Pettit J.E. (1993) Thrombosis and antithrombotic therapy. In: Essential Haematology, 3d edn. p. 356. Blackwell Scientific Publications, Boston. 116. Imagawa D.K., Busuttil R.W. & Farmer D.G. (1995) Role of cytokine in organ transplantation rejection. In: Human Cytokines: Their Role in Disease and Therapy (ed. B. B. Aggarwal & R. K. Puri), p. 165. Blackwell Scientific Publications, Cambridge. 117. Tocci M.J., Matkovich D.A., Collier K.A. , Kwok P., Dumont F., Lin S., Degudicibus S., Siekierka J.J., Chin J. & Hutchinson N.I. (1989) The immunosuppressant FK506 ^ selectively inhibits expression of early T cell activation genes. Journal of Immunology, 143, 718. 118. Mattila P.S. (1996) The actions of cyclosporin A and FK506 on T-lymphocyte activation. Biochemical Society Transactions, 24, 45. 119. Wesselborg S., Fruman D.A., Sagoo J.K., Bierer B.E. & Burakoff S.J. (1996) Identification of a physical interaction between calcineurin and nuclear factor of activated T cells (NFATp). Journal of Biological Chemistry, 271, 1274. 120. Gerez L. , Madar L. , Shkolnik T., Kristal B., Arad G., Reshef A., Steinberger A., Ketzinel M . , Sayar D. and Shasha S. (1991) Regulation of interleukin-2 and interferon-gamma gene expression in renal failure. Kidney International, 40, 266 121. Morita Y., Yamamura M . , Kashihara N. & Makino H. (1997) Increased production of interleukin 10 and inflammatory cytokines in blood monocytes of hemodialysis patients. Research Communications in Molecular Pathology & Pharmacology, 98, 19. 122. Weinstein W., Fishman P., Dialdetti M . and Levi J. (1993) Cytokine production by mononuclear cells from patients with chronic renal disease. Israel Journal of Medical Sciences, 29, 183. 123. Ha, S.K., Cho, H.S., Lee, H.Y., Kim, H.S., Choi, K.H. and Han, D.S. (1993) Studies on IL-2 production and T cell colony forming unit in patients with chronic renal failure. Korean Journal of Internal Medicine, 8, 86. 103 124. Manca F., Carrozzi S., A. K., Barocci S., Dessi V., Valente U., Fontana I. & Celada F. (1986) Difference in sensitivity to cyclosporine in vitro of human alloreactive lines and clones. Transplantation, 41, 199. 125. Kumagal N., Benedict S.H., Mills G.B. & Gelfand E.W. (1988) Cyclosporin A inhibits initiation but not progression of human T cell proliferation triggered by phorbol esters and calcium ionophores. Journal of Immunology, 141, 3747. 126. Batiuk T.D. & Ffalloran P.F. (1997) The downstream consequences of calcineurin inhibition. Transplantation Proceedings, 2 9, 1239. 127. Batiuk T.D., Kung L. & Halloran P.F. (1997) Evidence that calcineurin is rate limiting for primary human lymphocyte activation. Journal of Clinical Investigations, 100, 1894. 128. Mardiney III M . , Brown M.R. & Fleisher T.A. (1996) Measurement of T cell CD69 expression: a rapid and efficient means to assess mitogen or antigen induced proliferative capacity in normals. Cytometry, 26, 305. 129. Maino V.C. , Suni M.A. & Ruitenberg J.J. (1995) Rapid flow cytometric method for measuring lymphocyte subset activation. Cytometry, 20, 127. 130. Testi R., Phillips J.H. & Lanier L.L. (1989) T cell activation via leu 23 (CD69). Journal of Immunology, 143, 1123. 131. Testi R., D'Ambrosio D., De Maria R. & Santoni A. (1994) The CD69 receptor: a multipurpose cell surface trigger for hematopoietic cells. Immunology Today, 15, 479. 132. Taylor-Fishwick D.A. & J.N. S. (1995) Raf-1 provides a dominant but not exclusive signal for the induction of CD69 expression on T cells. European Journal of Immunology, 25, 3215. 133. D' Ambrosio D., Trotta R., Vacca A., Frati L. , Santoni A., Gulino A. & Testi R. (1993) Transcriptional regulation of interleukin-2 gene expression by CD69-generated signals. European Journal of Immunology, 23, 2993. 134. June C.H., Ledbetter J.A., Gillespie M.M. , Lindsten T. & Thompson C.B. (1987) T cell proliferation involving the CD28 pathway is associated with cyclosporine resistant interleukin 2 gene expression. Molecular and Cellular Biology, 7, 4472. 