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A novel T cell activating factor Williams, Laura Dawn 1987

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A NOVEL T CELL ACTIVATING FACTOR By LAURA DAWN WILLIAMS  B.Sc,  The U n i v e r s i t y o f B r i t i s h Columbia, 1985  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER IN SCIENCE  in  THE FACULTY OF GRADUATE STUDIES G e n e t i c s Program  We a c c e p t t h i s  t h e s i s as  to the required  conforming  standard  THE UNIVERSITY OF BRITISH COLUMBIA May, 1987 ® L a u r a Dawn W i l l i a m s  U6  In presenting degree  this  at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department publication  this or of  and study.  this  his  or  her  representatives.  Department of  G e n e t i c s Program  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 n a  .  DE-6(3/81)  July 23, 1987  that the  may be It  thesis for financial gain shall not  permission.  requirements  I further agree  thesis for scholarly purposes by  the  is  an  advanced  Library shall make it  that permission  for extensive  granted  head  by the  understood be  for  that  allowed without  of  my  copying  or  my written  11  ABSTRACT  The maturation of cytotoxic T lymphocyte (CTL) effectors from CTL precursors (CTLp) requires specific signals mediated through cellular interactions and soluble factors.  The most studied factor is T cell  growth factor (TCGF) which is also termed interleukin-2 (IL-2).  This  lymphokine is produced by T helper cells (T^) and induces activated CTLp to proliferate and differentiate.  However, in the absence of mitogen or  antigen stimulation, IL-2 alone cannot induce CTL (except in the case of very high c e l l density).  A factor is described that is found in the  supernatant of 4-B-phorbol-12-myristate-13-acetate  (PMA)-induced EL4 cells  that can polyclonally activate CTL in the presence of IL-2.  This factor  elutes at 27 kilodaltons (KDa) on a G-100 column, and its target cell includes T cells of the Thyl  +  Lyt2 L3T4~ phenotype. +  The factor  increases the frequency of IL-2 receptor expressing cells within a population, thereby increasing the response to IL-2.  It is suggested that  this factor acts through an alternative pathway of CTL activation which is independent of specific stimulation by antigen.  iii  T a b l e o f Contents  PAGE Abstract Abbreviations L i s t of Tables L i s t of Figures Acknowledgements  i i iv v vi v i i  Introduction  1  M a t e r i a l s and Methods  8  Results  14  Discussion  49  References  52  /  iv ABBREVIATIONS  CTL  c y t o l y t i c T lymphocyte(s)  CTLp  c y t o l y t i c T lymphocyte p r e c u r s o r ( s )  T  T helper  H  cell  cell  TCGF  T c e l l growth f a c t o r  (IL-2)  IL  interleukin  rIL-2  human recombinant IL-2  TcR  T cell  IL-2R  IL-2 r e c e p t o r  PMA  4-B-phorbol-12-myristate-13-acetate  ConA  Concanavalin  PKC  p r o t e i n kinase C  KDa  kilodalton  EL4SN  supernatant  FACS  fluorescence activated c e l l  MTT  3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazodium  receptor  (TPA)  A  o f PMA-induced EL4 c e l l s sorter bromide  V  LIST OF TABLES  PAGE Table 1  Ontogeny o f the response  Table 2  EL4SN e f f e c t  Table 3  IL-2 a l o n e does not induce CTL  Table 4  EL4SN e f f e c t  Table 5  EL4SN induces IL-2R e x p r e s s i o n and  i s not due  i s not due  responsiveness Table 6  The  t o F23.1  15  t o IL-2, but r e q u i r e s IL-2 i n t h i s system  t o IL-4 o r IL-5 IL-2  28 Thyl Lyt2 L3T4~ +  +  n y l o n wool nonadherent c e l l Table 7  A 27KDa f a c t o r  induces IL-2 r e s p o n s i v e n e s s  IL-2R e x p r e s s i o n  19 21  i n thymocytes  t a r g e t c e l l o f EL4SN i s a  18  33 and 40  vi  LIST OF  FIGURES  PAGE Figure 1  Classical  pathway o f CTL i n d u c t i o n  Figure 2  EL4SN i n c r e a s e s F23.1 e x p r e s s i o n on thymocytes  Figure 3  EL4SN induces  Figure 4  EL4SN p o l y c l o n a l l y a c t i v a t e s CTL  Figure 5  EL4SN induces  Figure 6  A 27KDa f a c t o r induces  Figure 7  FPLC-superose column e l u t i o n p r o f i l e f o r EL4SN  Figure 8  S-200 column e l u t i o n p r o f i l e :  e x p r e s s i o n o f the IL-2R on thymocytes  IL-2R e x p r e s s i o n on L y t 2  from C T L - i n d u c i n g Figure 9  2 24 ....  26 30  +  cells  CTL a c t i v i t y  35 37 42  t o remove albumin  activity  FPLC-Mono Q column on albumin-depleted  44 EL4SN  46  vii  ACKNOWLEDGEMENTS  I support  would  like  to  and g u i d a n c e ,  suggestions.  I  D.  Zecchini for  R.  Bauzon f o r  e x p r e s s my a p p r e c i a t i o n t o as w e l l  as D r s .  would a l s o l i k e operating  typing  this  the  to  FACS,  thesis.  R.  thank E.  Dr.  H.-S.  McMaster and F . P . C . Kwong f o r  Kwan f o r  Teh f o r Takei  all  running the  her  for  his their  help,  FPLC,  and  1  INTRODUCTION  i.  Classical Pathway of CTL Induction The purpose of this investigation was to study the process of  differentiation of CTL precursors into CTL effectors.  Generally, the  induction of CTL involves several specific signals mediated through cellular interactions and soluble factors.  In the classical pathway of  CTL induction, the allogeneic response can be depicted as in Figure 1. The MHC class II (Ia) expressing accessory cells secrete IL-1 which induces the activation of the T c e l l (1-4).  (For simplicity,  H  nonallogeneic accessory cells are not included in the diagram.) The activated T c e l l proliferates and secretes IL-2. H  IL-2 induces  activated CTLp to proliferate and differentiate into CTL effectors (5-10).  Activation of CTLp in this pathway involves the triggering of the  antigen receptor by the class I molecule on the accessory c e l l .  Of  course, CTLp may be activated by other allogeneic cells through recognition of the foreign class I molecule. There are instances in which CTLp cannot be activated through the classical pathway.  For example, CTLp which have not yet encountered  antigen or mitogen do not express IL-2 receptors (IL-2R), and therefore cannot respond to IL-2.  As well, murine fetal thymocytes prior to day  seventeen of gestation (dl7) do not express the T c e l l receptor (TcR) (11-13), and therefore cannot respond to antigen triggering.  In cases  such as these, IL-2 alone is insufficient to induce proliferation and differentiation of CTLp.  2  Figure 1.  The classical pathway of CTL activation.  3  4  ii.  The  A l t e r n a t i v e Pathway o f T C e l l  Activation  In a d d i t i o n to the c l a s s i c a l antigen-dependent pathway o f T a c t i v a t i o n , a l t e r n a t i v e antigen-independent mechanisms o f T a c t i v a t i o n have been r e p o r t e d Reinherz described the TcR  effect  binding  glycoprotein  T i l can i s IL-1  t r i g g e r T c e l l p r o l i f e r a t i o n and and  macrophage independent. l i g a n d f o r t h i s pathway.  IL-2  T i l is  l a c k the TcR,  a n t i - T i l stimulation a l t h o u g h the  IL-2  d i d not  receptors  Antibodies the  Presumably t h e r e i s an as  yet  I t i s also of i n t e r e s t  s u g g e s t i n g the  a l t e r n a t i v e pathway mediated through T i l .  in  p r o d u c t i o n , and  i s p r e s e n t on v i r t u a l l y a l l thymocytes i n c l u d i n g the  populations that  Unlike  that  l e s s mature  importance o f  the  peripheral T  r e s u l t i n p r o d u c t i o n o f IL-2  cells,  i n thymocytes,  are f u l l y f u n c t i o n a l i n t h a t p r o l i f e r a t i o n  merely depends on the presence o f exogenous  IL-2.  There are a l s o examples o f c e l l u l a r r e c e p t o r s can  (14-16).  d i f f e r e n t i a t i o n than the nominal a n t i g e n r e c e p t o r .  undefined n a t u r a l Til  human T c e l l a c t i v a t i o n mediated through e i t h e r  component o f an a l t e r n a t i v e pathway which appears e a r l i e r  intrathymic against  cell  (14-23).  or the T i l sheep e r y t h r o c y t e  a surface  cell  on murine T c e l l s which  respond to c r o s s l i n k i n g , r e s u l t i n g i n the a c t i v a t i o n o f r e s t i n g T  cells. (17).  C e r t a i n monoclonal a n t i - T h y - 1 a n t i b o d i e s Furthermore, the  on T and  B cells,  12-KDa TAP  can be  a c t i v a t i o n response  (18).  can  activate resting  CTL  p r o t e i n , which i s p r i m a r i l y expressed  c r o s s l i n k e d w i t h an ensuing s t r o n g  T  cell  5  It has recently been noted that the requirement of TcR triggering can be replaced by the phorbol ester, PMA (19-23).  PMA can induce an  increased expression of IL-2 receptor (IL-2R) on T cells, while PMA plus calcium ionophore can result in IL-2 production, followed by proliferation and differentiation into CTL.  The significance of this observation is  that i t demonstrates an alternative mechanism for T c e l l activation. Protein kinase C (PKC) is activated when a T c e l l is stimulated by antigen, mitogen, or PMA.  Classical activation involves the interaction  of a membrane receptor with a corresponding ligand, triggering the hydrolysis of phosphatidylinositol phosphates, which leads subsequently to the mobilization of calcium ions and PKC activation. also involved in the activation of PKC. antigen to activate PKC.  Accessory cells are  PMA bypasses the requirement for  This study is concerned with a factor that can  induce normal adult T cells to differentiate in the complete absence of antigen or mitogen.  