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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.S c , The Un i v e r s i t y of 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 i n THE FACULTY OF GRADUATE STUDIES Genetics Program We accept t h i s t hesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1987 ® Laura Dawn Williams U 6 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. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my 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 Genetics Program The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 n a . July 23, 1987 DE-6(3/81) 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 cell 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. i i i Table of Contents PAGE Abstract i i Abbreviations i v L i s t of Tables v L i s t of Figures v i Acknowledgements v i i Introduction 1 Materials and Methods 8 Results 14 Discussion 49 References 52 / ABBREVIATIONS i v 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 precursor(s) T H c e l l T helper c e l l TCGF T c e l l growth f a c t o r (IL-2) IL i n t e r l e u k i n rIL-2 human recombinant IL-2 TcR T c e l l receptor IL-2R IL-2 receptor PMA 4-B-phorbol-12-myristate-13-acetate (TPA) ConA Concanavalin A PKC protein kinase C KDa k i l o d a l t o n EL4SN supernatant of PMA-induced EL4 c e l l s FACS fluorescence activated c e l l sorter MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazodium bromide V LIST OF TABLES PAGE Table 1 Ontogeny of the response to F23.1 15 Table 2 EL4SN e f f e c t i s not due to IL-2, but requires IL-2 18 Table 3 IL-2 alone does not induce CTL i n t h i s system 19 Table 4 EL4SN e f f e c t i s not due to IL-4 or IL-5 21 Table 5 EL4SN induces IL-2R expression and IL-2 responsiveness i n thymocytes 28 Table 6 The target c e l l of EL4SN i s a Thyl +Lyt2 +L3T4~ nylon wool nonadherent c e l l 33 Table 7 A 27KDa f a c t o r induces IL-2 responsiveness and IL-2R expression 40 v i LIST OF FIGURES PAGE Figure 1 C l a s s i c a l pathway of CTL induction 2 Figure 2 EL4SN increases F23.1 expression on thymocytes 24 Figure 3 EL4SN induces expression of the IL-2R on thymocytes .... 26 Figure 4 EL4SN p o l y c l o n a l l y activates CTL 30 Figure 5 EL4SN induces IL-2R expression on L y t 2 + c e l l s 35 Figure 6 A 27KDa f a c t o r induces CTL a c t i v i t y 37 Figure 7 FPLC-superose column e l u t i o n p r o f i l e f o r EL4SN 42 Figure 8 S-200 column e l u t i o n p r o f i l e : to remove albumin from CTL-inducing a c t i v i t y 44 Figure 9 FPLC-Mono Q column on albumin-depleted EL4SN 46 v i i ACKNOWLEDGEMENTS I wou ld l i k e t o e x p r e s s my a p p r e c i a t i o n t o D r . H . - S . Teh f o r h i s s u p p o r t and g u i d a n c e , as w e l l as D r s . R. McMaster and F . T a k e i f o r t h e i r s u g g e s t i o n s . I wou ld a l s o l i k e t o thank P . C . Kwong f o r a l l h e r h e l p , D. Z e c c h i n i f o r o p e r a t i n g t h e F A C S , E . Kwan f o r r u n n i n g t h e F P L C , and R. Bauzon f o r t y p i n g t h i s t h e s i s . 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 cell (1-4). (For simplicity, H nonallogeneic accessory cells are not included in the diagram.) The activated T cell proliferates and secretes IL-2. IL-2 induces H 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 cel 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 cell 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 i i . The A l t e r n a t i v e Pathway of T C e l l A c t i v a t i o n In addition to the c l a s s i c a l antigen-dependent pathway of T c e l l a c t i v a t i o n , a l t e r n a t i v e antigen-independent mechanisms of T c e l l a c t i v a t i o n have been reported (14-23). Reinherz described human T c e l l a c t i v a t i o n mediated through e i t h e r the TcR or the T i l sheep erythrocyte binding glycoprotein (14-16). T i l i s a surface component of an a l t e r n a t i v e pathway which appears e a r l i e r i n intrathymic d i f f e r e n t i a t i o n than the nominal antigen receptor. Antibodies against T i l can 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 IL-2 production, and the e f f e c t i s IL-1 and macrophage independent. Presumably there i s an as yet undefined natural ligand f o r t h i s pathway. It i s also of i n t e r e s t that T i l i s present on v i r t u a l l y a l l thymocytes including the less mature populations that lack the TcR, suggesting the importance of the a l t e r n a t i v e pathway mediated through T i l . Unlike peripheral T c e l l s , a n t i - T i l stimulation d i d not r e s u l t i n production of IL-2 i n thymocytes, although the IL-2 receptors are f u l l y f u n c t i o n a l i n that p r o l i f e r a t i o n merely depends on the presence of exogenous IL-2. There are also examples of c e l l u l a r receptors on murine T c e l l s which can 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 of r e s t i n g T c e l l s . Certain monoclonal anti-Thy-1 antibodies can a c t i v a t e r e s t i n g CTL (17). Furthermore, the 12-KDa TAP protein, which i s p r i m a r i l y expressed on T and B c e l l s , can be crosslinked with an ensuing strong T c e l l a c t i v a t i o n response (18). 