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The Journal of Immunology, 2004, 173: 7062-7069.
Copyright © 2004 by The American Association of Immunologists

Self-Reactive T Cell Receptor-Reactive CD8+ T Cells Inhibit T Cell Lymphoma Growth In Vivo1

Marie Gonthier*, Régine Llobera*, Jacques Arnaud* and Bent Rubin2,{dagger}

* Centre National de la Recherche Scientifique-Unité Propre de Recherche 2163, Centre-Hopital-Universitaire Purpan, Toulouse, France; and {dagger} Unité Mixte Recherche 2587, Centre National de la Recherche Scientifique-Pierre Fabre, Toulouse, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Syngenic C57BL/6 mice (H-2b) vaccinated with mitomycin C-treated L12R4 T lymphoma cells develop protective immunity toward the MHC class II-negative tumor cells. In the present study, we characterize the nature, mode of function, and specificity of the effector cells in this immunity. These cells are TCR-specific CD8+ T lymphocytes with effector function in vitro as well as in vivo upon transfer to naive mice. They produce high levels of IFN-{gamma} and TNF-{alpha}, but little or no IL-4. By means of TCR{beta}-negative variant L12R4 cells, P3.3, and TCR-V{beta}2 cDNA-transfected and TCR-V{beta}2-expressing P3.3 lymphoma cells, we found that a significant part of the effector T cells are specific for the V{beta}12 region. The growth inhibition of L12R4 cells in vitro was inhibited by anti-H-2, anti-Kb, and anti-Db mAb. Furthermore, vaccination with V{beta}12 peptide p67–78, which binds to both Kb and Db MHC class I molecules, induces partial protection against L12R4 T lymphoma cells. Thus, self-reactive TCR-V{beta}-specific, Kb-, or Db-restricted CD8+ T cells mediate inhibition of T cell lymphoma growth in vitro and in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The immune system, normally characterized by specificity, enormous repertoire, efficiency, and fine-tuned regulation, does not always function optimally and beneficially to the host. Three examples are allergy, autoimmunity, and failure to protect against syngenic tumors. In the first case, the immune system reacts far too strongly. In the two latter cases, the system reacts inappropriately and insufficiently, respectively, against self-components (1, 2). Immune responses are initiated by the interaction between processed Ag presented by APC and the TCR complex on the surface of Th cells. Upon activation, these latter cells produce cytokines, which help the development of Ag-specific B cells and CTLs from precursor cells. The TCR molecule is a heterodimer of TCR{alpha} and TCR{beta} chains, the genes of which rearrange randomly in differentiating T cells. Therefore, developing T cells may express one of multiple combinations of TCR{alpha} and {beta}-chains and some of these combinations may be autoreactive (3, 4). Autoimmune reactions are normally avoided by negative selection of autoreactive immune cells (5, 6). Negative selection in the thymus is dependent on the avidity of the TCR for its ligand (formed as a function of the avidity of the MHC-encoded class I and class II molecules for self-peptides) as well as on the interaction of several coreceptors on both APC and Th cells (7). Thus, T cells with high-avidity TCR specific for TCR peptides associated with class I or class II proteins are assumed to be deleted.

However, immune cells with reactivity against variable and constant regions of self-TCR are present in the immunological repertoire of normal mice (8, 9, 10, 11, 12, 13, 14, 15, 16). B cell responses are directed against conformational TCR epitopes, producing anti-clonotypic Abs (8, 9, 10, 11, 12). In contrast, CD4+ and CD8+ T cell responses are specific for TCR peptides associated with MHC class II and class I molecules, respectively (13, 14, 15, 16). Nascent TCR chains may either be processed for MHC class I presentation within the T cell itself (17) or TCR proteins may somehow be internalized, processed, and associated with MHC class I or class II molecules by professional APC (18).

TCR-specific CD4+ T cells have been described as playing an important regulatory role in autoimmunity (13, 15, 16, 19, 20). CD8+ T cells have also been suggested to play such a role, but in these studies either the function, specificity, or MHC restriction of the CD8+ cells were not reported (14, 21, 22). We have found previously 1) that TCR-specific CD8+ T cells play a significant biological role in the regulation of experimental autoimmune encephalomyelitis (EAE)3 (23, 24) and 2) that mice vaccinated with mitomycin C (Mito-C)-treated T lymphoma cells were resistant to new challenge with the same T lymphoma cells and that this immunity may be TCR specific (24, 25). In the present study, we have tried to elucidate the different parameters of an immune response to TCR chains: 1) the lymphocytes involved in the initiation and effector phases of the response, 2) the mechanism of the effector phase, and 3) the fine specificity and MHC restriction of the effector cells. The protective immune response of C57BL/6 mice to the syngeneic L12R4 T lymphoma cells was used as a model system. Our data show that TCR-V{beta} peptides may be presented by L12R4 T lymphoma cells and that vaccination against the TCR-V{beta}12 region of the L12R4 TCR{alpha}{beta} heterodimer protects against L12R4 cells, but not against TCR{beta}-negative L12R4 cells, TCR-V{beta}2-expressing L12R4 cells, or C57BL/6 T lymphoma cells with other TCR{alpha}{beta} heterodimers (15). Furthermore, IFN-{gamma}- and TNF-{alpha}-producing CD8+ T cells with reactivity to TCR-V{beta} regions play an important role in protection against L12R4 T cell lymphomas. In contrast, CD4+ T cells appear important in certain stages of the immune response to L12R4 cells, but not for direct effector function against the T lymphoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, immunization, and cell transfer

C57BL/6 (B6), BALB/c, and B6-IFN-{gamma}–/– mice were bred in our in-house specific pathogen-free animal facility from stocks originally obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were immunized i.p. with 10 x 106 Mito-C-treated T lymphoma cells three times at weekly intervals (25). After 2–3 wk of rest, the mice were challenged with 102–107 live tumor cells (102–104 L12R4 cells kill normal B6 mice in 3–4 wk) (25). Naive mice were injected i.p. with 5–50 x 106 spleen cells, T cells, or T cell subsets 1 day before challenge with 103–104 live tumor cells. Ascites tumor growth was monitored every second day for up to 90 days.