IL-2 is the only additional requirement the factor  needs to induce CTL activity.  Furthermore, this factor can induce CTL in  fetal thymocytes which do not yet express the T c e l l receptors (TcR).  It  is suggested that this factor bypasses the requirement of antigen-triggering by mediating its effect through an unknown cellular receptor.  iii.  Antigen-Nonspecific Factors Raulet and Bevan were the f i r s t to describe a factor other than IL-2  that could activate CTLp (24).  The factor they described was called CDF  (CTL differentiation factor), and was present in the supematants (SN) of Concanavalin A (ConA) activated mouse spleen cells (ConASN).  CDF is a  6  pH2-sensitive molecule (unlike IL-2) that acted on resting CTL precursors of the Lyt2 phenotype and in the presence of IL-2 induced CTL +  activity.  Falk et a l . (25) also described a factor which they called TCF1  (T c e l l cytotoxicity inducing factor) which induced the proliferation and differentiation of thymocytes in an IL-2-dependent fashion.  They isolated  TCF1 from the supernatants of ConA activated spleen (32), K15 hybridoma cells (25) as well as PMA-induced EL4 cells (33).  They showed that TCF1  induced IL-2R expression on thymocytes in the presence of IL-2 (25). Wagner et a l . also studied CTL differentiation and described a factor CTDF (CTL differentiation factor) that converted proliferating CTLp into CTL effectors ConASN.  (26).  CTDF acted on Lyt2 cells and could be found in +  A final example from a rather large set of reported factors is  that of Takai et a l . (27).  They described a CDF activity that was present  in rat ConASN and synergized with IL-2 to induce CTL.  The factor eluted  at 31KDa on a G-100 column, and acted on the Lyt2 c e l l population. +  Recently, two new interleukins, IL-4 and IL-5, have been defined which have varied effects on T cells.  IL-4 (BSF-1) has several activities:  it  allows activated B lymphocytes to switch from IgM to IgG secretion; i t increases surface levels of l a antigens and increases the size of B cells; and i t supports the growth of some factor-dependent c e l l lines (28,29). In the presence of PMA, IL-4 induces differentiation of intrathymic precursors into CTL in an IL-2-independent mechanism (29).  IL-5 (T cell  replacing factor) has also been shown to have activity on T cells.  In the  presence of suboptimal levels of IL-2 and ConA, IL-5 can induce proliferation of resting thymocytes (30).  IL-5 has also been shown to act  7  on B c e l l s , inducing immunoglobulin secretion (30). This  investigation  involves a factor that can act on resting CTL, and has a variety of s i m i l a r i t i e s and differences with other described  factors (24-27; 31-41).  8  MATERIALS AND METHODS  1.  Mice C57BL/6J (B6), DBA/2J (D2), Balb/cJ, CBA/J (CBA), and B6 x D2)F1  (BDF1) mice were bred in the Animal Unit of this department.  2.  Culture Conditions Iscove's Modified Dvilbecco's Medium (Gibco, Grand Island, New York)  supplemented with 10% fetal calf serum (FCS) (Bockneck, Rexdale, Ontario), _5 5 x 10  M 2-mercaptoethanol (2-Me) (Sigma Chemical Co., St. Louis, MO),  50 U/ml penicillin and 50 yg/ml streptomycin (Gibco) was used for a l l cultures and assays.  Cultures were incubated at 37°C with 5% CO,, in  humid a i r . 3•  Cell Preparations Organs are asceptically dissected, then forced through a seive to form  a single c e l l suspension.  Thymocytes and lymph node cells were then  washed three times in cold phosphate buffered saline (PBS).  Spleen cells  were f i r s t treated with ammonium chloride to lyse red blood cells prior to washing.  4.  Lymphoklnes a.  To prepare factor for biological studies, 10^ EL4 cells/ml were  incubated for 24 hr in a total volume of 50 ml in a 250 ml flask (Falcon #3024, Becton Dickinson, Labware, CA). The medium used was RPMI 1640  9  (Gibco)  supplemented w i t h 5% FCS,  ng/ml PMA  (Sigma).  The  of  B6 o r i g i n and was  of  Biochemistry,  HB101 and  HEPES (Sigma), a n t i b i o t i c s , and  c e l l l i n e i s a benzopyridine-induced  a kind g i f t  o f Dr. Verner  s t u d i e s , 10^ EL4  Research P r o d u c t s )  b.  lymphoma  prepare  c e l l s / m l were i n c u b a t e d  i n the presence o f 10 ng/ml PMA  in  HB101  f o r 24  hr.  transferin  Both p r e p a r a t i o n s o f f a c t o r proved t o have the same  b i o l o g i c a l a c t i v i t i e s , u s i n g the assays Human recombinant IL-2  described i n this  pure as assayed  by SDS-PAGE and  x 10^ u n i t s .  One  study.  ( l o t #LP-315 from E. c o l i ) was  p r o v i d e d by Cetus C o r p o r a t i o n , E m e r y v i l l e , CA  1.5  To  i s a serum-free medium w i t h d e f i n e d amounts o f albumin, insulin.  10  Paetkau o f the Department  U n i v e r s i t y o f A l b e r t a , Edmonton, A l b e r t a .  f a c t o r f o r biochemical medium (NEN  EL4  10 mM  (42).  The  c o n t a i n s l e s s than 0.01  kindly 99%  r I L - 2 was ng endotoxin  u n i t i s d e f i n e d as the amount o f IL-2  per  required to  cause 30% maximal p r o l i f e r a t i o n o f an IL-2-dependent c e l l l i n e c u l t u r e d at 10  4  c e l l s / 0 . 2 ml f o r 48 c.  The  rIL-4  hr.  (BSF-1) was  a kind g i f t  DNAX Research I n s t i t u t e o f M o l e c u l a r and  o f Dr. Timothy Mosmann o f  the  C e l l u l a r Biology, Palo A l t o ,  CA  (28). d.  The  r I L - 5 was  k i n d l y p r o v i d e d by Dr. Vern Paetkau.  This  factor  has  i d e n t i c a l p r o p e r t i e s t o t h a t d e s c r i b e d by K i n a s h i e t a l . (30).  5.  C e l l L i n e s and Monoclonal A n t i b o d i e s a.  The  B c e l l hybridoma l i n e s p r o d u c i n g  m o l e c u l e ( J l j ) (42), the IL-2 m o l e c u l e (GK1.5) (45), and  r e c e p t o r (7D4)  MAbs a g a i n s t the Thy-1.2 (44), the L3T4  cell  surface  the mouse L y t 2 molecule (TIB105) (46) were  o b t a i n e d from the American Type C u l t u r e  Collection.  10  b.  The F23.1 hybridoma c e l l line, which produces an IgG2a MAb against  a variable region determinant of the 8 chain of the TcR was a gift of Dr. Mike Bevan of the Scripps Clinic and Research Foundation, La Jolla, CA (47,48). c.  The 11B11 hybridoma line which produces MAb against IL-4 was  kindly provided by Dr. B i l l Paul of the National Institute of Health, Bethesda, MD (49)-  Sources of these MAbs for assays and FACS analyses  include concentrated spent culture supernatant or the 50% ammonium sulfate precipitates of the relevant ascitic fluids.  Relative protein  concentrations were determined by the approximation: mg/ml protein.  ^280 ^'^  =  For proliferation assays, F23.1 was coupled to Affigel  beads according to the supplier's (Bio-Rad, Richmond, California) protocol. d.  The FITC labelled antibodies were purchased from Zymed  Laboratories, Dimension Labs, Mississauga, Ont.  Because the  FITS-rabbit-anti-mouse IgG (gamma chain specific) cross reacts extensively with rat IgG, i t was used to detect the binding of GK1.5 and Lyt 2, as well as F23.1.  FITC-rabbit-anti-rat IgM (v chain specific) was used to  detect the binding of 7D4.  6.  Proliferation Assays 4  a.  To determine units/ml of IL-2 activity, 10  02^/0.2 ml were  incubated 48 hr in flat bottomed wells (Flow Labs, Rockville, MD, #76-002-05) with varying dilutions of the sample to be titrated.  The  C2t^ are an IL-2 dependent mouse c e l l line, derived from day 15 fetal thymus, in this lab.  After 2 days, 100 ul of the supernatant was  11  removed from each well and replaced with 20 pi of 2.5 mg/ml MTT/ml (Sigma) (50).  Cultures were incubated 2-4 more hours, then 150 pi of  acidified isopropanol was added to each well with thorough mixing. 0Dg  Q0  values were measured, and the level of IL-2 activity was  determined using a standard curve, constructed with rIL-2.  This assay was  developed by Mosmann (50) as a quick and precise measurement of cellular proliferation.  The method detects living but not dead cells, and results  can be read quickly on a multiwell scanning spectrophotometer. of the assay is the MTT i t s e l f .  The basis  It is a tetrazolium salt, and the  tetrazolium ring is cleaved in active mitochondria resulting in a colour change. b.  Thus, only live cells are detected. 3 5 To determine the extent of c e l l proliferation, 10 to 10  cells were incubated in round bottomed wells (Flow Labs, #76-013-05) in a total volume of 0.2 ml for 2-4 days. 3 0.5 mCi/well of  For the last 6 hr of incubation,  H-thymidine was added to each well.  The cells were  harvested on glass filters (Whatman) and lysed with itt^O.  After drying,  scintillation fluid was added, and the level of incorporation was determined. 7.  Lectin Mediated Cytotoxic T Cell Assay A l l cultures were set up in conical microtitre plates (Flow Labs  #76-023-05) in 0.2 ml volumes, and incubated for 5 days.  One half of each  3 51 well was then tested for CTL by adding 3 x 10  Cr labelled P815  targets in a final concentration of 10 yg/ml PHA-P, and incubating for a  12  f u r t h e r 3-5 released  hr.  The  s u p e m a t a n t s were then h a r v e s t e d  and  tested for  "^Cr. % S p e c i f i c L y s i s = t e s t count - spontaneous count maximum r e l e a s a b l e count - spontaneous count  The  spontaneous counts were g e n e r a l l y l e s s than 10%  r e l e a s a b l e count; the maximum r e l e a s a b l e count was the t o t a l  8.  