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 it demonstrates an alternative mechanism for T cell activation. Protein kinase C (PKC) is activated when a T cell 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. Accessory cells are also involved in the activation of PKC. PMA bypasses the requirement for antigen to activate PKC. 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 cell receptors (TcR). It is suggested that this factor bypasses the requirement of antigen-triggering by mediating its effect through an unknown cellular receptor. i i i . Antigen-Nonspecific Factors Raulet and Bevan were the first 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 cel 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 (26). CTDF acted on Lyt2+ cells and could be found in ConASN. 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+ cell 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: i t allows activated B lymphocytes to switch from IgM to IgG secretion; it increases surface levels of la antigens and increases the size of B cells; and it supports the growth of some factor-dependent cell 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 cell s , inducing immunoglobulin secretion (30). This investigation involves a factor that can act on resting CTL, and has a variety of similarities 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 air. 3• Cell Preparations Organs are asceptically dissected, then forced through a seive to form a single cel l suspension. Thymocytes and lymph node cells were then washed three times in cold phosphate buffered saline (PBS). Spleen cells were first 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 with 5% FCS, 10 mM HEPES (Sigma), a n t i b i o t i c s , and 10 ng/ml PMA (Sigma). The EL4 c e l l l i n e i s a benzopyridine-induced lymphoma of B6 o r i g i n and was a kind g i f t of Dr. Verner Paetkau of the Department of Biochemistry, U n i v e r s i t y of Alberta, Edmonton, Alberta. To prepare f a c t o r f o r biochemical studies, 10^ EL4 c e l l s / m l were incubated i n HB101 medium (NEN Research Products) i n the presence of 10 ng/ml PMA f o r 24 hr. HB101 i s a serum-free medium with defined amounts of albumin, t r a n s f e r i n and i n s u l i n . Both preparations of f a c t o r proved to have the same b i o l o g i c a l a c t i v i t i e s , using the assays described i n t h i s study. b. Human recombinant IL-2 ( l o t #LP-315 from E. c o l i ) was kindly provided by Cetus Corporation, Emeryville, CA (42). The rIL-2 was 99% pure as assayed by SDS-PAGE and contains less than 0.01 ng endotoxin per 1.5 x 10^ u n i t s . One unit i s defined as the amount of IL-2 required to cause 30% maximal p r o l i f e r a t i o n of an IL-2-dependent c e l l l i n e cultured at 10 4 cells/0.2 ml f o r 48 hr. c. The rIL-4 (BSF-1) was a kind g i f t of Dr. Timothy Mosmann of the DNAX Research I n s t i t u t e of Molecular and C e l l u l a r Biology, Palo A l t o , CA (28). d. The rIL-5 was kindly provided by Dr. Vern Paetkau. This f a c t o r has i d e n t i c a l properties to that described by Kinashi et a l . (30). 5. C e l l Lines and Monoclonal Antibodies a. The B c e l l hybridoma l i n e s producing MAbs against the Thy-1.2 molecule ( J l j ) (42), the IL-2 receptor (7D4) (44), the L3T4 c e l l surface molecule (GK1.5) (45), and the mouse Lyt 2 molecule (TIB105) (46) were obtained from the American Type Culture C o l l e c t i o n . 10 b. The F23.1 hybridoma cell 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: ^280 ^'^ = mg/ml protein. 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 cel 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. 0DgQ0 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. The basis of the assay is the MTT itself. It is a tetrazolium salt, and the tetrazolium ring is cleaved in active mitochondria resulting in a colour change. Thus, only live cells are detected. 3 5 b. To determine the extent of cel 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. For the last 6 hr of incubation, 3 0.5 mCi/well of 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 Al 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 further 3-5 hr. The supematants were then harvested and tested f o r released "^Cr. % S p e c i f i c Lysis = test count - spontaneous count  maximum releasable count - spontaneous count The spontaneous counts were generally less than 10% of the maximum releasable count; the maximum releasable count was c a l c u l a t e d as 90% of the t o t a l incorporated counts. 