Cells, mutagenesis, and cDNA transfection

L12R4 T lymphoma cells (H-2b), P815 mastocytoma cells, and L1210 B lymphoma cells (H-2d) were used as described previously (24, 25). They were grown in vivo by passage of 106 live tumor cells every 10–14 days or in vitro in 8% FCS/RPMI 1640 (FCS/R) medium. X6310 cells produce mouse IL-2 in 8% FCS/R (26); these cells were generously provided by Dr. F. Melchors (Basel Institute for Immunology, Basel, Switzerland). L12R4 cells were mutagenized as described previously for human Jurkat T cells (27). Surface TCR/CD3-negative L12R4 cells were immunoselected, cloned at limiting dilution, and one clone (P3.3) was selected (see Results). P3.3 cells were transfected with either V{beta}2 or V{beta}12 DNA (27) and V{beta}2+ or V{beta}12+ transfectant cells were selected with specific mAb using flow cytometry. The V{beta}2+ P3.3 cells, called T142.4 cells, were used in the present experiments (see Fig. 3). Both P3.3 and T142.4 cells grow in B6 mice as L12R4 cells.



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  		FIGURE 3. Protection against wild-type L12R4 cells is dependent on the expression of the TCR-V{beta}12 chain. Twenty-four B6 mice were immunized three times at weekly intervals with 10 x 106 L12R4M cells ({blacksquare}, {blacktriangleup}, and •) and eight B6 mice were immunized with P3.3M cells ({square}). Two weeks later, the L12R4-immunized mice were divided into three groups of eight mice that were challenged with 103 live L12R4 cells (•), P3.3 cells ({blacktriangleup}), or P3.3-V{beta}2-transfected T142 cells ({blacksquare}). The P3.3-immunized mice ({square}) and eight normal mice ({circ}) were challenged with 103 live P3.3 cells. Tumor growth was monitored every second day throughout 90 days. The results from days 16 to 50 are shown, and they are expressed as percent survival. Seven mice from the L12R4M-restimulated group survived beyond day 90 (data not shown).

 
Reagents

The 2C11 anti-CD3{epsilon}, H57 anti-TCR.C{beta}, anti-V{beta}2, H129 anti-CD4, H58.55 anti-CD8, and H140.61 anti-Thy1 mAb were produced in vitro from hybridoma cells obtained from Drs. J. Bluestone, R. Kubo, B. Malissen, and M. Pierres, respectively (28). Anti-TCR.C{alpha}, anti-Kb, anti-Db, and anti-V{beta}12 mAb were obtained from BD Pharmingen (San Diego, CA). M1/42 rat anti-mouse MHC class I mAb and Ox3 anti-IAb mAb were provided by American Type Culture Collection (Manassas, VA) and Dr. A. Williams (Oxford, U.K.), respectively. MTT was purchased from Sigma-Aldrich (St. Louis, MO). Thirty microliters of a 5-mg/ml solution in Dulbecco’s PBS was added per well.

IL-4, IFN-{gamma}, and TNF-{alpha} contents in T cell supernatants (harvested on days 1, 2, or 3) were determined using BD Pharmingen ELISA kits 555232, 555138, and 558874, respectively, by means of the protocol indicated by the manufacturer. IL-2 activity was measured by growth of CTLL-2 cells (28).

The p67–78 peptide was produced and purified by Neosystems (Strasbourg, France). It was >95% pure.

T cell lines and cell separations

T and B cell separations were performed using either anti-Thy1 mAb or anti-mouse Ig (MIg) Ab-coated magnetic beads from Miltenyi Biotec (Auburn, CA). CD4+ or CD8+ T cells were obtained from cells first treated with anti-MIg beads, and then column nonadhering cells were incubated with either anti-CD4 mAb or anti-CD8 mAb beads. In different experiments, CD4+ and CD8+ T cells were prepared by both positive and negative selection. In some cases, T cells were purified on MIg anti-MIg columns as before (28).

T lymphoma cells used for immunization or for stimulator cells in vitro were treated with 100 µg/ml of Mito-C (Sigma-Aldrich) at 37°C for 1 h followed by three washings. Spleen cells were treated with 50 µg/ml Mito-C at 37°C for 30 min, and effector T cells were treated with 5–50 µg/ml Mito-C at 37°C for 30 min. Mito-C-treated cells are indicated by L12R4M or spleenM cells.

T cell lines were made by culturing 25 x 106 spleen cells with 106 L12R4M cells in 10 ml of 8% FCS/R medium. After ~10 days, 5 x 106 surviving cells (>90% T cells) were stimulated with 106 L12R4M cells and 10 x 106 spleenM cells; after another 10–14 days, 2 x 106 T cells were stimulated with 106 L12R4M cells and 20 x 106 spleenM cells, and the line was stimulated repeatedly in this manner. T cell lines grown in this way contained ~60–70% CD4+ and 30–40% CD8+ cells. CD8+ T cells were selected by growth in 10% of X6310 IL-2 supernatant and L12R4M cells only. The percentage of T cell subsets as well as the content of anti-TCR Abs in antiserums from vaccinated mice were determined by flow cytometry using fluorescent F(ab')2 of rabbit anti-MIg Abs as described previously (27, 28).