incorporated  C e l l Surface 0.5  - 1.0  incubated  surface antigen. at  min  w i t h 100  incubation,  c e l l s were washed e x t e n s i v e l y w i t h PBS,  ml o f medium c o n t a i n i n g a n t i b o d i e s  the  cell  For l a b e l l i n g w i t h F23-1, c e l l s were i n c u b a t e d  and  7D4  spent c u l t u r e supernatant  antibodies,  c e l l s were i n c u b a t e d  c e l l s were washed e x t e n s i v e l y i n c o l d PBS, 10 yg/ml F I T C - l a b e l l e d . a n t i b o d y was  p e l l e t , f o l l o w e d by a 45 min.  i n c u b a t i o n on i c e .  30  on i c e f o r  determine the e x t e n t  then one added t o  and  min.  (47,48).  After a final  washing, the c e l l s were suspended i n 0.1-1.0 ml PBS  9.  against  then  ug/ml ammonium s u l f a t e p r e c i p i t a t e d a s c i t i c p r o t e i n .  medium c o n t a i n i n g  of  counts.  37°C i n f i v e times c o n c e n t r a t e d  For L3T4, L y t 2,  c a l c u l a t e d as 90%  Labelling  x 10^  i n 1.0  o f the maximum  analyzed  45  After  ml  of  the thorough to  o f a n t i b o d y b i n d i n g on a FACS IV.  FACS A n a l y s i s Ten  thousand c e l l s o f each sample were t e s t e d f o r bound  a n t i b o d y , w i t h dead c e l l s and between the n e g a t i v e  d e b r i s gated out.  c o n t r o l and  The  the t e 3 t sample was  FITC-labelled  cross-over  point  a r b i t r a r i l y chosen as  13  the cut-off point between positive and negative c e l l s . i l l u s t r a t i o n , log of fluorescence was plotted against cell  number.  10.  FPLC  For purposes of l i n e a r or log of  Samples were loaded onto a Superose 12 column and 0.5 ml fractions were eluted with PBS at a rate of 0.5 ml/min.  S i m i l a r l y , samples were  loaded on a Mono Q column and 1.0 ml fractions were eluted at a rate of 1.0 ml/min i n t r i s - H C l with a l i n e a r gradient  of NaCl.  14  RESULTS  1.  Ontogeny of the Response to F23-1 In order to study the process of CTL induction, i t was of interest to  determine at what stage of development a CTLp becomes responsive to antigen receptor triggering.  The purpose was to discern whether the  ability to differentiate depended solely upon TcR expression, or i f other factors were involved.  A convenient system in which to follow the  maturation process utilized the monoclonal antibody F23-1. recognizes a determinant of the  v  This antibody  g_g chain on the TcR of most strains,  and in the presence of IL-2, can induce T cells to proliferate and differentiate (47,48).  The ontogeny of the TcR was followed as defined by  the ability of T cells to respond to F23.1.  Even though the TcR is  expressed on the surface of fetal thymocytes at dl7 of gestation (11-13), a measurable response to F23.1 was undetectable until approximately one week of postnatal l i f e :  Table 1 summarizes these findings.  The study was  then expanded to include the differentiation factors reported to exist in the supernatant of PMA-induced EL4 cells (EL4SN) (32,33,39,52,53).  As  indicated in Table 1, i t was observed that EL4SN could induce the activation of newborn thymocytes, which are normally nonresponsive to antigen-triggering by F23.1.  Furthermore, dl5 fetal thymocytes, which  were shown using FACS to have virtually no surface TcR (data not shown) could respond to the factor(s) in EL4SN and proliferate.  Of Interest is  the apparent synergy between EL4SN + F23•1 in the newborn thymocytes. This finding suggests that a factor or factor(s) in EL4SN allows the  Table 1. i.  Ontogeny of the response to F23.1  Proliferative Response Balb/C thymocytes 4 weeks  2 weeks  F23.1 rIL-2 F23.1 + rIL-2  2 215 ± 1165 2 947 ± 1398 16 700 ± 4349  2 493 ± 1318 4 634 + 1563 8 392 ± 1200  EL4SN EL4SN+F23.1  68 270 ± 778 73 586 ± 1414  98 931 ± 1200 45 940 ± 7332  ii.  1 week 3 317 ± 1521 4 096 ± 1652 12 191 ± 1466 52 257 ± 1030 55 973 ± 10834  newborn 766 ± 200 2 701 ± 221 3 730 ± 1458 37 032 ± 3501 61 099 ± 14584  Cytotoxic Responses*  5  4 weeks  2 weeks  F23.1 rIL-2 F23.1 + rIL-2  -0.1 ± 2.7 2.2 ± 3.3 61.5 ± 7.4  0.4 ± 1.3 1.9 ± 1.8 13.7 ± 3.5  0.2 ± 2.2 19.5 ± 6.1 27.6 ± 17.6  -2.4 ± 1.1 -1.5 ± 2.9 -1.6 ± 2.1  EL4SN EL4SN + F23.1  71.4 ± 3.5 67.9 ± 2.7  62.9 ± 4.7 62.7 ± 6.2  65.8 ± 6.3 59.9 ± 3.2  59.0 ± 5.0 63.1 ± 3.0  1 week  newborn  dl5 FT 1 471 ± 605 24 572 ± 4778  16  Table 1 (continued)  Thymocytes were c u l t u r e d w i t h 10 ug/ml F23.1 (bound t o A f f i g e l ) , 20 U/ml r I L - 2 , o r 15% v/v EL4SN f o r 4 days, and then t h e c u l t u r e d were assayed f o r p r o l i f e r a t i v e r e s p o n s e s .  cells  The r e s u l t s a r e expressed  as a mean o f 4 c u l t u r e s w i t h t h e s t a n d a r d d e v i a t i o n .  b  Thymocytes were c u l t u r e d w i t h f a c t o r s f o r 5 days, and then assayed f o r c y t o t o x i c responses i n a PHA-P mediated k i l l i n g a s s a y .  The r e s u l t s  are e x p r e s s e d as the mean o f 6 c u l t u r e s w i t h the s t a n d a r d e r r o r .  17  newborn thymocytes t o respond t o a n t i g e n t r i g g e r i n g by F23.1.  The  synergy  4 was  always observed a t low c e l l numbers ( l e s s than 3x10  mouse thymocytes,  a g a i n s u g g e s t i n g t h a t EL4SN i s expanding  p o p u l a t i o n t h a t can respond was  /well) of adult  t o F23.1.  a  cell  Because o f these o b s e r v a t i o n s , i t  d e c i d e d t h a t f u r t h e r study would be c a r r i e d out on the f a c t o r ( s ) i n  EL4SN.  2.  The  I n d u c t i o n o f CTL by EL4SN but not by IL-2, IL-4 o r IL-5  a.  EL4SN e f f e c t  i s not due  t o IL-2, but r e q u i r e s IL-2.  known t h a t EL4SN c o n t a i n s f a c t o r s o t h e r than IL-2 lab  It i s well  (32,33,39,52,53).  Our  has p r e v i o u s l y r e p o r t e d t h a t EL4SN can n o n s p e c i f i c a l l y a c t i v a t e CTL i n  the absence o f a n t i g e n o r mitogen i n an IL-2 dependent f a s h i o n ( 3 1 ) .  This  o b s e r v a t i o n i s confirmed i n T a b l e 2, i n which i t i s shown t h a t t h e r e i s a f a c t o r o t h e r than IL-2 i n EL4SN. e q u i v a l e n t o f 40 U/ml  In t h i s case, 10% EL4SN c o n t a i n e d the  IL-2 a c t i v i t y .  As shown, IL-2 c o u l d not  p r o l i f e r a t i o n or d i f f e r e n t i a t i o n of T c e l l s . high d i l u t i o n s ,  c o u l d a c t i v a t e CTLp.  induce  In c o n t r a s t , EL4SN, even a t  In a d d i t i o n , w h i l e IL-2 alone  cannot  mediate the e f f e c t observed w i t h EL4SN, i t i s c l e a r t h a t IL-2 i s r e q u i r e d i n t h i s process.  A d d i t i o n o f 7D4  a n t i b o d y (which b i n d s t o the IL-2R,  b l o c k i n g IL-2 b i n d i n g ) (44) t o the c u l t u r e s i n h i b i t e d the response  to  EL4SN, whereas e q u a l c o n c e n t r a t i o n s o f an i r r e l e v a n t IgM prepared i n the same way b.  had no e f f e c t on CTL  activity.  IL-2 a l o n e does not induce CTL  i n t h i s system.  It i s well  e s t a b l i s h e d t h a t a t h i g h c e l l d e n s i t y , IL-2 a l o n e can induce CTL (6).  T h i s phenomenon was  observed and i s shown i n T a b l e 3.  activity  However, as  18  Table 2.  B6 o r BDF1 spleen c e l l s  5 X 10  EL4SN e f f e c t  EL4SN % (v/v)  i s n o t due t o  rIL-2 U/ml  J  H-TdR Incorporation CPM ± SE  741 + 62  3  3  16 585 + 1275  5  3 251 + 589  2.5  1 112 + 223  3  + 3.1  2.6 + 0.8  1 0 3 5 + 655  20  2 925 + 1446  10  1 4 0 1 + 880  0.9 + 0.6 - 0 . 6 + 0.7 2.4 + 0.7  86  0.0 + 0.8  N o t done  14.9 + 8.1  p g / m l 7D4  Not done  45.8 + 4.0  10% E L 4 S N + 10 P g / m l 7D4  N o t .done  54.1 + 5.6  10% E L 4 S N + 100 p g / m l 10% E L 4 S N + 30  Culture  72.0 + 2.9  5 . 1 + 1.6  40  5  5 X 10  % Specific Lysis M ± SE  27.3  830 + 89  507 +  IL-2  1.4 4 0 . 5  10  1.25  5 X 10  I L - 2 , but r e q u i r e s  and a s s a y c o n d i t i o n s  7D4  as d e s c r i b e d i n Table  1.  19  TABLE 3.  IL-2 alone does not induce CTL in this system'  EL4SN  BDF1 spl  rIL-2  4 4 x 10 4 2 x 10* 1 x 10  CPM ± SE  % specific lysis  1 973  +  136  2.4 ± 1.8  886  +  58  1.2 ± 1.8  4  567  +  48  0.0 ± 1.5  3  1 262  +  786  1.7 ± 1.1  1 349  +  457  1.0 ± 1.1  10%  49 238  +  811  84.1 ± 5.4  592  81.7 ± 4.7  5 x 10  2.5 x 10  3  4 4 x 10 4 2 x 10* 1 x 10  10%  28 126  +  4  10%  17 867  +  725  81.7 ± 4.3  3  10%  9 196  +  128  77.0 ± 8.9  10%  5 384  +  536  54.8 ± 8.9  5 x 10  2.5 x 10  3  4 4 x 10 2 x 10  20 U/ml  5  015  +  450  58.0 ± 8.1  20 U/ml  1 107  +  67  31.2 ± 8.0  4  1 x 10  20 U/ml  847  +  321  8.0 ± 3.5  3  x 10  20 U/ml  975  +  41  6.0 ± 3.1  20 U/ml  499  +  38  0.0 ± 0.7  4  5  2.5 x 10  3  Culture and assay conditions as described in Table 1.  '  20  the c e l l number was lowered into the range used for the assays, 3  approximately 5 x 10 /well, this IL-2 effect was not observed.  This  observation suggests that IL-2 responsive cells exist in low number in normal spleen, and contribute l i t t l e or not at a l l to a proliferative or cytotoxic response in low density c e l l cultures.  It appears that EL4SN  induces IL-2 nonresponsive cells to become IL-2 responsive, and thus proliferate and differentiate in the presence of IL-2. c.  EL4SN effect is not due to IL-4 or IL-5. As mentioned, IL-4 and  IL-5 have recently been shown to have some effects upon T cells.  As well,  it is reported that these interleukins exist in EL4SN (Paetkau, unpublished results, 54).  