8. C e l l Surface L a b e l l i n g 0.5 - 1.0 x 10^ c e l l s were washed extensively with PBS, then incubated i n 1.0 ml of medium containing antibodies against the c e l l surface antigen. For l a b e l l i n g with F23-1, c e l l s were incubated 30 min. at 37°C i n f i v e times concentrated spent culture supernatant (47,48). For L3T4, Lyt 2, and 7D4 antibodies, c e l l s were incubated on i c e f o r 45 min with 100 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 protein. A f t e r incubation, c e l l s were washed extensively i n cold PBS, then one ml of medium containing 10 yg/ml FITC-labelled. antibody was added to the p e l l e t , followed by a 45 min. incubation on i c e . A f t e r a f i n a l thorough washing, the c e l l s were suspended i n 0.1-1.0 ml PBS and analyzed to determine the extent of antibody binding on a FACS IV. 9. FACS Analysis Ten thousand c e l l s of each sample were tested f o r bound FITC-labelled antibody, with dead c e l l s and debris gated out. The cross-over point between the negative control and the te3t sample was 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 . For purposes of illus t r a t i o n , log of fluorescence was plotted against linear or log of c e l l number. 10. FPLC 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. Similarly, samples were loaded on a Mono Q column and 1.0 ml fractions were eluted at a rate of 1.0 ml/min in tris-HCl with a linear 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. This antibody recognizes a determinant of the vg_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 fe: 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. Ontogeny of the response to F23.1 i . Proliferative Response 4 weeks Balb/C thymocytes 2 weeks 1 week newborn dl5 FT F23.1 rIL-2 F23.1 + rIL-2 2 215 ± 1165 2 947 ± 1398 16 700 ± 4349 2 493 ± 1318 3 4 634 + 1563 4 8 392 ± 1200 12 317 ± 1521 096 ± 1652 191 ± 1466 766 ± 200 2 701 ± 221 3 730 ± 1458 1 471 ± 605 EL4SN EL4SN+F23.1 68 270 ± 778 73 586 ± 1414 98 931 ± 1200 45 940 ± 7332 52 257 ± 1030 55 973 ± 10834 37 032 ± 3501 61 099 ± 14584 24 572 ± 4778 i i . Cytotoxic Responses*5 4 weeks 2 weeks 1 week newborn 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 71.4 ± 3.5 62.9 ± 4.7 65.8 ± 6.3 59.0 ± 5.0 EL4SN + F23.1 67.9 ± 2.7 62.7 ± 6.2 59.9 ± 3.2 63.1 ± 3.0 16 Table 1 (continued) Thymocytes were cultured with 10 ug/ml F23.1 (bound to A f f i g e l ) , 20 U/ml rIL-2, or 15% v/v EL4SN f o r 4 days, and then the cultured c e l l s were assayed f o r p r o l i f e r a t i v e responses. The r e s u l t s are expressed as a mean of 4 cultures with the standard deviation. b Thymocytes were cultured with f a c t o r s f o r 5 days, and then assayed f o r cytotoxic responses i n a PHA-P mediated k i l l i n g assay. The r e s u l t s are expressed as the mean of 6 cultures with the standard e r r o r . 17 newborn thymocytes to respond to antigen t r i g g e r i n g by F23.1. The synergy 4 was always observed at low c e l l numbers (less than 3x10 /well) of adult mouse thymocytes, again suggesting that EL4SN i s expanding a c e l l population that can respond to F23.1. Because of these observations, i t was decided that further 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 Induction of CTL by EL4SN but not by IL-2, IL-4 or IL-5 a. EL4SN e f f e c t i s not due to IL-2, but requires IL-2. It i s well known that EL4SN contains factors other than IL-2 (32,33,39,52,53). Our lab has previously reported that 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 of antigen or mitogen i n an IL-2 dependent fashion (31). This observation i s confirmed i n Table 2, i n which i t i s shown that there i s a fa c t o r other than IL-2 i n EL4SN. In t h i s case, 10% EL4SN contained the equivalent of 40 U/ml IL-2 a c t i v i t y . As shown, IL-2 could not induce 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 . In contrast, EL4SN, even at high d i l u t i o n s , could a c t i v a t e CTLp. In addition, while IL-2 alone cannot mediate the e f f e c t observed with EL4SN, i t i s c l e a r that IL-2 i s required i n t h i s process. Addition of 7D4 antibody (which binds to the IL-2R, blocking IL-2 binding) (44) to the cultures i n h i b i t e d the response to EL4SN, whereas equal concentrations of an i r r e l e v a n t IgM prepared i n the same way had no e f f e c t on CTL a c t i v i t y . b. IL-2 alone does not induce CTL i n t h i s system. It i s well established that at high c e l l density, IL-2 alone can induce CTL a c t i v i t y (6). This phenomenon was observed and i s shown i n Table 3. However, as 18 T a b l e 2 . EL4SN e f f e c t i s n o t due t o I L - 2 , bu t r e q u i r e s I L - 2 B6 o r BDF1 EL4SN r I L - 2 J H - T d R I n c o r p o r a t i o n % S p e c i f i c L y s i s s p l e e n c e l l s % ( v / v ) U / m l CPM ± SE M ± SE 5 X 1 0 3 741 + 62 1.4 4 0 . 5 10 16 585 + 1275 7 2 . 0 + 2 . 9 5 3 251 + 589 2 7 . 3 + 3 . 1 2 . 5 1 112 + 223 5 . 1 + 1.6 1 .25 830 + 89 2 . 6 + 0 . 8 5 X 1 0 3 40 1 035 + 655 0 . 