L12R4 cell growth inhibition assay

To get optimal conditions for the L12R4 growth inhibition assay, we first studied the concentration of tumor cells per well (200 µl). It was found that 103–104 L12R4 cells gave optimal incorporation of MTT dye (measured as A540 in a Titertech microtiter plate spectrophotometer) during 1- to 3-day assays. In most experiments, 104 L12R4 cells were used. Effector cells were used up to 106 cells/well (ratio 1:100). Normal spleen cells and effector T cells cultured alone or with L12R4M cells incorporated little MTT. However, immune effector cells from in vivo incorporated appreciable amounts of MTT. Therefore, effector cells were treated with different concentrations of Mito-C as described above. Ten to 20 µg/ml Mito-C inhibited effectively cell proliferation without affecting the growth inhibitory activity. Each experimental point was the mean of A540 values from four cultures (these values varied <10%).

T cell proliferation and cytotoxicity assays

Proliferation of effector T cells against different T lymphoma cells and splenic APC was measured by [3H]thymidine incorporation (the radioactivity was present for 18 h between days 4 and 5), and the 51Cr release assay was performed as described previously (28, 29)

cDNA cloning and sequence analysis

Total RNA was prepared from 107 L12R4 cells by means of the RNA PLUS method (Bioprobe Systems, France); cDNA production, PCR analysis, cloning of PCR products with the pCTTM II vector kit (Invitrogen Life Technologies, San Diego, CA), and nucleotide sequencing using the T7 kit (Pharmacia, Uppsala, Sweden) were all performed as described in detail elsewhere (27).

Immunoprecipitations

L12R4 T cells, variant cells, and transfectant cells were labeled with [35S]methionine/cysteine (for 90 min), solubilized in 1% digitonin or 1% Nonidet P-40 detergent lysis buffer, immunoprecipitated with anti-TCR/CD3 mAb and protein G-Sepharose beads, and analyzed by SDS-PAGE as described previously (27, 30, 31).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of protective immunity against L12R4 cells

Injection of live T lymphoma cells like L12R4 or C6VL cells (100–1000/mouse) kill C57BL/6 mice (B6) in 3–4 wk. In contrast, as many as 50 x 106 L12R4 cells grow only transiently in BALB/c mice, which totally reject the tumor cells around day 10 (data not shown; Ref.25). However, if syngeneic mice are preimmunized with Mito-C-treated lymphoma cells, they become resistant to the tumor cells used for vaccination (23, 24, 25). To get more insight into the mechanism of induction of the protective immune response, B6 mice were immunized with different doses of L12R4M cells as described in Materials and Methods. Three weeks after the last vaccination, the mice were challenged with 103 live L12R4 cells. As can be seen in Fig. 1A, mice immunized three times with 10 x 106 L12R4M cells were protected. Two of 10 mice immunized with 20 x 106 L12R4M cells died of tumors; the kinetics of tumor cell growth in these two mice indicated that tumor cell growth was due to residual live L12R4 cells in the Mito-C-treated population used for immunization. In contrast, immunization with 0.5–1 x 106 L12R4M cells had little effect and immunization with 2–5 x 106 L12R4M cells had suboptimal effects. The same results were obtained in three similar experiments.



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  		FIGURE 1. Effect of the L12R4M vaccination dose on protective immune responses and on CD4 T cell proliferative responses. A, Groups of 16 B6 mice were vaccinated with either Dulbecco’s PBS or 0.5–20 x 106 L12R4M cells. Three weeks after the last immunization, 10 mice of each group were challenged with 103 live L12R4 cells and tumor cell-induced death was monitored. The x-axis is the same for Fig. 2, i.e., mice immunized three times with different doses of L12R4M cells (x106). The y-axis is percent dead mice 6 wk after live L12R4 cell challenge. B, Unfractionated spleen cells from immune mice were stimulated (5 x 105 cells/well) directly with L12R4M cells (105/well) in vitro (•); alternatively, CD4+ T cells were purified with anti-CD4 mAb-coated magnetic beads and then stimulated in vitro (2 x 105/well) either with L12R4M cells alone ({triangleup}), L12R4M cells and 2 x 105/well normal spleen cellsM ({blacksquare}), or immune spleen cellsM (5 x 105/well) from mice vaccinated with different doses of L12R4M cells without additional L12R4M cells ({diamond}). The y-axis is [3H]thymidine uptake expressed as percentage of the maximal response obtained, i.e., the proliferative response of immune spleen cells from 10 x 106 L12R4M-vaccinated mice was taken as 100% (123,900 cpm; background = 2,109 cpm).

 
To investigate the nature of the L12R4-reactive effector cells, we studied the proliferative responses and IL-2 production of spleen cells from mice immunized with different numbers of L12R4M cells. The data in Fig. 1B illustrate that spleen cells from mice immunized with 10–20 x 106 L12R4M cells showed strong proliferative responses; spleen cells from mice immunized with 0.5–5 x 106 L12R4M cells showed increasing, intermediate responses. Similar results were obtained using IL-2 production as an assay system (data not shown). Thus, the in vitro proliferative results correlated with the protective responses observed in Fig. 1A.