Both these factors were tested for the ability  to induce CTL in the presence of large amounts of rIL-2, and were ineffective (Table 4).  The IL-5 that we received was shown to be active 3  using BCL1 cells in a 2 day assay with a 4 hr not shown).  H-thymidine label (data  Although the EL4SN could induce BCL1 cells to proliferate,  this activity can be attributed to the IL-2 present in the EL4SN.  When  one compared various dilutions of EL4SN with the equivalent concentrations of rIL-2, there was no difference in the extent of BCL1 proliferation.  To  ensure that the IL-5 and IL-4 did not require any trace amounts of PMA that may remain in the EL4SN, this aspect was also investigated (Table 4).  It is evident that the PMA + IL-2 effect (11.0% specific lysis) is  minimal compared to the effect of the EL4SN (46.4%).  Addition of IL-4 or  IL-5 to PMA, or PMA + IL-2, had no effect above the minimal CTL activity observed with PMA + IL-2 alone.  Finally, a monoclonal antibody against  IL-4, 11B11, did not have any effect on the ability of EL4SN to induce CTL activity.  Table 4. BDF1 s p l  rIL-2  rIL-5  EL4SN effect i s not due to IL-4 or I L - 5 rIL-4  C  HBll  d  EL4SN  U/ml  PMA ng/ml  % specific lysis M ± SE  10%  46.4 + 10.0  25% SN  10%  41.0 + 12.6  12.5% SN  10%  38.4 + 11.0  10 vg/ml asc  10%  43.4 + 13.3  3 pg/ ml asc  10%  46.8 + 10.6 2.2  +  2.4  2.1  +  1.7  2.8  +  3.7  1/10  -0.3  +  1.3  1/10  -1.1  +  2.9  20 100 20  10  a  100  10  0.1  11.0  +  3.9  10  1.0  5.7  +  2.4  10  30  0.1  15.1  +  7.4  10  30  1.0  17.6  +  6.2  10  1/30  0.1  5.9  +  1.0  10  1/30  1.0  6.4  +  7.2  22  Table 4 (continued)  C u l t u r e and assay c o n d i t i o n s as d e s c r i b e d i n T a b l e 1.  ^ r I L - 5 was o b t a i n e d by t r a n s l a t i o n o f a c l o n e d cDNA i n COS c e l l s and the COS supernatant, when used a t 10% v/v, was a b l e t o induce the  5 p r o l i f e r a t i o n o f BCL  cells  (30); Acpm = 51,985 f o r 1.5 x 10  c e l l s / 0 . 2 ml a f t e r a 2 day c u l t u r e  period.  COS supernatant o f t h e IL-5  cDNA c l o n e , t r a n s c r i b e d i n t h e r e v e r s e d i r e c t i o n , had no a c t i v i t y BCL^  assay.  The a c t i v i t y o f r I L - 4 was determined  d  i n the  as p r e v i o u s l y d e s c r i b e d ( 6 1 ) .  T h e 11B11 a n t i b o d y (49) was used as e i t h e r spent c u l t u r e  supernatant  (SN) o r as t h e ammonium s u l f a t e p r e c i p i t a t e o f a s c i t i c p r o t e i n  (asc).  The d a t a p r e s e n t e d i s r e p r e s e n t a t i v e o f s e v e r a l e x t e n s i v e t i t r a t i o n s . ( r I L 5 was t e s t e d a t d i l u t i o n s v a r y i n g from 1/10 t o 1/100. t e s t e d a t 1.25 U/ml t o 100 07ml).  r I L 4 was  23  3.  Mechanism o f A c t i o n o f EL4SN a.  EL4SN induces the e x p r e s s i o n  o f IL-2R and  apparent a b i l i t y o f EL4SN to induce IL-2 population, cellular  i t was  Because o f  responsiveness  the  in a cell  o f i n t e r e s t to determine the e f f e c t o f EL4SN upon  receptor expression.  frequency and  TcR.  The  first  d e n s i t y o f the F23.1  o b s e r v a t i o n was  that  the  determinant on thymocytes  upon i n c u b a t i o n w i t h  15% EL4SN.  F i g u r e 2.  days c u l t u r e s i n 15% EL4SN, 30-40% o f the  A f t e r 2-3  The  increased  FACS a n a l y s i s i s r e p r e s e n t e d  in  original  5 5x10  c e l l s / m l remained v i a b l e .  were 12.9% one  F 2 3 . 1 , and were 25.2% +  week o l d B a l b / c  culture. cells  I n i t i a l l y , 4 week o l d B a l b / c F23.1  +  after culture.  thymocytes i n c r e a s e d from 6.4%  An a d d i t i o n a l p o i n t was  i n the 4 week (12.9%) and  The  Similarly,  t o 20.0%  the d i f f e r e n t f r e q u e n c i e s  w i t h age  F23.1  +  the  shown t h a t the b l a s t s e x p r e s s e d the IL-2R.  c u l t u r e d i n IL-2  alone had  +  IL-2. to  Selective gating  used t o d i s t i n g u i s h between the l a r g e a c t i v a t e d b l a s t s , and  cells  of F23.1  c e l l s a l s o i n c r e a s e d from 2.8  when the c e l l s were c u l t u r e d i n EL4SN ( F i g u r e 3 ) .  i t was  after  +  accounted f o r the  i n c r e a s e i n response t o s t i m u l a t i o n w i t h  number o f IL-2R e x p r e s s i n g  c e l l s , and  F23.1  1 week o l d (6.4%) mouse thymocytes.  Perhaps the i n c r e a s e i n frequency o f F23.1 corresponding  thymocytes  7.2% was  small  In c o n t r a s t ,  no e f f e c t on c e l l s i z e or IL-2R  expression. b.  EL4SN induces r e s p o n s i v e n e s s  EL4SN f o r 2-3 IL-2  days had  t o IL-2.  i n c r e a s e d IL-2R e x p r e s s i o n ,  i n a p r o l i f e r a t i v e f a s h i o n (Table 5 ) .  little,  i f any,  Thymocytes i n c u b a t e d  IL-2R (5,55,56) and  and  c o u l d respond to  Normal f r e s h c e l l s  c o u l d not  in  respond t o IL-2.  expressed As  24  Figure 2.  EL4SN increases F23.1 expression on thymocytes.  In a l l FACS analysis, the crossover point between the control and test curves was arbitrarily chosen as the cut-off point for positive and negative cells.  The X-axis of these analyses is defined as the log of  fluorescence, with the Y-axis as the linear of c e l l number.  4 week old Balb/c thymocytes were 12.9% F23.1 (i) which became 1.8% +  after 2 day culture in 10 U/ml rIL-2 ( i i ) and 25.2% after 2 day culture in 15% EL4SN ( i i i ) .  1 week old Balb/c thymocytes were 6.4% F23.1 (iv) which changed to 2.2% +  after 2 day culture in IL-2 (v) and 20.0% after 2 day culture in EL4SN (vi).  (i)  (iv)  •  u\ •  fa*  (iii)  (vi)  i  26  Figure 3.  a.  EL4SN induces the expression of the IL-2R on thymocytes.  In this figure, the X-axis is defined as the log of fluoresence and the Y-axis as the log of c e l l number.  4 week old Balb/c thymocytes were cultured 3 days in 15% EL4SN resulting in 7.2% of the surviving cells expressing IL-2R ( i ) .  EL4SN  induced blast formation, and 15.9% of these large cells expressed the IL-2R, whereas 0.9% of the small cells did ( i i ) .  Culturing cells in  IL-2 alone had no effect on the IL-2R (only 13% are 7D4 ) ( i i i ) . +  6.0% of one week old Balb/c thymocytes expressed the IL-2R after culture in 15% EL4SN for 3 days (iv), whereas 1.3% expressed the IL-2R after culture in rIL-2 (v).  27 (i)  V  (iv)  *AV.  ••JV/iV. . t  (v)  (ii)  1  (iii)  -^JP  '  TABLE 5.  EL4SN induces IL-2R expression and IL-2 responsiveness in thymocytes  Balb/c  FACS Analysis  10 U/ml IL-2  15% EL4SN  F23.1  fresh  12.6  2.8  6 318 ± 7 698  53 819 ± 6 123  1.8  1.3  3 758 ± 3 063  43 807 ± 9 421  30.0  7.2  56 094 ± 8 112  74 837 ± 2 595  cult. 2d in IL-2 cult. 2d in EL4SN large blasts small cells  7D4  H-TdR Incorporation in response to:  thymocytes  +  +  15.9 0.9  thymocytes were cultured in either 10 U/ml rIL-2 or 15% v/v EL4SN for 2 days. The cells were then tested for surface markers and for the ability to respond to IL-2 or EL4SN.  29  expected, c u l t u r i n g c e l l s to respond t o IL-2.  i n IL-2 a l o n e had no e f f e c t on the c e l l s  ability  In c o n t r a s t t o these r e s u l t s , the i n c r e a s e i n  F23.1  determinant had no c o r r e s p o n d i n g i n c r e a s e i n a b i l i t y t o respond t o F23.1  +  IL-2.  I t may  be i n f e r r e d t h a t EL4SN i s a c t i v a t i n g the T c e l l s t o  express more h i g h a f f i n i t y IL-2R, and F23.1 as a r e s u l t o f t h i s a c t i v a t i o n .  e x p r e s s i o n was  I t i s u n c l e a r whether EL4SN was  i n d u c i n g the e x p r e s s i o n o f IL-2R i n CTLp, o r i f i t was IL-2R  +  CTLp, expanding  also increased  this population.  actually  a c t i n g on the  Both p o s s i b i l i t i e s  result  few  i n an  i n c r e a s e i n IL-2 r e s p o n s i v e c e l l s . c.  EL4SN p o l y c l o n a l l y a c t i v a t e s CTLs.  The above d a t a suggested  a l a r g e p r o p o r t i o n o f the p o p u l a t i o n can respond t o EL4SN: supernatant was  r e p r e s e n t a t i v e experiment, 100% o f thymocytes, 7D4 . +  r a t ConA  o n l y s l i g h t l y more e f f e c t i v e i n a c t i v a t i n g T c e l l s  express IL-2R as compared t o EL4SN ( d a t a not shown).  that  to  In one  ConA SN induced b l a s t f o r m a t i o n i n v i r t u a l l y  which a f t e r 2 day c u l t u r e i n ConASN were  17.9%  S i m i l a r l y , 2 day c u l t u r e i n EL4SN induced a p p r o x i m a t e l y 40% o f  thymocytes  t o form b l a s t s , and o f t h e s e , 15.9%  expressed the IL-2R.  To  determine whether CTLp o f a l l s p e c i f i c i t i e s were a c t i v a t e d by EL4SN, B6 4 s p l e e n c e l l s were c u l t u r e d a t 10 /0.2  ml i n c o n i c a l w e l l s (the low  cell  d e n s i t y ensures no a c t i v a t i o n due t o I L - 2 ) , and then t e s t e d f o r the a b i l i t y to k i l l  a f t e r 5 days, ( F i g u r e 4 ) . 3 51  v a r i o u s r a t i o s t o 5 x 10 cytotoxic a c t i v i t y w e l l as P815 total  d  C r - l a b e l l e d t a r g e t s , and assayed f o r  against self/B6  (H-2 ).  The e f f e c t o r s were added i n  (H-2 ), b  CBA  A d d i t i o n o f PHA-P t o P815  ( H - 2 ) , D2 k  (H-2 ), d  t a r g e t s determines  number o f k i l l e r s p r e s e n t (57,58), which was  as the  found t o be over a  30  F i g u r e 4.  EL4SN p o l y c l o n a l l y a c t i v a t e s  B6 s p l e e n c e l l s were c u l t u r e d plates  4 a t 10 /0.2 ml.  