9 + 0 . 6 20 2 925 + 1446 - 0 . 6 + 0 . 7 10 1 401 + 880 2 . 4 + 0 . 7 5 507 + 86 0 . 0 + 0 . 8 5 X 1 0 3 10% EL4SN + 100 p g / m l 7D4 Not done 1 4 . 9 + 8 . 1 10% EL4SN + 30 p g / m l 7D4 Not done 4 5 . 8 + 4 . 0 10% EL4SN + 10 P g / m l 7D4 Not . done 5 4 . 1 + 5 . 6 C u l t u r e and a s s a y 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 . 19 TABLE 3. IL-2 alone does not induce CTL in this system' BDF1 spl EL4SN rIL-2 CPM ± SE % specific lysis 4 4 x 10 1 973 + 136 2.4 ± 1.8 4 2 x 10* 886 + 58 1.2 ± 1.8 1 x 104 567 + 48 0.0 ± 1.5 5 x 103 1 262 + 786 1.7 ± 1.1 2.5 x 103 1 349 + 457 1.0 ± 1.1 4 4 x 10 10% 49 238 + 811 84.1 ± 5.4 4 2 x 10* 10% 28 126 + 592 81.7 ± 4.7 ' 1 x 104 10% 17 867 + 725 81.7 ± 4.3 5 x 103 10% 9 196 + 128 77.0 ± 8.9 2.5 x 103 10% 5 384 + 536 54.8 ± 8.9 4 4 x 10 20 U/ml 5 015 + 450 58.0 ± 8.1 2 x 104 20 U/ml 1 107 + 67 31.2 ± 8.0 1 x 104 20 U/ml 847 + 321 8.0 ± 3.5 5 x 103 20 U/ml 975 + 41 6.0 ± 3.1 2.5 x 103 20 U/ml 499 + 38 0.0 ± 0.7 Culture and assay conditions as described in Table 1. 20 the cel 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 cell 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 H-thymidine label (data not shown). 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. EL4SN effect i s not due to IL-4 or IL-5 a BDF1 spl rIL-2 rIL-5 rIL-4 C H B l l d EL4SN PMA % specific l y s i s U/ml ng/ml 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 20 2.2 + 2.4 100 2.1 + 1.7 20 100 2.8 + 3.7 1/10 -0.3 + 1.3 10 1/10 -1.1 + 2.9 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) Culture and assay conditions as described i n Table 1. ^rIL-5 was obtained by t r a n s l a t i o n of a cloned cDNA i n COS c e l l s and the COS supernatant, when used at 10% v/v, was able to induce the 5 p r o l i f e r a t i o n of BCL c e l l s (30); Acpm = 51,985 f o r 1.5 x 10 cells/0.2 ml a f t e r a 2 day culture period. COS supernatant of the IL-5 cDNA clone, transcribed i n the reverse d i r e c t i o n , had no a c t i v i t y i n the BCL^ assay. The a c t i v i t y of rIL-4 was determined as previously described (61). dThe 11B11 antibody (49) was used as e i t h e r spent culture supernatant (SN) or as the ammonium s u l f a t e p r e c i p i t a t e of a s c i t i c p rotein (asc). The data presented i s representative of several extensive t i t r a t i o n s . (rIL5 was tested at d i l u t i o n s varying from 1/10 to 1/100. rIL4 was tested at 1.25 U/ml to 100 07ml). 23 3. Mechanism of Action of EL4SN a. EL4SN induces the expression of IL-2R and TcR. Because of the apparent a b i l i t y of EL4SN to induce IL-2 responsiveness i n a c e l l population, i t was of i n t e r e s t to determine the e f f e c t of EL4SN upon c e l l u l a r receptor expression. The f i r s t observation was that the frequency and density of the F23.1 determinant on thymocytes increased upon incubation with 15% EL4SN. The FACS analysis i s represented i n Figure 2. A f t e r 2-3 days cultures i n 15% EL4SN, 30-40% of the o r i g i n a l 5 5x10 c e l l s / m l remained v i a b l e . I n i t i a l l y , 4 week old Balb/c thymocytes were 12.9% F23.1 +, and were 25.2% F23.1 + a f t e r c u l t u r e . S i m i l a r l y , one week old Balb/c thymocytes increased from 6.4% to 20.0% F23.1 + a f t e r cu l t u r e . An a d d i t i o n a l point was the d i f f e r e n t frequencies of F23.1 + c e l l s i n the 4 week (12.9%) and 1 week o l d (6.4%) mouse thymocytes. Perhaps the increase i n frequency of F23.1 with age accounted f o r the corresponding increase i n response to stimulation with F23.1 + IL-2. The number of IL-2R expressing c e l l s also increased from 2.8 to 7.2% when the c e l l s were cultured i n EL4SN (Figure 3). S e l e c t i v e gating was used to d i s t i n g u i s h between the large activated b l a s t s , and the small c e l l s , and i t was shown that the b l a s t s expressed the IL-2R. In contrast, c e l l s cultured i n IL-2 alone had no e f f e c t on c e l l s i z e or IL-2R expression. b. EL4SN induces responsiveness to IL-2. Thymocytes incubated i n EL4SN f o r 2-3 days had increased IL-2R expression, and could respond to IL-2 i n a p r o l i f e r a t i v e fashion (Table 5). Normal fresh c e l l s expressed l i t t l e , i f any, IL-2R (5,55,56) and could not respond to IL-2. 