To demonstrate whether the increased proliferative and protective responses were attributable to increased frequency of L12R4-specific CD4+ T cells and/or of APC-expressing L12R4 Ag in association with MHC class II molecules, we purified CD4+ T cells from vaccinated mice. Such T cells alone do not respond to L12R4M cells (Fig. 1B). However, if normal, Mito-C-treated splenic APC were added to the cultures, the CD4+ T cells proliferated in a manner similar to immune spleen cells (Fig. 1B). Thus, the activity of L12R4-specific CD4+ T cells elevates with increasing doses of vaccination. Furthermore, if L12R4-reactive CD4+ T cells were cultured with splenic APC from mice vaccinated with different doses of L12R4M cells but without addition of L12R4M cells, only APC from mice immunized with 10–20 x 106 L12R4M cells could activate the L12R4-specific CD4+ T cells by themselves. These data indicate that such immune spleen cells contain sufficient amounts of APC-expressing L12R4 Ag to activate L12R4-specific CD4+ T cells.

On the specificity of the protective immune responses to T lymphoma cells

Since both L12R4 and C6VL cells were of B6 origin but with different TCR{alpha}{beta} heterodimers (15, 25), one possible target Ag for the protective immunity was the TCR{alpha}{beta} variable regions. To get more direct evidence for a V{alpha}/V{beta}-specific immunity, we induced mutagenesis in L12R4 cells (27) and isolated variant cells with no TCR/CD3 expression at the cell surface (FACS analysis with anti-CD3{epsilon} mAb). One resulting clone, P3.3, was used for subsequent experiments (Fig. 2A). [35S]Methionine/cysteine-labeled TCR/CD3 proteins were isolated from digitonin cell lysates by precipitation with anti-TCR.C{alpha}, anti-TCR.C{beta}, or anti-CD3{epsilon} mAb. Precipitated material was separated by SDS-PAGE under reducing conditions. As can be seen in Fig. 2B, surface membrane TCR/CD3-negative P3.3 variants (lanes 3) lacked expression of TCR{beta} chains, whereas TCR{alpha} chains and CD3 molecules were present in the variant cells. P3.3 cells transfected with unrelated TCR-V{beta}2 cDNA regained surface expression of TCR-CD3 complexes (T142 cells, Fig. 2A), indicating that the lack of TCR/CD3 membrane expression is due to the absence of autologous TCR{beta} chains only. TCR-V{alpha} and TCR-V{beta} cDNA from L12R4 cells were cloned and sequenced: L12R4 cells express V{alpha}10 and V{beta}12J{beta}2.3 variable regions. We confirmed that P3.3 cells indeed lacked autologous TCR{beta} chains by biosynthetic labeling and immunoprecipitation with anti-V{beta}12 mAb, and we could find no TCR{beta} mRNA in P3.3 cells using PCR (data not shown). Transfection of P3.3 cells with V{beta}12 cDNA also reconstituted surface TCR/CD3 membrane expression (data not shown). Thus, a useful model system for the study of TCR{beta}-specific immunity was produced: V{alpha}10+/V{beta}12+ L12R4 wild-type cells, V{alpha}10+/V{beta}12– P3.3 cells, and V{alpha}10+/V{beta}2+ T142 cells.



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  		FIGURE 2. Characterization of surface TCR/CD3-negative L12R4 variant cells. A, Percent positive cells in flow cytometry using mAb to surface markers: CD3{epsilon}, V{beta}2, V{beta}12, H-2Kb, or H-2IAb with L12R4 wild-type cells, surface TCR/CD3-negative L12R4 cells (P3.3), and P3.3 cells transfected with TCR-V{beta}2.C{beta} cDNA. B, Immunoprecipitation (IP) using anti-TCR.C{alpha} or anti-TCR.C{beta} mAb on digitonin lysates from [35S]methionine/cysteine-labeled L12R4 (lane 2) or P3.3 cells (lane 3). L1210 B lymphoma cells served as negative control (lane 1). To the left is indicated molecular mass markers (in KDa) and to the right is indicated partially and totally glycosylated (glyc) TCR{beta} chains, TCR{alpha} chains, and CD3{delta}{epsilon} chains. SDS-PAGE was performed under reducing conditions in a 10% polyacrylamide gel. It can be seen that P3.3 cells synthesize very little, if any, TCR{beta} chains.

 
Next, we immunized B6 mice with either Mito-C-treated L12R4 or P3.3 cells. The L12R4-primed mice were divided into three subgroups challenged with lethal doses of L12R4, P3.3, or T142 cells. Development of tumors was monitored every second day. The data in Fig. 3 show that L12R4-vaccinated mice were resistant to live L12R4 cells but not to P3.3 or T142 cells. In contrast, P3.3-vaccinated mice, compared with normal B6 mice, were only partially protected against live P3.3 cells (Fig. 3). Furthermore, B6 mice vaccinated with T142 cells (V{beta}2+) induced protective immunity to T142 cells but not to L12R4 or P3.3 cells (data not shown). These results and those from five independent experiments (24) suggest that V{beta}12 epitopes play an important role in the protective immunity to L12R4 cells. Moreover, antisera were isolated from vaccinated mice on the day of live tumor cell challenge and again 4 wk later when the tumor cells were rejected. Abs reactive against L12R4 cells but not against P3.3 cells were looked for using flow cytometry, but were not found (data not shown).