CTL.  w i t h 15% EL4SN i n V-bottomed m i c r o t i t r e  A f t e r 5 days, c e l l s from e l e v e n p l a t e s  were  51 pooled and assayed f o r c y t o t o x i c i t y a g a i n s t target density  cells.  Blast  the i n d i c a t e d  c e l l s were p r e p a r e d by i n c u b a t i n g  spleen c e l l s at a  o f l x l O ^ / m l w i t h 2 ug/ml ConA, two days p r i o r t o u s e .  where added, was a t a f i n a l c o n c e n t r a t i o n o f 10 ug/ml. 3 51 5x10  Cr-labelled  Cr-labelled targets  were added t o each w e l l .  PHA-P,  A t o t a l of  3 1  Effector/Target  ratio  32  hundred f o l d more f r e q u e n t than those t h a t c o u l d k i l l target.  As expected,  any  specific  k i l l e r s a c t i v a t e d a g a i n s t s e l f were r a r e i n the  p o p u l a t i o n , and r e l a t i v e l y e q u a l numbers o f k i l l e r s were a c t i v a t e d a g a i n s t the s p e c i f i c t a r g e t s .  These r e s u l t s were s i m i l a r t o those expected f o r  ConA a c t i v a t e d e f f e c t o r s , s u g g e s t i n g t h a t EL4SN can p o l y c l o n a l l y CTL  i n the absence o f a n t i g e n o r mitogen (58).  4.  The T a r g e t C e l l o f EL4SN a.  The  target c e l l  nonadherent c e l l .  The  is a Thyl  +  activate  L y t 2 * L3T4~ n y l o n wool  t a r g e t c e l l f o r EL4SN was  determined  lymph node (LN) c e l l s over a n y l o n wool column (NW)  by p a s s i n g  (59), and  B6  then  t r e a t i n g the nonadherent c e l l s w i t h monoclonal a n t i b o d i e s (MAbs) a g a i n s t the s u r f a c e molecules  T h y l , L y t 2 , o r L3T4, i n the presence  ( C ) , t o s e l e c t i v e l y remove c e r t a i n T c e l l a n t i b o d y p l u s C treatment 100% e f f e c t i v e  was  subsets.  t e s t e d u s i n g FACS, and  shown t o be  removal o f n y l o n wool adherent induction.  L3T4  +  Lyt2  the response. +  As shown i n T a b l e  c e l l s and/or L 3 T 4  In c o n t r a s t , treatment  a n t i b o d i e s , abrogated EL4SN i s a T h y l  virtually +  These t r e a t e d c e l l s were c u l t u r e d 3 days i n 15% EL4SN and  then t e s t e d f o r CTL a c t i v i t y i n a 5 day assay.  b.  e f f i c i e n c y o f the  (treatment w i t h <xL3T4 + C r e s u l t e d i n o n l y 3.0%  contaminants).  CTL  The  o f complement  c e l l s had no e f f e c t  with a n t i - L y t 2 or  I t was  L3T4~ n y l o n wool (NW)  concluded  +  cells.  o b t a i n e d when NW  on  anti-Thy-1  t h a t the t a r g e t o f  nonadherent  EL4SN induces IL-2R e x p r e s s i o n on L y t 2  c o n f i r m a t i o n o f the above d a t a was  +  6,  cell. Further  nonadherent c e l l s were  t r e a t e d w i t h a n t i - L 3 T 4 + C, and then c u l t u r e d 3 days i n EL4SN.  In the  33  Table 6. B6 LN  The target c e l l for EL4SN is a Thy-1 Lyt2 L3T4 +  Treatment  EL4SN  H-TdR incorporation  10% (v/v)  (CPM ± S.E.)  +  cell  % specific  lysis  (M ± S.E.)  3 X io  4  -  282 + 123  -1.3 + 1.2  1 X io  4  -  710 + 117  -0.7 + 1.2  3 X io  3  -  1,164 + 120  -0.2 + 2.2  3 X 10*  Untreated  +  17,426 + 1311  54.0 + 6.6  1 X 10  4  Untreated  +  5,021 + 579  34.9 + 9.6  3 X 10  3  +  2,272 + 203  3.0 + 2.1  3 X 10  4  +  14,065 + 5746  39.8 + 4.3  1 X 10  4  +  3,236 + 578  12.2 + 3.9  3 X io  3  +  2,379 + 459  0.4 + 1.0  3 X io  4  +  2,347 + 1019  -1.7 + 1.2  1 X 10  4  +  3,309 + 1616  -2.1 + 0.9  3 X 10  3  +  1,914 + 1255  -1.6 + 1.6  3 X 10  4  +  2,194 + 54  -1.2 + 1.9  1 X 10  4  +  2,033 + 706  -1.1 + 1.5  +  1,080 + 478  0.2 + 0.4  +  18,562 + 978  35.1 + 1.1  3 X IO  NW, C  NW, aThy-1 + C  NW, aLyt2 + C  3  NW, aL3T4+C  3 X 10  4  1 X 10  4  +  5,827 + 1050  5.0 + 2.3  3 X io  3  +  1,923 + 123  -0.6 + 1.3  B6 lymph node cells were passed over a nylon wool column to remove adherent cells. The indicated number of nonadherent cells were then treated with MAbs to Thy-1.2, Lyt2 or L3T4 and low-tox rabbit complement (Cedarlane, Hornby, Ontario) before culturing with 10% EL4SN.  The proliferative and cytotoxic responses were  determined as described in Table 1. Nylon wool column was run by C. Hamilton.  34  course o f t h i s i n c u b a t i o n , the c e l l number i n c r e a s e d 8 f o l d ,  indicating  t h a t the c e l l s  +  L 3 T 4 , 2.7% +  a n a l y s i s was  t h a t s u r v i v e d the a n t i b o d y treatment  7D4 , 14.4%  F23.1 ) responded  +  performed  +  w e l l , 32.0%  were 7D4  +  w e l l t o EL4SN.  on the c u l t u r e d c e l l s  c u l t u r e d c e l l s expressed and  (84% L y t 2 ,  17.9%  FACS  (Figure 5).  the Lyt2 marker and o n l y 2.6% were F 2 3 . 1 . +  78.0%  +  o f the  expressed L3T4.  I t appeared  c o u l d induce the e x p r e s s i o n o f the IL-2R on L y t 2  3.0%  L3T4  -  As  t h a t EL4SN cells,  a l l o w i n g them t o p r o l i f e r a t e and d i f f e r e n t i a t e i n t o CTL.  In c o n t r a s t ,  c e l l s c u l t u r e d i n IL-2 a l o n e under the same c o n d i t i o n s d i d not  proliferate  o r d i f f e r e n t i a t e , and d i d not g i v e r i s e t o IL-2R e x p r e s s i n g c e l l s  (data  not shown). 5.  B i o c h e m i s t r y o f BL4SN a.  The  ml o f 20X  CTL  i n d u c i n g a c t i v i t y i s a 27KDa IL-2-dependent f a c t o r .  c o n c e n t r a t e d EL4SN was  ml/hr, c o l l e c t i n g 1.2 f r a c t i o n was  loaded onto a G-100  ml f r a c t i o n s  measured, and then f i l t e r  t e s t e d f o r IL-2 a c t i v i t y i n the MTT an apparent CTL  (Figure 6).  sterilized.  assay, and  m o l e c u l a r weight o f 40 KDa.  i n d u c i n g a c t i v i t y w i t h and without  i n the assay, the CTL weight o f 27 KDa.  The  One  column and run a t 0D  O Q r i  o f each  Each f r a c t i o n  i t was  1.3  was  shown t o e l u t e w i t h  F r a c t i o n s were a l s o t e s t e d f o r exogenous IL-2.  With IL-2 i n c l u d e d  i n d u c i n g a c t i v i t y e l u t e d w i t h an apparent  When no exogenous IL-2 was  molecular  added, CTL were o n l y  induced  by the f r a c t i o n s t h a t c o n t a i n e d both the EL4SN a c t i v i t y and IL-2  ( d a t a not  shown).  7D4  T h i s o b s e r v a t i o n supports the r e s u l t s o b t a i n e d w i t h the  a n t i b o d y c o n c l u d i n g t h a t the CTL a c t i v i t y r e q u i r e s IL-2. b.  The  27KDa f a c t o r induces CTL and  IL-2 r e s p o n s i v e n e s s .  been shown t o c o n t a i n numerous f a c t o r s w i t h v a r i o u s a c t i v i t i e s .  EL4SN has It  was  35  Figure  5.  EL4SN induces IL-2R e x p r e s s i o n  Nylon wool nonadherent B6  LN  on L y t 2  +  cells.  c e l l s were t r e a t e d w i t h GK1.5  mAb  and  5 complement, and  then c u l t u r e d at 10  m i c r o t i t r e p l a t e s w i t h 10% h a r v e s t e d and  c e l l s / 0 . 2 ml  EL4SN added.  tested for c e l l  +  L3T4  +  and  17.9%  A f t e r 3 days, the c e l l s were  s u r f a c e molecules as d e s c r i b e d  A f t e r c u l t u r e FACS a n a l y s i s showed 78.0% 7D4 , 2.6%  i n round bottomed  F23.1 . +  o f the c e l l s  i n Figure  t o be L y t 2 , +  2.  32.0%  37  F i g u r e 6.  A 27KDa f a c t o r induces CTL  EL4SN was  prepared  activity.  (as d i s c u s s e d i n M a t e r i a l s and Methods) i n  medium, c o n c e n t r a t e d 20 times, and 1 ml was column. 1.2  The  column was  ml f r a c t i o n s .  e l u t e d w i t h PBS  HB101  loaded onto a 74 x 1 cm  a t a r a t e o f 1.3  F r a c t i o n s were t e s t e d f o r CTL  ml/hr,  G-100  collecting  inducing a c t i v i t y  by  3 c u l t u r i n g 5 x 10  B6 s p l e e n c e l l s i n 0.2  f r a c t i o n s added at a 1 i n 5 d i l u t i o n .  ml V-bottomed w e l l s w i t h  C y t o t o x i c a c t i v i t y was  tested after  4 5 days.  IL-2 a c t i v i t y was  d e t e c t e d by c u l t u r i n g 10  f l a t bottomed w e l l w i t h f r a c t i o n s added a t a 1 i n 100 P r o l i f e r a t i o n was  The  measured 2 days l a t e r i n an MTT  column was  C2t c e l l s / 0 . 2  ml  dilution.  assay.  s t a n d a r d i z e d u s i n g b l u e d e x t r a n t o determine  Vo i n one  3 run, then  H-thymidine, alubmin,  run t o determine  V  66 KDa,  c a r b o n i c anhydrase and cytochome c were  29 KDa  and  12.4  KDa,  respectively.  39  important t o determine  whether the CTL  i n d u c i n g a c t i v i t y was  mediated  through the same f a c t o r t h a t induced the e x p r e s s i o n o f the IL-2R. the a c t i v e G-100  f r a c t i o n s , the 27KDa f a c t o r was  induce IL-2 r e s p o n s i v e n e s s i n thymocytes. (v/v) p o o l e d G-100  fractions  e q u i v a l e n t o f 100 U/ml  Using  t e s t e d f o r the a b i l i t y  to  Thymocytes were c u l t u r e d i n 15%  (which were t i t r a t e d and shown t o have the  IL-2 a c t i v i t y ) f o r 3 days, and then t e s t e d f o r the  a b i l i t y t o respond t o IL-2  (Table 7).  As shown, the f a c t o r i n the  f r a c t i o n s had the a b i l i t y t o render thymocytes r e s p o n s i v e t o IL-2, i n d i c a t i n g t h a t e x p r e s s i o n o f the IL-2R had been induced. P r e v i o u s s t u d i e s u s i n g r a d i o l a b e l l e d PMA has a low m o l e c u l a r weight,  have shown t h a t a l t h o u g h  i t can b i n d t o l a r g e molecules  in fetal  serum and e l u t e s a t the v o i d volume when chromatographed ( 6 0 ) . r e p o r t , bound PMA pooled G-100  was  c l e a r l y s e p a r a t e d from the IL-2 peak.  f r a c t i o n s s h o u l d be d e v o i d o f PMA,  CTL a c t i v i t y and IL-2R e x p r e s s i o n remained. it  PMA  calf  In t h a t  Hence, the  y e t the a b i l i t y  t o induce  The d a t a above suggest  that  i s u n l i k e l y t h a t the e f f e c t s o f CTL i n d u c t i o n and IL-2R e x p r e s s i o n  i n d u c t i o n were due  t o any r e s i d u a l PMA  i n the EL4SN.  