135. Young H.A. (1996) Regulation of interferon-gamma gene expression. Journal of Interferon and Cytokine Research, 16, 563. 136. Rafig K., Kasran A., Peng X., Warmerdam P.A.M., Coorevits L. , Ceuppens J.L. & Van Gool S.W. (1998) Cyclosporin A increases IFNy production by T cells when co-stimulated through CD28. European Journal of Immunology, 28, 1481. 104 137. Fauci A.S. & Lane H.C. (1994) Human immunodeficiency virus (HIV) disease: Aids and related disorders. In: Harrison's Principles of Internal Medicine, 13th edn, p. 1580. McGraw-Hill, New York. 138. Fan J., Bass H.Z. & Fahey J.L. (1993) Elevated IFN-gamma and decreased IL-2 gene expression are associated with HIV infection. Journal of Immunology, 151, 5031. 139. Maggi E. , Mazzetti M . , Ravina A., Annunziato F., De Carli M . , Piccinni M.P., Manetti R., Carbonari M. , Pesce A . M . , Del Prete G. & Romagnani S. (1994) Ability of HIV to promote a Thl to ThO shift and to replicate perferentially in Th2 and ThO cells. Science, 265, 244. 140. Hagiwara E., Sacks T., Leitman-Klinman S.F. & Klinman D.M. (1996) Effect of HIV infection on the frequency of cytokine-secreting cells in human peripheral blood. AIDS Research & Human Retroviruses, 12, 127. 141. Baiter M . (1995) Cytokines move from the margins into the spotlight. In: First International Symposium on HIV and Cytokines, Reims, France. 142. Dower S.K. (1992) Interleukin-1. In: Human Cytokines: Handbook for Basic and Clinical Research (ed. B. B. Aggarwal & J. U. Gutterman), p. 48. Blackwell Scientific Publications, Cambridge. 143. De Vries, J. (1992) Interleukin-4. In: Human Cytokines: Handbook for Basic and Clinical Research (ed. B. B. Aggarwal & J. U. Gutterman), p. 114. Blackwell Scientific Publications, Cambridge. 144. Cox, G. & Gauldie, J. (1997) Interleukin-6. In: Cytokines in Health and Disease (ed. D. G. Remick & J. S. Friedkand), 2nd edn, p. 81. Marcel Dekker Inc., New York. 145. Taga, T. & Kishimoto, T. (1992) Interleukin-6. In: Human Cytokines: Handbookfor Basic and Clinical Research (ed. B. B. Aggarwal & J. U. Gutterman), p. 145. Blackwell Scientific Publications, Cambridge. 146. Zachariae, C.O.C. & Matsushima, K. (1992) Interleukin-8. In: Human Cytokines: Handbookfor Basic and Clinical Research (ed. B. B. Aggarwal & J. U. Gutterman), p. 182. Blackwell Scientific Publications, Cambridge. 147. Renauld J. (1997) Interleukin-9. In: Cytokines in Health and Disease (ed. D. G. Remick & J. S. Friedkand), 2nd edn, p. 134. Marcel Dekker Inc., New York. 148. Powrie, F., Bean, A. & Moore, K.W. (1997) Interleukin-10. In: Cytokines in Health and Disease (ed. D. G. Remick & J. S. Friedkand), 2nd edn, p. 144. Marcel Dekker Inc., New York. 149. Gately, M.K., Wu, C Y . & Faherty, D.A. (1997) Interleukin-12. In: Cytokines in Health and Disease (ed. D. G. Remick & J. S. Friedkand), 2nd edn, p. 168. Marcel 105 Dekker Inc., New York. 150. Minty, A.J. (1997) Interleukin-13. In: Cytokines in Health and Disease (ed. D. G. Remick & J. S. Friedkand), 2nd edn, p. 185. Marcel Dekker Inc., New York. 151. Carson, W. & Caligiuri, M. (1997) Interleukin-15. In: Cytokines in Health and Disease (ed. D. G. Remick & J. S. Friedkand), 2nd edn, p. 210. Marcel Dekker Inc., New York. 152. Spriggs M.K. (1997) Interleukin-17 and its receptor. Journal of Clinical Immunology, 17, 366. 153. Gillespie M.T. & Horwood N.J. (1998) Interleukin-18: perspectives on the newest interleukin. Cytokines and Growth Factor Reviews, 9, 109. 154. Svensson A., Schad E.M. , Sunstrom M . , Antonsson P., Kalland T. & Dohlsten M . (1997) Staphylococcal Enterotoxin A, D, and E. In: Super antigens: Molecular biology, Immunology, and Relevance to Human Disease (ed. D. Y. M . Leung, B. T. Huber & P. M . Schlievert), p. 199. Marcel Dekker Inc., New York. 155. Tracey, K.J. (1997) Tumor Necrosis Factor. In: Cytokines in Health and Disease (ed. D. G. Remick & J. S. Friedkand), 2nd edn, p. 225. Marcel Dekker Inc., New York. 106 

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