Characterization of the L12R4-specific effector cells

Immune cells in a protective tumor-immune response in vivo can inhibit proliferation of the same tumor cells in vitro (32). Since we were unable previously to demonstrate V{beta}8.2-specific CTLs with reactivity in a standard 51Cr release assay (23), we established a growth inhibition assay with L12R4 cells. L12R4 (104) cells were cultured for 1, 2, or 3 days and proliferation was measured by uptake of MTT dye. As negative control effector cells, we used normal spleen cells and BALB/c anti-B6 immune cells served as positive controls. Effector spleen cells from mice protected against L12R4 cells inhibited L12R4 cell growth, whereas P3.3 cell proliferation was affected much less (data not shown). Growth inhibition could be due to either induction of cell death or inhibition of one or more stages in the cell growth cycle induced by cytokines. Therefore, we cultured L12R4 cells with either normal spleen cells, L12R4-immune spleen cells, or BALB/c anti-L12R4 immune cells. The number of live V{beta}12+ L12R4 cells was measured on days 1, 2, or 3 by flow cytometry. Seventy, 95, and 100% of V{beta}12+ L12R4 cells were eliminated by L12R4-immune cells on days 1, 2, and 3, respectively. All L12R4 cells were eliminated by allogeneic CTLs on day 1 and V{beta}12+ L12R4 cells were not affected by normal spleen cells (Fig. 4). These data strongly suggest that L12R4 cell proliferation is arrested due to induction of cell death. Consequently, we tried to test the cytotoxic activity of either BALB/c anti-L12R4 T cells or L12R4-immune T cells from protected mice in a 51Cr release assay. However, only allogeneic CTLs kill L12R4 cells efficiently in 4–6 h; some specific cytotoxicity was observed with L12R4-immune T cells but only after 18 h, where the spontaneous 51Cr release from L12R4 cells was ~40–50% (data not shown). Thus, it appears that our effector T cells may be long-term killer cells (see Discussion).



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  		FIGURE 4. In vitro inhibition of L12R4 cell growth by L12R4-immune and alloantigen-specific T cells. Briefly, 2 x 105 L12R4 cells were cultured in the presence of 106 normal spleen cells ({blacksquare}), 106 BALB/c anti-L12R4 cells ({cjs2108}), or 106 B6 anti-L12R4M cells ({square}) for 0, 1, 2, or 3 days (y-axis). The number of V{beta}12+ cells was determined by flow cytometry using FITC-labeled anti-V{beta}12 mAb. The data are expressed as percentage of the number of V{beta}12+ L12R4 cells at the time of cell mixture (x-axis).

 
To better characterize the effector cells, we made T cell lines from L12R4-immune spleen cells. The cell lines contained almost exclusively T cells on day 10. Tumor cell growth inhibitory activity increased during T cell culture, in particular if the cell line was supplemented with IL-2 during stimulation; in parallel, the proportion of CD8+ T cells increased from ~30% to almost 100%. The data in Fig. 5 clearly demonstrate that the effector cells in the L12R4 growth inhibition assay were CD8+ T cells and that they reacted autonomously. Furthermore, the CD8+ T cells inhibited the growth of L12R4 cells, but not (or much less) the growth of P3.3, T142, or L1210 (H-2d lymphoma cells). The results in Fig. 6 show that the CD8+ T cells produced high amounts of IFN-{gamma} and TNF-{alpha} only when stimulated with L12R4 cells; they never produced detectable levels of IL-4 (or IL-2, data not shown).



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  		FIGURE 5. CD8+ L12R4-immune T cells eliminate L12R4 tumor cells autonomously. Briefly, 104 tumor cells were cultured alone (group 0) or in the presence of different effector cells (groups 1–6). E:T ratios were 20:1 for normal spleen cells (group 1), 5:1 for BALB/c anti-B6 CTL (group 6), and 10:1 for all other effector cell combinations: total L12R4-immune T cells (group 2), CD4+ T cells (group 3), CD8+ T cells (group 4), and CD4+ plus CD8+ T cells (group 5). Groups 2–5 are derived from a T cell line specific for L12R4 cells (sixth restimulation in vitro). White bars represent L12R4 cells, light gray bars represent P3.3 cells, dark gray bars indicate T142 cells, and black bars indicate L1210 cells. The x-axis shows A540 values of MTT uptake. Effector cells were treated for 20 min with 10 µg/ml Mito-C at 37°C before assay as described in Materials and Methods. The assay was terminated after 2 days of culture.

 


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  		FIGURE 6. Cytokine production by L12R4-specific CD8+ T cell line and its capacity to transfer protection to naive mice. Briefly, 106 L12R4-specific CD8+ T cells were stimulated with 5 x 104 L12R4M cells (open columns), P3.3M cells (light gray columns), T142M cells (medium gray columns), or medium alone (gray columns, next to control). Supernatants were harvested after 24, 48, or 72 h. The results are expressed as percentage of values obtained from a 36-h Con A-supernatant (black columns) from BALB/c spleen cell cultures (2.5 x 106/ml). Content of IFN-{gamma}, TNF-{alpha}, and IL-4 was determined by capture ELISA (BD Pharmingen). The values are in the picogram per milliliter range for all three cytokines; data from 48-h supernatants are shown.

 
L12R4-specific CD8+ T cells transfer the protective state to naive recipients

Previously, we have shown that T cells from L12R4-protected mice could transfer protection to naive recipient mice (24). In the present study, we asked whether CD8+ T cells could transfer protection alone or whether CD4+ T cells were also needed for effective protective immunity (Fig. 1). The results in Fig. 7 demonstrate that L12R4-specific CD8+ T cells from either L12R4-immune mice, or from L12R4-specific T cell lines, transferred complete immunity toward live L12R4 cells (but not to P3.3 cells, data not shown). Thus, V{beta}12-specific CD8+ T cells appear to be the actual effector cells in passive transfer of protective immunity to L12R4 cells in vivo as well as in inhibition of L12R4 growth in vitro.