F u r t h e r e v i d e n c e t o support these f i n d i n g s was medium, supplemented w i t h 10 ng/ml PMA,  1.2  ml was  loaded onto a G100  PBS  a t 1.3  ml/hr and t e s t e d a t 1 i n 20 d i l u t i o n f o r CTL r I L - 2 ( d a t a not shown).  s u g g e s t i n g t h a t i f any PMA  s e p a r a t e from the IL-2 and CTL  remained  then  ml f r a c t i o n s were e l u t e d w i t h inducing a c t i v i t y  A c t i v i t y was  i n f r a c t i o n s c o n t a i n i n g molecules w i t h m o l e c u l a r weights KDa,  HB101  c o n c e n t r a t e d 50 f o l d and  1.0  i n the presence o f 10 U/ml  column.  was  o b t a i n e d when  detected  g r e a t e r than  66  a c t i v e i n EL4SN, i t would e l u t e  inducing a c t i v i t i e s .  T a b l e 7.  27KDa f a c t o r induces IL-2 responsiveness and IL-2R e x p r e s s i o n  B6 thymocytes  3  FACS A n a l y s i s  c u l t u r e c o n d i t i o n sa  % 7D4  H-TdR I n c o r p o r a t i o n i n response t o :  +  10 U/ml IL-2  fresh  9.3  10 U/ml r I L - 2  5.4  15% EL4SN  52.5  15% G-100 f r a c t i o n s  1 3  15% S-200 f r a c t i o n s  a  2,094 ± 1267  0  N.D.  5  23,429 ± 704  10,130 ± 684  48,119 ± 1271  15,611 ± 1261  85,373 ± 6620  23,740 ± 2837  57,659 ± 3132  N.D.  N.D.  19.3  B6 thymocytes were c u l t u r e d a t 5 x 10  15% v/v EL4SN  c e l l s / m l i n l a r g e f l a s k s f o r 3 days.  c e l l s were then t e s t e d f o r t h e a b i l i t y t o p r o l i f e r a t e i n response  The  to various factors,  and f o r the e x p r e s s i o n of c e l l u l a r r e c e p t o r s .  G-100 f r a c t i o n s 39-48 were pooled and found t o have 15 U/ml IL-2 a c t i v i t y a t 15% v/v, and when used a t 10%, 36.0 ± 5.2% s p e c i f i c l y s i s was o b t a i n e d i n a s t a n d a r d lectin-mediated  c  assay.  S-200 f r a c t i o n s 53-64 were pooled and found t o have 80 U/ml IL-2 a c t i v i t y a t 15% v/v, and when used a t 10%, approximately  22% s p e c i f i c l y s i s was o b t a i n e d .  41  c.  Further biochemical analyses.  p u r i f i c a t i o n was  The  initial  purpose o f f u r t h e r  t o s e p a r a t e the CTL a c t i v i t y from IL-2 a c t i v i t y ,  such  t h a t the b i o l o g i c a l a c t i v i t i e s d e t e c t e d i n column f r a c t i o n s c o u l d be c o r r e l a t e d w i t h bands on SDS-PAGE. was  The  first  step i n t h i s  t o l o a d 50 x c o n c e n t r a t e d EL4SN on an FPLC-Superose 12 column  s e p a r a t e s p r o t e i n s on the b a s i s o f s i z e ) and t o e l u t e 0.5 0.5  purification  ml/min w i t h PBS.  activity  (Figure 7).  Each f r a c t i o n was The  t e s t e d f o r CTL  ml f r a c t i o n s at  i n d u c i n g and  a c t i v i t i e s e l u t e d with s i m i l a r molecular An i o n exchange column was inducing a c t i v i t y .  a l s o used  The  i n the attempt  I n i t i a l experiments  the albumin,  an S-200 column was  i n d u c i n g and the IL-2 a c t i v i t i e s .  loaded on the column and ran a t 12.9 (Figure 8).  i n the HB101  medium e l u t e d i n a  immunosuppressive  To overcome the d i f f i c u l t y  ml o f 50X  ml  not s e p a r a t e w e l l ( F i g u r e 9 ) .  false positives.  l i t t l e OD,  however, the  CTL  was  fractions the  activities  I t has been r e p o r t e d b e f o r e (54) t h a t t o f a c t o r s o t h e r than IL-2, r e s u l t i n g i n  However, i n the case o f the EL4SN, the f a c t o r  have an i n h e r e n t q u a l i t y o f m u l t i p l e charged  be s e p a r a t e d i n t h i s manner.  and  with  from the  c o n c e n t r a t e d EL4SN  ml/hr, c o l l e c t i n g 2.15  T h i s run had extremely  IL-2 dependent l i n e s can respond  to  purify  F r a c t i o n s 53-64 were pooled, and 10 ml were l o a d e d onto  Mono Q column. did  to f u r t h e r  used t o s e p a r a t e the albumin 2.55  CTL  w i t h FPLC-Mono Q column  l a r g e c o n c e n t r a t i o n o f p r o t e i n was  i n t e r f e r e d w i t h the IL-2 and CTL a s s a y s .  G-100  weights.  i n d i c a t e d t h a t the l a r g e amount o f albumin s i n g l e peak.  IL-2  r e s u l t s c o n f i r m e d those observed w i t h the  column because IL-2 i s r e q u i r e d i n the CTL assay, and the IL-2 and  the CTL  (which  forms,  appeared  and thus c o u l d not  42  Figure 7.  FPLC-Superose column elution profile for EL4SN.  EL4SN was prepared as described in Figure 6 and then concentrated 50 fold.  Ten ml were loaded onto the column, and 0.5 ml fractions were  eluted with PBS.  Fractions were tested for IL-2 and CTL-inducing  activities as described in Figure 6, except that the fractions were diluted 1 in 20 for the cytotoxicity test.  43  Fraction number  F i g u r e 8.  S-200 column e l u t i o n p r o f i l e : CTL-inducing  EL4SN was prepared concentrate  t o remove albumin from  activity.  as d e s c r i b e d i n F i g u r e 7 and 2.55 ml o f t h e 50 f o l d  were loaded  onto a 1.6 x 100 cm S-200 column.  The column  e l u t e d w i t h 20 mM T r i s HCl a t a r a t e o f 12.9 ml/hr c o l l e c t i n g 2.15 ml f r a c t i o n s which were t e s t e d as d e s c r i b e d i n F i g u r e 7.  46  F i g u r e 9.  FPLC-Mono Q column e l u t i o n p r o f i l e f o r albumin-depleted  S-200 f r a c t i o n s 53-64 were pooled Q column.  and 10 ml were loaded  EL4SN.  onto t h e Mono  The column was e l u t e d w i t h 20 mM T r i s HCl ( w i t h a l i n e a r  g r a d i e n t o f NaCl from 0 t o 0.3 molar) i n 1 ml f r a c t i o n s which were t e s t e d as d e s c r i b e d i n F i g u r e 7.  hi  48  F u r t h e r e v i d e n c e t h a t PMA was not r e s p o n s i b l e f o r the EL4SN e f f e c t was o b t a i n e d activity  u s i n g t h e S-200 f r a c t i o n s 53-64.  and IL-2R e x p r e s s i o n  i n B6 thymocytes.  These f r a c t i o n s induced CTL A f t e r 3 day c u l t u r e i n  15% v/v S-200 f r a c t i o n s , 10.3% o f t h e c e l l s e x p r e s s e d t h e 7D4 determinant. and  ( F r a c t i o n s 53-64 c o n t a i n no h i g h m o l e c u l a r weight  t h e r e f o r e should  proteins  be f r e e o f PMA).  In summary, t h e f a c t o r i n EL4SN t h a t a c t i v a t e s CTLp i n t h e presence o f IL-2  e l u t e d from a G-100 column w i t h an apparent m o l e c u l a r weight o f  27KDa.  I t i s t h e same f a c t o r t h a t mediates the i n d u c t i o n o f IL-2  r e s p o n s i v e n e s s ( i e , induces IL-2R e x p r e s s i o n ) .  The G-100 and S-200 column  d a t a a l s o suggest t h a t PMA i s not i n v o l v e d t o any s i g n i f i c a n t extent i n the e f f e c t s s t u d i e d , because f r a c t i o n s v o i d o f any PMA had b o t h  activities.  49  DISCUSSION  In t h i s t h e s i s , a f a c t o r i s d e s c r i b e d t h a t i s p r e s e n t supernatant  o f PMA-induced EL4  n o n s p e c i f i c a l l y induce killers.  The  c e l l s and  induced  mediates i t s e f f e c t through the L y t 2  t o IL-2  r e q u i r e s IL-2.  s t i m u l a t i o n o f the t a r g e t c e l l s . the e x p r e s s i o n o f the IL-2  CTL  effect.  t o r u l e out these N e i t h e r IL-4  w i t h or without  PMA  I t can induce  (the frequency The  effect  [It  Lyt2~  of  i s not  due  s p e c i f i c a n t i g e n or mitogen  T h i s same f a c t o r i s capable  of  inducing  and  IL-5 on T c e l l s made i t  lymphokines as the mediators of the EL4SN c o u l d induce  CTL  i n the presence o f  In a d d i t i o n , a n t i - I L - 4 a n t i b o d i e s had  e f f e c t on the a b i l i t y o f EL4SN t o a c t . EL4SN c o n t a i n s a unique lymphokine. r e p o r t s o f f a c t o r s which can induce the p r e s e n c e o f IL-2  L3T4~  receptor.  nor IL-5  added.  +  i n d u c t i o n i s mediated by a 27KDa  r e c e n t r e p o r t s o f the e f f e c t s o f IL-4  necessary  Lyt2  i n mature lymph nodes, EL4SN  L3T4~ c e l l s  +  f a c t o r t h a t a c t s i n the absence o f any  The  activity.  i n lymph nodes i s very l o w ) ] .  a l o n e , but  +  t o respond t o IL-2 upon c u l t u r e i n EL4SN.  d l 5 thymocytes t o p r o l i f e r a t e and  cells  to  r e s t i n g CTLp t o become p o l y c l o n a l l y a c t i v a t e d  i s p o s s i b l e t h a t EL4SN has more than one  L y t 2 ~ L3T4  the a b i l i t y  t a r g e t c e l l o f the f a c t o r i n c l u d e s T h y - 1  c e l l s , which can be  L3T4  has  i n the  (24-27; 31-41).  Thus, i t can be concluded  IL-2, no that  As mentioned, t h e r e have been s e v e r a l CTL by a c t i v a t i n g the L y t 2 In a d d i t i o n t o those  d i s c u s s e d , Garman e t a l . d e s c r i b e d a 30KDa and  a 17KDa CTL  from murine a n t i - v i r u s s p l e e n c e l l c u l t u r e (38).  +  CTLp i n  already helper f a c t o r  E r a r d e_t a l . d e s c r i b e d  a  50  12-18 KDa factor from rat Con A SN with CDF activity (39).  Most recently,  Garman and Raulet described a T cell-activating factor present in the supernatant of ConA activated murine spleen cells (41). 30-35 KDa, as determined by gel chromatography.  