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 cel 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* i ( i i i ) (vi) 26 Figure 3. EL4SN induces the expression of the IL-2R on thymocytes. a. In this figure, the X-axis is defined as the log of fluoresence and the Y-axis as the log of cell 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) (iv) V *AV. ••JV/iV. . t ( i i ) (v) 1 -^JP ' ( i i i ) TABLE 5. EL4SN induces IL-2R expression and IL-2 responsiveness in thymocytes Balb/c FACS Analysis thymocytes F23.1+ 7D4+ fresh 12.6 2.8 cult. 2d in IL-2 1.8 1.3 cult. 2d in EL4SN 30.0 7.2 large blasts 15.9 small cells 0.9 H-TdR Incorporation in response to: 10 U/ml IL-2 15% EL4SN 6 318 ± 7 698 53 819 ± 6 123 3 758 ± 3 063 43 807 ± 9 421 56 094 ± 8 112 74 837 ± 2 595 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 i n IL-2 alone had no e f f e c t on the c e l l s a b i l i t y to respond to IL-2. In contrast to these r e s u l t s , the increase i n F23.1 determinant had no corresponding increase i n a b i l i t y to respond to F23.1 + IL-2. I t may be i n f e r r e d that EL4SN i s a c t i v a t i n g the T c e l l s to express more high a f f i n i t y IL-2R, and F23.1 expression was also increased as a r e s u l t of t h i s a c t i v a t i o n . I t i s unclear whether EL4SN was a c t u a l l y inducing the expression of IL-2R i n CTLp, or i f i t was acting on the few IL-2R + CTLp, expanding t h i s population. Both p o s s i b i l i t i e s r e s u l t i n an increase i n IL-2 responsive c e l l s . c. EL4SN p o l y c l o n a l l y activates CTLs. The above data suggested that a large proportion of the population can respond to EL4SN: rat ConA supernatant was only 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 to express IL-2R as compared to EL4SN (data not shown). In one representative experiment, ConA SN induced b l a s t formation i n v i r t u a l l y 100% of thymocytes, which a f t e r 2 day culture i n ConASN were 17.9% 7D4 +. S i m i l a r l y , 2 day culture i n EL4SN induced approximately 40% of thymocytes to form b l a s t s , and of these, 15.9% expressed the IL-2R. To determine whether CTLp of a l l s p e c i f i c i t i e s were activated by EL4SN, B6 4 spleen c e l l s were cultured at 10 /0.2 ml i n c o n i c a l wells (the low c e l l density ensures no a c t i v a t i o n due to IL-2), and then tested f o r the a b i l i t y to k i l l a f t e r 5 days, (Figure 4). The e f f e c t o r s were added i n 3 51 various r a t i o s to 5 x 10 C r - l a b e l l e d targets, and assayed f o r cytotoxic a c t i v i t y against self/B6 (H-2 b), CBA (H-2 k), D2 (H-2 d), as well as P815 (H-2 d). Addition of PHA-P to P815 targets determines the t o t a l number of k i l l e r s present (57,58), which was found to be over a 30 Figure 4. EL4SN p o l y c l o n a l l y activates CTL. B6 spleen c e l l s were cultured with 15% EL4SN i n V-bottomed m i c r o t i t r e 4 plates at 10 /0.2 ml. Aft e r 5 days, c e l l s from eleven plates were 51 pooled and assayed f o r c y t o t o x i c i t y against the indicated C r - l a b e l l e d target c e l l s . Blast c e l l s were prepared by incubating spleen c e l l s at a density of lxlO^/ml with 2 ug/ml ConA, two days p r i o r to use. PHA-P, where added, was at a f i n a l concentration of 10 ug/ml. A t o t a l of 3 51 5x10 C r - l a b e l l e d targets were added to each w e l l . 3 1 Effector/Target ratio 32 hundred f o l d more frequent than those that could k i l l any s p e c i f i c target. As expected, k i l l e r s activated against s e l f were rare i n the population, and r e l a t i v e l y equal numbers of k i l l e r s were activated against the s p e c i f i c targets. These r e s u l t s were s i m i l a r to those expected f o r ConA activated e f f e c t o r s , suggesting that EL4SN can p o l y c l o n a l l y a c t i v a t e CTL i n the absence of antigen or mitogen (58). 4. The Target C e l l of EL4SN a. The target c e l l i s a T h y l + Lyt2* L3T4~ nylon wool  nonadherent c e l l . The target c e l l f o r EL4SN was determined by passing B6 lymph node (LN) c e l l s over a nylon wool column (NW) (59), and then t r e a t i n g the nonadherent c e l l s with monoclonal antibodies (MAbs) against the surface molecules Thyl, Lyt2, or L3T4, i n the presence of complement (C), to s e l e c t i v e l y remove c e r t a i n T c e l l subsets. The e f f i c i e n c y of the antibody plus C treatment was tested using FACS, and shown to be v i r t u a l l y 100% e f f e c t i v e (treatment with <xL3T4 + C resulted i n only 3.0% L3T4 + contaminants). These treated c e l l s were cultured 3 days i n 15% EL4SN and then tested f o r CTL a c t i v i t y i n a 5 day assay. As shown i n Table 6, removal of nylon wool adherent c e l l s and/or L3T4 + c e l l s had no e f f e c t on CTL induction. In contrast, treatment with anti-Lyt2 or anti-Thy-1 antibodies, abrogated the response. I t was concluded that the target of EL4SN i s a T h y l + L y t 2 + L3T4~ nylon wool (NW) nonadherent c e l l . b. EL4SN induces IL-2R expression on L y t 2 + c e l l s . Further confirmation of the above data was obtained when NW nonadherent c e l l s were treated with anti-L3T4 + C, and then cultured 3 days i n EL4SN. In the 33 Table 6. The target cel l for EL4SN is a Thy-1+ Lyt2+ L3T4 cell B6 LN Treatment EL4SN H-TdR incorporation % specific lysis 10% (v/v) (CPM ± S.E.) (M ± S.E.) 3 X i o 4 Untreated - 282 + 123 -1.3 + 1.2 1 X i o 4 - 710 + 117 -0.7 + 1.2 3 X i o 3 - 1,164 + 120 -0.2 + 2.2 3 X 10* Untreated + 17,426 + 1311 54.0 + 6.6 1 X 104 + 