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  		FIGURE 7. CD8+ L12R4-specific T cells transfer protection to naive B6 mice. Groups of six naive B6 mice were injected i.p. with 10 x 106 effector cells 1 day before challenge with 103 live L12R4. Effector cells were either L12R4-immune spleen cells from B6 mice ({blacksquare} and {square}) or an L12R4-specific T cell line ({blacktriangleup} and {triangleup}). The L12R4-specific T cell line contained 63% CD4+ and 37% CD8+ T cells. The L12R4-immune spleen cells were depleted of B cells (see Materials and Methods) and enriched for CD8+ ({blacksquare} and {blacktriangleup}) or CD4+ ({square} and {triangleup}) by incubation with anti-CD8 mAb-coated magnetic beads. Tumor growth was observed every second day for 90 days (only the results from days 18 to 40 are shown), and the results are expressed as percent survival. Six mice injected with the CD8+ in vitro L12R4-specific T cell line and five mice injected with CD8+ in vivo L12R4-immuneT cells were alive on day 90.

 
Role of IFN-{gamma} in the protective immunity

IFN-{gamma} production by the V{beta}12-specific, CD8+ T cells appears to be an important part of the effector function of these cells. Therefore, we investigated whether protective immunity against L12R4 cells could be induced in B6 mice lacking functional IFN-{gamma} genes. Normal and IFN-{gamma}–/– B6 mice were immunized three times with L12R4M cells. After 2 wk, the mice were challenged with 103 live L12R4 cells. As before, almost 100% of normal B6 mice were protected by vaccination, whereas none of the IFN-{gamma}–/– B6 mice was protected by vaccination (data not shown). We then isolated T cells from the L12R4-immune mice and transferred these T cells to naive B6 mice of either normal or IFN-{gamma}–/– genotype. The data in Fig. 8 clearly show that L12R4-immune T cells from primed B6 mice can transfer protective immunity to both normal and IFN-{gamma}–/– B6 mice, although this protection is less long-lasting in IFN–/– mice. These results suggest that IFN-{gamma} production is necessary for L12R4-specific CD8+ T cells to exert their protective activity against tumor cell growth (see Discussion).



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  		FIGURE 8. CD8+ L12R4-specific T cells from immune B6 mice can transfer protective immunity to IFN-{gamma}-deficient B6 mice. Normal ({circ} and •) or IFN-{gamma}-deficient ({triangleup} and {blacktriangleup}) naive mice were injected with 10 x 106 L12R4-immune T cells from protected B6 mice ({circ} and {triangleup}) 1 day before challenge with 104 live L12R4 cells. Groups of normal and IFN-{gamma}-deficient B6 mice (• and {blacktriangleup}, full lines) were challenged with 104 live L12R4 cells as controls. Tumor growth was monitored every second day for 90 days (data from days 18 to 40 are shown), and the results are expressed as percent survival. All six normal mice receiving L12R4-immune T cells were alive on day 90. In contrast, the last four IFN-{gamma}-deficient mice died on days 51, 65, 73, and 73.

 
On the fine specificity of the V{beta}12-reactive CD8+ T cells

The peptide-binding motifs of H-2Kb and H-2Db molecules are known (33). Therefore, we asked whether the V{beta}12J{beta}2.3 sequence contained peptides with binding capacity to H-2b class I molecules. The binding avidity of peptides to MHC class I molecules is directly proportional to the efficiency of peptide immunization in different model systems including vaccination against carcinomas (29, 34). It was found that one V{beta}12 peptide, p70–78, binds well to Kb molecules (score ~24), whereas three V{beta}12 peptides may interact with Db molecules (p26–34, p67–76, and p94–102 with scores of 15, 14 and 19, respectively (33) (Table I). We concentrated our studies on a peptide, p67–78, that binds to both Kb and Db molecules. This peptide induces a partially protective immune response to L12R4 cells, but not to T142 cells (Fig. 9). These results are in accordance with the observation that p67–78 peptide has no significant homology to the equivalent V{beta}2 sequence (Table I). The effector cells in p67–78 immune, L12R4-protected B6 mice were CD8+ T cells with specificity for the p67–78 peptide. Such p67–78 peptide-reactive CD8+ T cells transferred specific protection to naive B6 mice (Fig. 10). CD8+ T cell lines were produced from spleen cells of p67–78 peptide-vaccinated and L12R4-protected mice. Such cell lines inhibit L12R4 cell growth in vitro and this growth inhibition is almost abolished by the addition of anti-Kb, anti-Db, or anti-H-2 mAb to the cultures (Fig. 11). Anti-IAb mAb had no effect. Two more vaccination experiments were conducted, one in which the mice were immunized with 10, 30, 100, or 300 µg of p67–78 peptide/CFA and another where the mice were immunized with 100 µg of either p67–76 or p70–78 peptide in CFA. In both experiments, 40–60% of the mice were protected against L12R4 tumor cell growth, and there was no difference among groups of mice immunized with a different type or dose of V{beta}12 peptide (data not shown).


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  		Table I. H-2Kb and H-2Db motifs in V{beta}12/J{beta}2.3 and V{beta}2J{beta}2.3 sequencesa

 


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  		FIGURE 9. TCR-V{beta}12 peptide p67–78 induces partial L12R4-specific protection against live L12R4 cells. Two groups of 10 B6 mice ({triangleup} and {circ}) were immunized with 50 µg p67–78 peptide in CFA (s.c. in hind footpads, at the base of the tail, and under the dorsal skin). Two groups of six B6 mice ({blacktriangleup} and •) were immunized with CFA only. Four weeks later, the mice were challenged with 103 live L12R4 cells (• and {circ}) or with 103 live T142 cells ({blacktriangleup} and {triangleup}). Tumor growth was measured every second day for 90 days. The data from days 12 to 40 are shown and expressed as percent survival. The four p67–78 immune and protected mice were alive on day 90.