Their factor is  It is difficult to  determine whether these and the other factors mentioned in the introduction are a l l the same molecule, with the variations in assays and sources of factor accounting for the different molecular weights, or i f there are numerous molecules which perform the same function. As mentioned, Mannel and Falk (33) described TCF1, an activity present in EL4SN.  In that report they stated that any PMA remaining in the  supernatant would be inhibitory rather than stimulatory.  While i t has  been shown that InM - lOOnM PMA inhibited CTL activity (22,23), in the assays described in this thesis, lower concentrations of PMA actually stimulate CTL in the presence of IL-2.  Levels of PMA at 10 ng/ml were  inhibitory, but 0.3 to 1.0 ng/ml plus rIL-2 optimally stimulated CTL activity.  However, even at these concentrations, PMA + IL-2 could not  replace the effect observed with EL4SN (ie. PMA + IL-2 was much less efficient in CTL induction).  In the course of this study, there have been  several reports on the effects of phorbol esters on CTL induction (20-23).  However, i t is highly unlikely that PMA is the mediator of the  effect of EL4SN, or even involved with the effect.  G-100 column fractions  containing molecules of approximately 27 KDa were presumably void of any PMA activity, and yet retained a l l ability to induce CTL activity and IL-2R expression.  Similarly, the S-200 column fractions were void of high  molecular weight material, and could induce CTL and IL-2R expression.  51  Thus, i t i s proposed t h a t t h e f a c t o r p r e s e n t  i n EL4SN i s a unique and new  mediator o f CTL a c t i v a t i o n t h a t a c t s through an a l t e r n a t i v e pathway o f CTL induction.  The w e l l a c c e p t e d pathway o f CTL i n d u c t i o n  a n t i g e n - t r i g g e r i n g l e a d i n g t o IL-2R e x p r e s s i o n , and  d i f f e r e n t i a t i o n i n response t o IL-2.  not o n l y  involves  f o l l o w e d by p r o l i f e r a t i o n  I n the case o f t h e EL4SN f a c t o r ,  i s t h e r e no a n t i g e n - t r i g g e r i n g , the c e l l s do not even need t o  express the a n t i g e n this factor.  receptor,  as d l 5 f e t a l thymocytes were r e s p o n s i v e t o  There a r e s e v e r a l examples o f o t h e r  receptors  on T c e l l s  t h a t can be t r i g g e r e d w i t h t h e e n s u i n g response o f p r o l i f e r a t i o n and differentiation.  F o r example, MAbs t o the Thy-1 and TAP c e l l  molecules can a c t i v a t e T c e l l s  (17,18).  Thus, i t i s c o n c e i v a b l e  molecules such as Thy-1 o r TAP c o u l d p o t e n t i a l l y serve receptors,  surface that  as lymphokine  and t h a t T c e l l a c t i v a t i o n s i g n a l s c o u l d be d e l i v e r e d as a  r e s u l t o f such an i n t e r a c t i o n . In c o n c l u s i o n , cell  a l t h o u g h many f a c t o r s have been observed i n d i f f e r e n t  s u p e r n a t a n t s , and have been assayed i n v a r i o u s  possess remarkably s i m i l a r p r o p e r t i e s . activated T c e l l s , Lyt2  +  L3T4  -  still  A l l seem t o be a product o f  and a c t i n synergy w i t h IL-2.  They a l l induce t h e  subset o f T c e l l s , and most appear t o have an e f f e c t on  the IL-2 r e c e p t o r . described  systems, they  The simple f a c t t h a t CTL i n d u c i n g  f a c t o r s have been  so o f t e n i n such d i f f e r e n t systems suggests t h a t the mechanism  through which they a c t i s important t o t h e f u n c t i o n o f t h e immune system.  52  REFERENCES 1.  F a r r a r , W.L., S.B. M i z e l and J . J . F a r r a r . 1980. P a r t i c i p a t i o n o f lymphokine a c t i v a t i n g f a c t o r ( I n t e r l e u k i n 1) i n the i n d u c t i o n o f c y t o l y t i c T c e l l r e s p o n s e s . J . Immunol. 124:1371.  2.  Kaye, J . , S. G i l l i s , S.B. M i z e l , E.M. Shevach, T.R. Malek, C A . D i n a r e l l o , L.B. Lachman and C R . Janeway, J r . 1984. Growth o f a c l o n e d h e l p e r T c e l l l i n e induced by a monoclonal a n t i b o d y s p e c i f i c f o r the a n t i g e n r e c e p t o r : I n t e r l e u k i n 1 i s r e q u i r e d f o r the e x p r e s s i o n o f r e c e p t o r s f o r I n t e r l e u k i n 2. J . Immunol. 133:1339.  3.  Rock, Kenneth, L. o f T lymphocytes.  4.  S c a l a , G. and J . J . Oppenheim. 1983. A n t i g e n p r e s e n t a t i o n by human monocytes: E v i d e n c e f o r s t i m u l a n t p r o c e s s i n g and requirement f o r I n t e r l e u k i n 1. J . Immunol. 131:1160.  5.  Robb, R.J., A. Munck and K.A. Smith. receptors. J . Exp. Med. 154:1455.  6.  E r a r d , F., P. Corthesy, M. Nabolz, J.W. Lowenthal, P. Zaech, G. P l a e t i n c k and H.R. MacDonald. 1985. I n t e r l e u k i n 2 i s both n e c e s s a r y and s u f f i c i e n t f o r the growth and d i f f e r e n t i a t i o n o f l e c t i n - s t i m u l a t e d c y t o l y t i c T lymphocyte p r e c u r s o r s . J . Immunol. 134:1644.  7.  Smith, K e n d a l l A.  8.  Robb, R.J. 1984. I n t e r l e u k i n 2.: The molecule and i t s f u n c t i o n . Immunol. Today 5:203.  9.  P i l a r s k i , L.M. 1977. A requirement f o r a n t i g e n s p e c i f i c h e l p e r T c e l l s i n the g e n e r a t i o n o f c y t o t o x i c T c e l l s from thymocyte precursors. J . Exp. Med. 145:709.  10.  Smith, K.A. and D.A. C a n t r e l l . 1986. I n t e r l e u k i n 2 r e g u l a t e s i t s own r e c e p t o r s . P r o c . N a t l . Acad. S c i . USA 83:1857-  11.  K i s i e l o w , P., W. L e i s e r s o n and H. von Boehmer. 1984. D i f f e r e n t i a t i o n o f thymocytes i n f e t a l organ c u l t u r e : a n a l y s i s o f p h e n o t y p i c changes accompanying the appearance o f c y t o l y t i c and Interleukin-2-producing c e l l s . J . Immunol. 133:1117.  12.  Snodgrass, H.R., P. Kieslow, M. K i e f e r , M. Steinmetz and H. von Boehmer. 1985. Ontogeny o f the T c e l l a n t i g e n r e c e p t o r w i t h i n the thymus. Nature 313:592.  1982. The r o l e o f I a m o l e c u l e s i n the a c t i v a t i o n J . Immunol. 129:1360.  1984-  1981.  I n t e r l e u k i n 2.  T c e l l growth f a c t o r  Ann. Rev. Immunol.  2:319.  53  13.  14.  von Boehmer, H., A. C r i s a n t i , P. K i s i e l o w and W. Haas. 1985. Absence o f growth by most r e c e p t o r - e x p r e s s i n g f e t a l thymocytes presence o f I n t e r l e u k i n - 2 . Nature 314:539.  i n the  Fox, D.A., R.E. Hussey, K.A. F i t z g e r a l d , A. Bensussan, J.F. Daley, S.F. Schlossman and E.L. R e i n h e r z . 1985. A c t i v a t i o n o f human thymocytes v i a t h e 40KD T i l sheep e r y t h r o c y t e b i n d i n g p r o t e i n induces the e x p r e s s i o n o f I n t e r l e u k i n 2 r e c e p t o r s on b o t h T 3 and T3~ populations. J . Immunol. 134:330. +  15.  R e i n h e r z , E l l i s L. 1985. A m o l e c u l a r b a s i s f o r thymic s e l e c t i o n : R e g u l a t i o n o f T i l induced thymocyte expansion by the T 3 - T i antigen/MHC r e c e p t o r pathway. Immunol. Today 6:75.  16.  Fox, D.A, S.T. Schlossman and E.L. R e i n h e r z . 1986. Regulation of the a l t e r n a t i v e pathway o f T c e l l a c t i v a t i o n by a n t i - T 3 monoclonal antibody. J . Immunol. 136:1945.  17.  Kroczek, R.A., K.C. Gunter, R.N. Germain and E.M. Shevah. 1986. Thy-1 f u n c t i o n s as a s i g n a l t r a n s d u c t i o n m o l e c u l e i n T lymphocytes and t r a n s f e c t e d B lymphocytes. Nature 322:181.  18.  Malek, T.R., G. Ortega, C. Chan, R.A. Kroczek and E.M. Shevach. 1986. Role o f Ly-6 i n lymphocyte a c t i v a t i o n . J . Exp. Med. 164:709.  19.  R u s s e l , J.H. 1984. P h o r b o l e s t e r s i n a c t i v a t e the l y t i c c y t o t o x i c T lymphocytes. J . Immunol. 133:907.  20.  d'Angeac, A.D., M. Monis and T. Reme. 1986. Mitogenic e f f e c t of and IL-2 on s u b p o p u l a t i o n s o f c o r t i c o r e s i s t a n t thymocytes. J . Immunol. 137:3501.  21.  Depper, J.M., W.J. Leonard, M. Kronke, P.D. Noguchi, R.E. Cunningham, T.A. Waldman and W.G. Greene. 1984. Regulation of Interleukin 2 r e c e p t o r e x p r e s s i o n : E f f e c t s o f p h o r b o l d i e s t e r , p h o s p h o l i p a s e C, and reexposure t o l e c t i n o r a n t i g e n . J . Immunol. 133:3054.  22.  Bensussan, A., B. T o u r v i e t t e , L.-K. Chen, J . Dausset and M. Sasportes. 1985. P h o r b o l e s t e r induces a d i f f e r e n t i a l e f f e c t on the e f f e c t o r f u n c t i o n o f human a l l o s p e c i f i c c y t o t o x i c T lymphocytes and natural k i l l e r clones. P r o c . N a t l . Acad. S c i . USA 82:6642.  23.  Droge, W. 1986. P r o t e i n k i n a s e C i n T- c e l l Immunol. Today 7:340.  24.  R a u l e t , D.H. and Bevan, M.J. 1982. A differentiation factor r e q u i r e d f o r the e x p r e s s i o n o f c y t o t o x i c T c e l l f u n c t i o n . Nature 296:754.  apparatus o f  regulation.  PMA  54  25.  F a l k , W., D.N. Mannel, B. K a t z e r , B. Kaltman, P.H. Krammer, T. Diamanstein and W. Droge. 1985. I n d u c t i o n o f IL-2 r e c e p t o r e x p r e s s i o n and c y t o t o x i c i t y o f thymocytes by s t i m u l a t i o n w i t h TCF1. J . Immunol. 135:1160.  26.  Wagner, H., C. Hardt, B.T. Rouse, M. R o l l i n g h o f f , P. S h e v r i c h and K. Pfizenmaier. 