5,021 + 579 34.9 + 9.6 3 X 103 + 2,272 + 203 3.0 + 2.1 3 X 104 NW, C + 14,065 + 5746 39.8 + 4.3 1 X 104 + 3,236 + 578 12.2 + 3.9 3 X i o 3 + 2,379 + 459 0.4 + 1.0 3 X i o 4 NW, aThy-1 + C + 2,347 + 1019 -1.7 + 1.2 1 X 104 + 3,309 + 1616 -2.1 + 0.9 3 X 103 + 1,914 + 1255 -1.6 + 1.6 3 X 104 NW, aLyt2 + C + 2,194 + 54 -1.2 + 1.9 1 X 104 + 2,033 + 706 -1.1 + 1.5 3 X IO3 + 1,080 + 478 0.2 + 0.4 3 X 104 NW, aL3T4+C + 18,562 + 978 35.1 + 1.1 1 X 104 + 5,827 + 1050 5.0 + 2.3 3 X i o 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 of t h i s incubation, the c e l l number increased 8 f o l d , i n d i c a t i n g that the c e l l s that survived the antibody treatment (84% L y t 2 + , 3.0% L3T4 +, 2.7% 7D4 +, 14.4% F23.1 +) responded well to EL4SN. FACS analysis was performed on the cultured c e l l s (Figure 5). 78.0% of the cultured c e l l s expressed the Lyt2 marker and only 2.6% expressed L3T4. As well, 32.0% were 7D4 + and 17.9% were F23.1 +. It appeared that EL4SN could induce the expression of the IL-2R on L y t 2 + L3T4 - c e l l s , allowing them to 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 into CTL. In contrast, c e l l s cultured i n IL-2 alone under the same conditions d i d not p r o l i f e r a t e or d i f f e r e n t i a t e , and d i d not give r i s e to IL-2R expressing c e l l s (data not shown). 5. Biochemistry of BL4SN a. The CTL inducing a c t i v i t y i s a 27KDa IL-2-dependent f a c t o r . One ml of 20X concentrated EL4SN was loaded onto a G-100 column and run at 1.3 ml/hr, c o l l e c t i n g 1.2 ml f r a c t i o n s (Figure 6). The 0D O Q r i of each f r a c t i o n was measured, and then f i l t e r s t e r i l i z e d . Each f r a c t i o n was tested f o r IL-2 a c t i v i t y i n the MTT assay, and i t was shown to elute with an apparent molecular weight of 40 KDa. Fractions were also tested f o r CTL inducing a c t i v i t y with and without exogenous IL-2. With IL-2 included i n the assay, the CTL inducing a c t i v i t y eluted with an apparent molecular weight of 27 KDa. When no exogenous IL-2 was added, CTL were only induced by the f r a c t i o n s that contained both the EL4SN a c t i v i t y and IL-2 (data not shown). This observation supports the r e s u l t s obtained with the 7D4 antibody concluding that the CTL a c t i v i t y requires IL-2. b. The 27KDa f a c t o r induces CTL and IL-2 responsiveness. EL4SN has been shown to contain numerous factors with various a c t i v i t i e s . I t was 35 Figure 5. EL4SN induces IL-2R expression on L y t 2 + c e l l s . Nylon wool nonadherent B6 LN c e l l s were treated with GK1.5 mAb and 5 complement, and then cultured at 10 cells/0.2 ml i n round bottomed m i c r o t i t r e plates with 10% EL4SN added. A f t e r 3 days, the c e l l s were harvested and tested f o r c e l l surface molecules as described i n Figure 2. A f t e r culture FACS analysis showed 78.0% of the c e l l s to be L y t 2 + , 32.0% 7D4 +, 2.6% L3T4 + and 17.9% F23.1 +. 37 Figure 6. A 27KDa f a c t o r induces CTL a c t i v i t y . EL4SN was prepared (as discussed i n Materials and Methods) i n HB101 medium, concentrated 20 times, and 1 ml was loaded onto a 74 x 1 cm G-100 column. The column was eluted with PBS at a rate of 1.3 ml/hr, c o l l e c t i n g 1.2 ml f r a c t i o n s . Fractions were tested f o r CTL inducing a c t i v i t y by 3 c u l t u r i n g 5 x 10 B6 spleen c e l l s i n 0.2 ml V-bottomed wells with f r a c t i o n s added at a 1 i n 5 d i l u t i o n . Cytotoxic a c t i v i t y was tested a f t e r 4 5 days. IL-2 a c t i v i t y was detected by c u l t u r i n g 10 C2t cells/0.2 ml f l a t bottomed well with f r a c t i o n s added at a 1 i n 100 d i l u t i o n . P r o l i f e r a t i o n was measured 2 days l a t e r i n an MTT assay. The column was standardized using blue dextran to determine Vo i n one 3 run, then H-thymidine, alubmin, carbonic anhydrase and cytochome c were run to determine V 66 KDa, 29 KDa and 12.4 KDa, r e s p e c t i v e l y . 39 important to determine whether the CTL inducing a c t i v i t y was mediated through the same f a c t o r that induced the expression of the IL-2R. Using 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 tested f o r the a b i l i t y to induce IL-2 responsiveness i n thymocytes. Thymocytes were cultured i n 15% (v/v) pooled G-100 f r a c t i o n s (which were t i t r a t e d and shown to have the equivalent of 100 U/ml IL-2 a c t i v i t y ) f o r 3 days, and then tested f o r the a b i l i t y to respond to 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 to render thymocytes responsive to IL-2, i n d i c a t i n g that expression of the IL-2R had been induced. Previous studies using r a d i o l a b e l l e d PMA have shown that although PMA has a low molecular weight, i t can bind to large molecules i n f e t a l c a l f serum and elutes at the void volume when chromatographed (60). In that report, bound PMA was c l e a r l y separated from the IL-2 peak. Hence, the pooled G-100 f r a c t i o n s should be devoid of PMA, yet the a b i l i t y to induce CTL a c t i v i t y and IL-2R expression remained. The data above suggest that i t i s u n l i k e l y that the e f f e c t s of CTL induction and IL-2R expression induction were due to any r e s i d u a l PMA i n the EL4SN. Further evidence to support these findings was obtained when HB101 medium, supplemented with 10 ng/ml PMA, was concentrated 50 f o l d and then 1.0 ml was loaded onto a G100 column. 