 


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  		FIGURE 10. TCR-V{beta}12 peptide p67–78-specific T cells transfer specific protection against live L12R4 cells. p67–78 peptide-immune CD8+ T cells (from the four surviving mice in Fig. 10) were injected into naive C57BL/6 mice 1 day before challenge with 103 live L12R4 cells (10 mice, •) or with 103 live T142 (V{beta}12-expressing) cells (10 mice, {square}). Twenty normal B6 mice were challenged with 103 live L12R4 cells ({blacktriangleup}) or 103 live T142 cells ({triangleup}). The y-axis represents percent surviving mice. The 10 naive mice injected with T cells from p67–78 peptide-immunized mice were all alive on day 90.

 


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  		FIGURE 11. The activity of L12R4-specific effector T cells is inhibited by anti-MHC class I mAb. Briefly, 104 L12R4 cells were cultured for 48 h either alone or with 2 x 105 CD8+ T cells from a T cell line obtained from spleen T cells of p67–78 peptide-immunized B6 mice alone or with the addition of 10 µg/ml Ox3, M1/42, anti-Kb, or anti-Db mAb. The mAb were present during the 48 h of culture. The data are expressed as percent inhibition of L12R4 cell growth by the CD8+ T cells.

 
Thus, our results demonstrate that three V{beta}12 peptides that bind to Kb or Db molecules are capable of inducing protective immunity to L12R4 cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Syngeneic tumor-specific immune effector cells can be directed against overexpressed self-Ags; cryptic self Ags; mutated cell cycle control/signal transduction proteins; and, in the case of virally induced tumors, various viral peptides (35, 36). Negative selection processes in the thymus normally eliminate self-reactive T cells (5, 6). However, in some circumstances self-reactive T cells (e.g., TCR-V{beta}-reactive T cells) may emigrate from the thymus because of insufficient interactions between APC and T cells. In the present study, we used syngeneic T lymphoma cells to obtain more information about the nature and specificity of self-reactive TCR-specific T cells. T lymphoma cells present only a single TCR{alpha}{beta} heterodimer at the cell surface, and epitopes on these TCR{alpha}{beta} molecules could serve as a form of tumor-specific Ag(s) (15, 23, 24, 37, 38).

L12R4 T lymphoma cells express a V{alpha}10/V{beta}12 TCR heterodimer and no other TCR{alpha} or TCR{beta} sequences have been identified. A surface TCR/CD3-negative L12R4 variant, P3.3, produced no TCR{beta} chains as demonstrated by 1) direct immunoprecipitation and internal FACS analysis with anti-V{beta}12 or anti-TCR.C{beta} mAb, 2) indirect immunoprecipitation with anti-CD3{epsilon} mAb, and 3) the absence of TCR{beta} mRNA. Thus, P3.3 cells can have no TCR{beta} peptides associated with their MHC class I molecules. Transfection of P3.3 cells with V{beta}2.C{beta} cDNA (very different from V{beta}12, Table I) restores TCR/CD3 surface membrane expression (T142 cells). Transfection of P3.3 with V{beta}12.C{beta} cDNA also reconstitutes TCR/CD3 surface expression, thus strongly suggesting that the lack of TCR{beta} chain expression is the only defect in P3.3 cells related to TCR/CD3 expression (Fig. 2).

Vaccination of B6 mice with Mito-C-treated L12R4 cells induces specific protection, i.e., vaccinated mice reject L12R4 cells but not syngeneic C6VL (V{alpha}9/V{beta}19) cells (see Refs. 15 and 24), P3.3 cells, or T142 cells (Fig. 3). Vaccination against T142 cells induces protection against T142 cells and not against L12R4 cells (data not shown), whereas vaccination against P3.3 induces partial protection against P3.3, L12R4, and T142 cells but not C6VL cells (Fig. 4 and data not shown). Thus, TCR-V{beta}12-specific immunity plays an important role in protection against L12R4 cells, though other self-tumor Ags may also be involved. Although protective immunity correlated with the proliferative activity of L12R4-reactive CD4+ T cells, the effector cells in the protective immune response are CD8+ V{beta}12-reactive T cells. These cells have cytotoxic activity (Figs. 4 and 5) and they produce IFN-{gamma} and TNF-{alpha} but not IL-4 (Fig. 6). The production of IFN-{gamma} appears crucial for protection (38, 39), as no protective immunity is induced in IFN-{gamma}–/– mice upon vaccination with Mito-C-treated L12R4 cells. However, CD8+ V{beta}12-reactive T cells (in contrast to CD4+ L12R4-reactive T cells) can transfer protection to naive normal B6 mice as well as to naive IFN-{gamma}–/– B6 mice (Figs. 7 and 8). Thus, it appears that CD8+ V{beta}12-reactive T cells play a major role in the protective immune response against L12R4 cells. Studies similar to ours have indicated that CD8+, V{beta}-specific T cells may be possible effector cells in T lymphoma cell rejection, but T lymphoma-specific Abs of the IgG2c isotype also play a significant role in normal as well as in IFN-{gamma}–/– B6 mice (38). As mentioned in Results, we find no evidence for specific anti-L12R4 Abs (data not shown). The antisera from the present study are being analyzed for content of anti-TCR peptide Abs (12).