1982. D i s s e c t i o n o f the p r o l i f e r a t i v e and d i f f e r e n t i a t i v e s i g n a l s c o n t r o l l i n g murine c y t o t o x i c T lymphocyte r e s p o n s e s . J . Exp. Med. 155:1876.  27.  T a k a i , Y., S.H. Herrmann, J . L . G r e e n s t e i n , G.L. S p i t a l n y and S.J. B u r a k o f f . 1986. Requirement f o r t h r e e d i s t i n c t lymphokines f o r the i n d u c t i o n o f c y t o t o x i c T lymphocytes. J . Immunol. 137:3494.  28.  Lee, F., T. Yokota, F. Otsuka, P. Meyerson, D. V i l l a r e t , R. Coffman, T. Mosmann, D. Rennick, N. Roehm, C. Smith, A. Z l o t n i k and K. A r a i . 1986. I s o l a t i o n and c h a r a c t e r i z a t i o n o f a mouse i n t e r l e u k i n cDNA c l o n e t h a t expresses B - c e l l s t i m u l a t o r y f a c t o r 1 a c t i v i t i e s and T - c e l l - and m a s t - c e l l - s t i m u l a t i n g a c t i v i t i e s . P r o c . N a t l . Acad. S c i . USA 83:2061.  29.  P a l a c i o s , R., P. S i d e r a s and H. von Boehmer. 1987. Recombinant I n t e r l u e k i n 4/BSF-l promotes growth and d i f f e r e n t i a t i o n o f i n t r a t h y m i c T c e l l p r e c u r s o r s from f e t a l mice i n v i t r o . EMBO J o u r n a l 6:91.  30.  K i n a s h i , T., N. Harada, E. S e v e r i n s o n , T. T a n a b l e , P. S i d e r a s , M. K o n i s h i , C. Azuma, A. Tominaga, S. B e r g s t e d t - L i n d q u i s t , M. Takahashi, F. Matsuda, Y. Y a o i t a , K. T a k a t s u and T. Honjo. 1986. Cloning of complementary DNA e n c o d i n g T - c e l l r e p l a c i n g f a c t o r and i d e n t i t y w i t h B c e l l growth f a c t o r I I . Nature 324:70.  31.  Teh, H.-S. and Yu, M. 1983. A c t i v a t i o n of nonspecific k i l l e r c e l l s by I n t e r l e u k i n - 2 c o n t a i n i n g s u p e m a t a n t s . J . Immunol. 131:1827.  32.  F a l k , W., D.N. Mannel and W. Droge. 1983. A c t i v a t i o n of cytotoxic T lymphocytes r e q u i r e s a t l e a s t two s p l e e n c e l l - d e r i v e d h e l p e r f a c t o r s b e s i d e s I n t e r l e u k i n 2. J . Immunol. 130:2214.  33.  Mannel, D.N. and W. F a l k . 1985. T c e l l c y t o t o x i c i t y inducing f a c t o r (TCF1) i s a T c e l l product t h a t induces thymic d i f f e r e n t i a t i o n In " C e l l u l a r and M o l e c u l a r B i o l o g y o f Lymphokines". Sorg, C , A. Schimpl and M. Lanoy, eds. Academic P r e s s , p. 93.  34.  Reddehase, M., W. Svessmuth, C. Moyers, W. F a l k and W. Droege. 1982. I n t e r l e u k i n 2 i s not s u f f i c i e n t as h e l p e r component f o r the a c t i v a t i o n o f c y t o t o x i c T lymphocytes but s y n e r g i z e s w i t h a l a t e h e l p e r e f f e c t t h a t i s p r o v i d e d by i r r a d i a t e d I - r e g i o n - i n c o m p a t i b l e stimulator c e l l s . J . Immunol. 128:61.  55  35-  Garman, R.D. and D.P. Fan. 1983. C h a r a c t e r i z a t i o n of helper d i s t i n c t from I n t e r l e u k i n 2 n e c e s s a r y f o r the g e n e r a t i o n o f a l l o s p e c i f i c c y t o l y t i c T lymphocytes. J . Immunol. 130:756.  factors  36.  F i n k e , J.H., J . S c o t t , S. G i l l i s and M.L. H i l f i k e r . 1983. G e n e r a t i o n o f a l l o r e a c t i v e c y t o t o x i c T lymphocytes: Evidence f o r a d i f f e r e n t i a t i o n f a c t o r d i s t i n c t from IL-2. J . Immunol. 130:763.  37.  Kanagawa, Osami. 1983. Three d i f f e r e n t s i g n a l s are r e q u i r e d f o r i n d u c t i o n o f c y t o l y t i c T lymphocytes from r e s t i n g p r e c u r s o r s . J . Immunol. 131:606.  38.  Garman, R.D., A. Taku and D.P. Fan. 1984. Chromatographic s e p a r a t i o n from known c y t o k i n e s o f a h e l p e r f a c t o r n e c e s s a r y f o r the g e n e r a t i o n o f murine c y t o l y t i c T lymphocytes. J . Immunol. 132:1879.  39.  E r a r d , F., P. Corthesy, K.A. Smith, W. F i e r s , A. Conzelmann and M. Nabholz. 1984. C h a r a c t e r i z a t i o n o f s o l u b l e f a c t o r s t h a t induce the c y t o l y t i c a c t i v i t y and the e x p r e s s i o n o f T c e l l growth f a c t o r receptors of a T c e l l hybrid. J . Exp. Med. 160:584.  40.  T a k a t s u , K., Y. K i k u c h i , T. K a n a t a n i , K. Okuno, T. Hamaoka, A. Tominaga and Y. Sanyo. 1986. G e n e r a t i o n o f c y t o t o x i c T lymphocytes from thymocyte p r e c u r s o r s o f t r i n i t r o p h e n y l - m o d i f i e d s e l f - a n t i g e n s . J . Immunol. 136:116.  41.  Garman, R.D. and D.H. R a u l e t . 1987. C h a r a c t e r i z a t i o n of a novel murine T c e l l a c t i v a t i n g f a c t o r . J . Immunol. 138:1121.  42.  Wang, A., S.-D. Lu and D.F. Mark. 1984. S i t e s p e c i f i c mutagenesis o f the human I n t e r l e u k i n - 2 gene: S t r u c t u r e - f u n c t i o n a n a l y s i s of the cysteine residues. S c i e n c e 224:1431.  43.  Bruce, J . , F.W. Symington, T . J . McKearn and J . S p r e n t . 1981. A monoclonal a n t i b o d y documentary between subsets o f T and B c e l l s . J . Immunol. 127:2496.  44.  Malek, T.R., R.J. Robb and E.M. Shevach. 1983. I d e n t i f i c a t i o n and i n i t i a l c h a r a c t e r i z a t i o n o f a r a t monoclonal a n t i b o d y r e a c t i v e w i t h the murine i n t e r l e u k i n - 2 r e c e p t o r - l i g a n d complex. P r o c . N a t l . Acad. S c i . USA 80:5694.  45.  D i a l y n a s , D.P., D.B. Wilde, P. Marrack, A. P i e r r e s , K.A. Wall, W. Hauran, G.N. Otten, M.R. Loker, M. P i e r r e s , J . K a p p l e r and F.W. F i t c h . 1983. C h a r a c t e r i z a t i o n o f the murine a n t i g e n i c determinant d e s i g n a t e d L3T4a by f u n c t i o n a l T c e l l c l o n e s appears t o c o r r e l a t e p r i m a r i l y w i t h c l a s s I I MHC a n t i g e n - r e a c t i v i t y . Immunol. Rev. 74:29.  the  56  46.  L e d b e t t e r , J.A. and L.A. Herzenberg. 1979. Xenogeneic monoclonal a n t i b o d i e s t o mouse lymphoid d i f f e r e n t i a t i o n a n t i g e n s . Immunol. Rev. 47:64.  47.  S t a e r z , U.D., H.-G. Rammernsee, J.D. Benedetto and M.J. 1985. C h a r a c t e r i z a t i o n o f a murine monoclonal a n t i b o d y an a l l o t y p i c determinant on a T c e l l a n t i g e n r e c e p t o r . J . Immunol. 134:3994.  48.  S t a e r z , U.D. and M.J. Bevan. 1986. lymphocytes by a monoclonal a n t i b o d y determinant on the T c e l l r e c e p t o r .  49.  Ohara, J . and W.E. P a u l . 1985. Production to and m o l e c u l a r c h a r a c t e r i z a t i o n o f B c e l l Nature 315:333-  50.  Mosmann, T. 1983. Rapid c o l o r i m e t r i c assay f o r c e l l u l a r growth s u r v i v a l : a p p l i c a t i o n t o p r o l i f e r a t i v e and c y t o t o x i c i t y a s s a y s . J . Immunol. Methods 65:55.  51.  Wakasugi, H., J . B e r t o g l i o , T. T u r s e z and D. F r a d e l i z . 1985. r e c e p t o r i n d u c t i o n on human T lymphocytes: Role f o r IL-2 and monocytes. J . Immunol. 135:321.  52.  Ohara, J . , S. Lahet, J . Inman and W.E. P a u l . 1985. Partial p u r i f i c a t i o n o f murine B c e l l s t i m u l a t o r y f a c t o r ( B S F ) - l . J . Immunol. 135:2518.  53.  H i l f i k e r , M.L., R.N. Moore and J.L. F a r r a r . 1981. Biologic p r o p e r t i e s o f c h r o m a t o g r a p h i c a l l y separated murine thymoma d e r i v e d I n t e r l e u k i n 2 and c o l o n y - s t i m u l a t i n g f a c t o r . J . Immunol. 127:1983.  54.  G r a b s t e i n , K., J . Eisenman, D. M o c h i z u k i , K. Shanebeck, P. Conlon, T. Hopp, C. March and S. G i l l i s . 1986. P u r i f i c a t i o n t o homogeneity o f B c e l l s t i m u l a t i n g f a c t o r . J . Exp. Med. 163:1405.  55.  Robb, R.J., W.C. Greene and C M . Rusk. 1984. Low and h i g h r e c e p t o r s f o r I n t e r l e u k i n 2. J . Exp. Med. 160:1120.  56.  Reem, G.H., N.-H. Yeh, D.L. U r d a l , P.L. K i l i a n and J . J . F a r r a r . 1985. I n d u c t i o n and u p r e g u l a t i o n by I n t e r l e u k i n 2 o f h i g h a f f i n i t y I n t e r l e u k i n 2 r e c e p t o r s on thymocytes and T c e l l s . P r o c . N a t l . Acad. S c i . USA 82:8663.  57.  Bevan, M.J. and M. Conn. 1975. C y t o t o x i c e f f e c t s o f a n t i g e n and mitogen induced T c e l l s on v a r i o u s t a r g e t s . J . Immunol. 114:559.  Bevan. specific for  A c t i v a t i o n of r e s t i n g T d i r e c t e d a g a i n s t an a l l o t y p i c Eur. J . Immunol. 16:263. o f a monoclonal a n t i b o d y s t i m u l a t o r y f a c t o r 1.  and  IL-2  affinity  {  57  58.  Teh, H.-S., R.A. P h i l i p s and R.G. M i l l e r . 1977. Quantitative s t u d i e s on t h e p r e c u r s o r s o f c y t o t o x i c lymphocytes. J . Immunol. 118:1057.  59.  J u l i u s , M.H., E. Simpson and L. Herzenberg. 1973. A r a p i d method f o r t h e i s o l a t i o n o f f u n c t i o n a l thymus-derived murine lymphocytes. Eur. J . Immunol. 3:645.  60.  F a r r a r , J . J . , J . F u l l e r - F a r r a r , M.L. H i l f i k e r , B.M. S t a d l e r and W.L. F a r r a r . 1980. Thymoma p r o d u c t i o n o f T c e l l growth f a c t o r ( I n t e r l e u k i n 2 ) . J . Immunol. 125:2555.  61.  Mosmann, T.R., M.W. Bond, R.L. Cuffman, J . Ohara and W.E. P a u l . 1986. T - c e l l and mast c e l l l i n e s respond t o B c e l l stimulatory f a c t o r 1. P r o c . N a t l . Acad. S c i . USA 83:5654.  

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