1.2 ml f r a c t i o n s were eluted with PBS at 1.3 ml/hr and tested at 1 i n 20 d i l u t i o n f o r CTL inducing a c t i v i t y i n the presence of 10 U/ml rIL-2 (data not shown). A c t i v i t y was detected i n f r a c t i o n s containing molecules with molecular weights greater than 66 KDa, suggesting that i f any PMA remained a c t i v e i n EL4SN, i t would elute separate from the IL-2 and CTL inducing a c t i v i t i e s . Table 7. 27KDa factor induces IL-2 responsiveness and IL-2R expression B6 thymocytes culture conditions a FACS Analysis % 7D4 + 3 H-TdR Incorporation i n response t o : 10 U/ml IL-2 15% v/v EL4SN fresh 9.3 10 U/ml rIL-2 5.4 15% EL4SN 52.5 15% G-100 f r a c t i o n s 1 3 N.D. 15% S-200 f r a c t i o n s 0 19.3 2,094 ± 1267 10,130 ± 684 15,611 ± 1261 23,740 ± 2837 N.D. 23,429 ± 704 48,119 ± 1271 85,373 ± 6620 57,659 ± 3132 N.D. a 5 B6 thymocytes were cultured at 5 x 10 c e l l s / m l i n large f l a s k s f o r 3 days. The c e l l s were then tested f o r the a b i l i t y to p r o l i f e r a t e i n response to various f a c t o r s , and f o r the expression of c e l l u l a r receptors. G-100 f r a c t i o n s 39-48 were pooled and found to have 15 U/ml IL-2 a c t i v i t y at 15% v/v, and when used at 10%, 36.0 ± 5.2% s p e c i f i c l y s i s was obtained i n a standard lectin-mediated assay. cS-200 f r a c t i o n s 53-64 were pooled and found to have 80 U/ml IL-2 a c t i v i t y at 15% v/v, and when used at 10%, approximately 22% s p e c i f i c l y s i s was obtained. 41 c. Further biochemical analyses. The i n i t i a l purpose of further p u r i f i c a t i o n was to separate the CTL a c t i v i t y from IL-2 a c t i v i t y , such that the b i o l o g i c a l a c t i v i t i e s detected i n column f r a c t i o n s could be correlated with bands on SDS-PAGE. The f i r s t step i n t h i s p u r i f i c a t i o n was to load 50 x concentrated EL4SN on an FPLC-Superose 12 column (which separates proteins on the basis of size) and to elute 0.5 ml f r a c t i o n s at 0.5 ml/min with PBS. Each f r a c t i o n was tested f o r CTL inducing and IL-2 a c t i v i t y (Figure 7). The r e s u l t s confirmed those observed with the G-100 column because IL-2 i s required i n the CTL assay, and the IL-2 and CTL a c t i v i t i e s eluted with s i m i l a r molecular weights. An ion exchange column was also used i n the attempt to fur t h e r p u r i f y the CTL inducing a c t i v i t y . I n i t i a l experiments with FPLC-Mono Q column indicated that the large amount of albumin i n the HB101 medium eluted i n a s i n g l e peak. The large concentration of protein was immunosuppressive and i n t e r f e r e d with the IL-2 and CTL assays. To overcome the d i f f i c u l t y with the albumin, an S-200 column was used to separate the albumin from the CTL inducing and the IL-2 a c t i v i t i e s . 2.55 ml of 50X concentrated EL4SN was loaded on the column and ran at 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 (Figure 8). Fractions 53-64 were pooled, and 10 ml were loaded onto the Mono Q column. This run had extremely l i t t l e OD, however, the a c t i v i t i e s d i d not separate well (Figure 9). It has been reported before (54) that IL-2 dependent l i n e s can respond to factors other than IL-2, r e s u l t i n g i n f a l s e p o s i t i v e s . However, i n the case of the EL4SN, the f a c t o r appeared to have an inherent q u a l i t y of multiple charged forms, and thus could not be separated i n t h i s manner. 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 Figure 8. S-200 column e l u t i o n p r o f i l e : to remove albumin from CTL-inducing a c t i v i t y . EL4SN was prepared as described i n Figure 7 and 2.55 ml of the 50 f o l d concentrate were loaded onto a 1.6 x 100 cm S-200 column. The column eluted with 20 mM T r i s HCl at a rate of 12.9 ml/hr c o l l e c t i n g 2.15 ml fr a c t i o n s which were tested as described i n Figure 7. 