H-2Kb and H-2Db class I molecules can react with several V{beta}12J{beta}2.3 peptides (Table I). Our experiments with V{beta}12 peptide, p67–78 (which binds to both Kb and Db), and with p67–76 (Db) or p70–78 (Kb) peptides have shown that upon immunization with such peptides in CFA, protection against L12R4 cells can be induced in ~50% of the immunized mice (29, 34) (Fig. 9). Immune CD8+ T cells from the protected mice transfer complete protection to naive mice, suggesting that once established, V{beta}12 peptide-specific immunity confers efficient protection (Fig. 10). L12R4-reactive CD8+ T cell lines derived from p67–78 immune mice can be propagated in vitro with peptide and IL-2, and the L12R4-specific growth inhibition is abolished by Abs to Kb or Db molecules (Fig. 11). In addition, such T cells can convey L12R4-specific immunity autonomously in vivo (R. Llobera and B. Rubin, unpublished data). Thus, V{beta}12 peptide-specific CD8+ T cells restricted to either Kb or Db MHC class I molecules are responsible for a major part of the TCR{beta}-specific growth inhibition of L12R4 tumor cells. Future studies may investigate whether alternative Db-binding V{beta}12 peptides as well as V{alpha}10 octamer peptides binding to Kb or V{alpha}10 nonamer/decamer peptides binding to Db may induce protective immunity. It is interesting to note that in four independent studies with different H-2 haplotypes and TCR-V{beta} regions, a V{beta} peptide around p65–80 shows immunogenicity and capacity to induce tumor rejection (Figs. 9 and 10) or down-regulation of autoimmunity, i.e., EAE (16, 21, 23).

There is as yet no direct evidence for the association of V{beta}12 peptides with Kb or Db molecules at the L12R4 cell surface (24). This could be obtained by peptide elution from L12R4 and HPLC analysis (33). However, immunization with p67–78/CFA induces protection against L12R4 cells, but not against T142 cells, and the protective immunity is mediated by CD8+ T cells and not anti-TCR Abs. The only known difference between L12R4 and T142 cells is the expression of V{beta}12 and V{beta}2, respectively, and these two V{beta} families show low homology, in particular in the p67–78 region (Table I). Other indirect arguments come from studies on T cell biosynthesis. Only 1–5% of TCR{alpha}{beta} heterodimers reach the cell surface. Ninety-five percent or more of these molecules are degraded in a proteasome-dependent process (24, 31). Consequently, TCR{alpha}{beta} peptides may be available for the MHC class I biosynthetic pathway (17), and thus may be present on the T cell surface in association with MHC class I molecules. It is interesting to note that non-specific pathogen-free mice are more easily protected against L12R4 cells upon vaccination compared with specific pathogen-free mice. Thus, a certain "background" immunity may help to mount immune responses to tumor Ags. Such background immunity may consist of natural Abs and/or self-reactive T cells with specificity for TCR structures or peptides (12).

An open question concerns the mechanism by which the CD8+ V{beta}-specific T cells kill/eliminate their target cells. On one hand, TCR peptide-specific CTLs have been clearly demonstrated in vitro but the in vivo function of such cells was not assessed (14). In contrast, CD8+ TCR-specific T cells with in vivo function have been described previously (21, 22, 23, 24). However, these killer cells do not lyse TCR peptide-expressing target cells in short-term lytic assays in vitro. This could be due to the absence of sufficient quantities of TCR peptide-MHC complexes on the target cell surface and/or to the mechanism of lysis used by the different CD8+ T killer cells. The former possibility is supported by the studies of Reimann and colleagues (14): TCR-peptide-loaded A20 target cells were killed by and could restimulate TCR peptide-specific CTL lines, whereas the same target cells transfected with relevant TCR{beta} cDNA (and producing high levels of intracellular TCR{beta} chains) were poorly lysed and they could not restimulate TCR peptide-specific CTL lines. The latter idea is supported by Ratner and Clark (40), who found that short-term "acute" lysis is mainly mediated by perforin/granzyme mechanisms, whereas long-term "slow" lysis may be due to membrane-bound TNF-{alpha} on the CTLs.

In conclusion, we have described TCR peptide-specific, MHC class I-restricted CD8+ T cells which play an important role in syngeneic T lymphoma cell rejection. Such T cells resemble TCR-specific CD8+ T cells in autoimmune models of EAE (16, 21, 22, 23). Although almost certainly CD8+ T cells recognize V{beta} peptide-MHC class I complexes on target L12R4 cells during in vitro and in vivo effector functions, it remains to be shown whether they gain sensitization to V{beta} peptide/MHC on L12R4 cells (and other T lymphoma cells) or on some other APC. Given the stringent requirements for CD8 activation, the latter seems more likely.


   Acknowledgments
 
Many of the ideas in this article were developed during my sabbatical year in the laboratory of Prof. Eli Sercarz. His hospitality and generosity are greatly appreciated. We thank the animal facility of Institut Fédéral de Recherche 30 for breeding and maintenance of our mice. Profs. Bill Clark (University of California, Los Angeles, CA) and Alexandra Franco (Torrey Pines Institute for Molecular Studies, San Diego, CA) have provided indispensable help in the project, in the preparation of this manuscript, and in the interpretations of our results; we give them our sincere thanks.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The present work was supported by the Centre National de la Recherche Scientifique, the University Paul Sabatier of Toulouse, and the Association pour la Recherche Contre le Cancer, Contract 5979. Back

2 Address correspondence and reprint requests to Dr. Bent Rubin, Unité Mixte de Recherche 2587, Centre National de la Recherche Scientifique-Pierre Fabre, 3 Rue des satellites, 31400 Toulouse, France. E-mail address: bent.rubin{at}istmt.cnrs.fm Back

3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MIg, mouse Ig; Mito-C, mitomycin C; FCS/R, FCS/RPMI 1640. Back

Received for publication June 25, 2004. Accepted for publication September 24, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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