46 Figure 9. FPLC-Mono Q column e l u t i o n p r o f i l e f o r albumin-depleted EL4SN. S-200 f r a c t i o n s 53-64 were pooled and 10 ml were loaded onto the Mono Q column. The column was eluted with 20 mM T r i s HCl (with a l i n e a r gradient of NaCl from 0 to 0.3 molar) i n 1 ml f r a c t i o n s which were tested as described i n Figure 7. hi 48 Further evidence that PMA was not responsible f o r the EL4SN e f f e c t was obtained using the S-200 f r a c t i o n s 53-64. These f r a c t i o n s induced CTL a c t i v i t y and IL-2R expression i n B6 thymocytes. Af t e r 3 day culture i n 15% v/v S-200 f r a c t i o n s , 10.3% of the c e l l s expressed the 7D4 determinant. (Fractions 53-64 contain no high molecular weight proteins and therefore should be free of PMA). In summary, the f a c t o r i n EL4SN that activates CTLp i n the presence of IL-2 eluted from a G-100 column with an apparent molecular weight of 27KDa. I t i s the same f a c t o r that mediates the induction of IL-2 responsiveness ( i e , induces IL-2R expression). The G-100 and S-200 column data also suggest that PMA i s not involved to any s i g n i f i c a n t extent i n the e f f e c t s studied, because f r a c t i o n s void of any PMA had both a c t i v i t i e s . 49 DISCUSSION In t h i s t h e s i s , a f a c t o r i s described that i s present i n the supernatant of PMA-induced EL4 c e l l s and has the a b i l i t y to n o n s p e c i f i c a l l y induce r e s t i n g CTLp to become p o l y c l o n a l l y activated k i l l e r s . The target c e l l of the f a c t o r includes Thy-1 + L y t 2 + L3T4~ c e l l s , which can be induced to respond to IL-2 upon culture i n EL4SN. [It i s possible that EL4SN has more than one a c t i v i t y . I t can induce Lyt2~ L3T4 dl5 thymocytes to p r o l i f e r a t e and i n mature lymph nodes, EL4SN mediates i t s e f f e c t through the L y t 2 + L3T4~ c e l l s (the frequency of Lyt2~ L3T4 c e l l s i n lymph nodes i s very low)]. The e f f e c t i s not due to IL-2 alone, but requires IL-2. CTL induction i s mediated by a 27KDa fa c t o r that acts i n the absence of any s p e c i f i c antigen or mitogen stimulation of the target c e l l s . This same f a c t o r i s capable of inducing the expression of the IL-2 receptor. The recent reports of the e f f e c t s of IL-4 and IL-5 on T c e l l s made i t necessary to r u l e out these lymphokines as the mediators of the EL4SN e f f e c t . Neither IL-4 nor IL-5 could induce CTL i n the presence of IL-2, with or without PMA added. In addition, anti-IL-4 antibodies had no e f f e c t on the a b i l i t y of EL4SN to act. Thus, i t can be concluded that EL4SN contains a unique lymphokine. As mentioned, there have been several reports of f a c t o r s which can induce CTL by a c t i v a t i n g the L y t 2 + CTLp i n the presence of IL-2 (24-27; 31-41). In addition to those already discussed, Garman et a l . described a 30KDa and a 17KDa CTL helper f a c t o r from murine a n t i - v i r u s spleen c e l l culture (38). Erard e_t a l . described 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). Their factor is 30-35 KDa, as determined by gel chromatography. 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 that the f a c t o r present i n EL4SN i s a unique and new mediator of CTL a c t i v a t i o n that acts through an a l t e r n a t i v e pathway of CTL induction. The well accepted pathway of CTL induction involves antigen-triggering leading to IL-2R expression, followed by p r o l i f e r a t i o n and d i f f e r e n t i a t i o n i n response to IL-2. In the case of the EL4SN fa c t o r , not only i s there no antigen-triggering, the c e l l s do not even need to express the antigen receptor, as dl5 f e t a l thymocytes were responsive to t h i s f a c t o r . There are several examples of other receptors on T c e l l s that can be triggered with the ensuing response of p r o l i f e r a t i o n and d i f f e r e n t i a t i o n . For example, MAbs to the Thy-1 and TAP c e l l surface molecules can a c t i v a t e T c e l l s (17,18). Thus, i t i s conceivable that molecules such as Thy-1 or TAP could p o t e n t i a l l y serve as lymphokine receptors, and that T c e l l a c t i v a t i o n signals could be delivered as a re s u l t of such an i n t e r a c t i o n . In conclusion, although many factors have been observed i n d i f f e r e n t c e l l supernatants, and have been assayed i n various systems, they s t i l l possess remarkably s i m i l a r properties. A l l seem to be a product of activated T c e l l s , and act i n synergy with IL-2. They a l l induce the Ly t 2 + L3T4 - subset of T c e l l s , and most appear to have an e f f e c t on the IL-2 receptor. The simple f a c t that CTL inducing factors have been described so often i n such d i f f e r e n t systems suggests that the mechanism through which they act i s important to the function of the immune system. 52 REFERENCES 1. Farrar, W.L., S.B. Mizel and J . J . Farrar. 1980. P a r t i c i p a t i o n of lymphokine a c t i v a t i n g f a c t o r (Interleukin 1) i n the induction of c y t o l y t i c T c e l l responses. J . Immunol. 124:1371. 2. Kaye, J . , S. G i l l i s , S.B. Mizel, E.M. Shevach, T.R. Malek, CA. Di n a r e l l o , L.B. Lachman and CR. Janeway, J r . 1984. 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