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A Large Number of T Lymphocytes Recognize Moloney-Murine Leukemia Virus-Induced Antigens, but a Few Mediate Long-Lasting Tumor Immunosurveillance ¹

Antonella Facchinetti^*, Silvia Dalla Santa^*, Silvio Mezzalira^*, Antonio Rosato^* and Giovanni Biasi^2,

^* Department of Oncology and Surgical Sciences, University of Padova, Padova, Italy; and Department of Molecular Pathology and Experimental Therapies, Polytechnic University of the Marche, Ancona, Italy

	Abstract

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The CD8⁺ T cell response to Moloney-murine leukemia virus (M-MuLV)-induced Ags is almost entirely dominated by the exclusive expansion of lymphocytes that use preferential TCRV chain rearrangements. In mice lacking T cells expressing these TCRV, we demonstrate that alternative TCRV can substitute for the lack of the dominant TCRV in the H-2-restricted M-MuLV Ag recognition. We show that, at least for the H-2^b-restricted response, the shift of TCR usage is not related to a variation of the immunodominant M-MuLV epitope recognition. After virus immunization, all the potentially M-MuLV-reactive lymphocytes are primed, but only the deletion of dominant V rescues the alternative V response. The mechanism of clonal T cell "immunodomination" that guides the preferential V expansion is likely the result of a proliferative advantage of T cells expressing dominant V, due to differences in TCR affinity and/or cosignal requirements. In this regard, a CD8 involvement is strictly required for the virus-specific cytotoxic activity of CTL expressing alternative, but not dominant, V gene rearrangements. The ability of T cells expressing alternative TCRV rearrangements to mediate tumor protection was evaluated by a challenge with M-MuLV tumor cells. Although T cells expressing alternative V chains were activated and expanded, they were not able to control tumor growth in a long-lasting manner due to their incapacity of conversion and accumulation in the T central memory pool.

	Introduction

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Pathogen infection is followed by the expansion of activated CD8⁺ CTLs that recognize antigenic peptides bound to self-MHC class I molecules. Single cell assays of MHC/peptide tetramer staining or of peptide-induced cytokine release have also shown that the response to virus infection is much greater than previously estimated and may involve a large proportion of peripheral CD8⁺ T cells (1, 2).

Although a large number of virus-specific CD8 T cells are detectable during both primary and secondary infection, only few virus gene products are recognized in an MHC-restricted fashion (3, 4). Furthermore, T cell responses often appear limited to a single encoded immunodominant peptide which generally stimulates a large number of specific CTL, while subdominant epitopes elicit T cell response that do not offer a high protection level against pathogens (5, 6). Clonotypic analysis of CD8 T cell response in many cases provides evidence that immunodominant determinants are recognized by a restricted T cell repertoire that uses a limited number of TCRV and/or V domains (7, 8, 9, 10, 11).

Immunodominance and TCRV restriction seem to represent a general characteristic of T cell responses and have been previously reported for the CD4 T cell response to complex proteins (12, 13). However, the MHC class II-restricted response remains in the absence of the dominant TCRV elements because a shift of TCRV usage permits maintenance of the fine specificity pattern of response (14, 15, 16). TCR flexibility seems to occur also in the CD8⁺ T cell response to Moloney-murine leukemia virus (M-MuLV) ³-induced Ags; while C57BL/6 (B6) mice preferentially use TCRV5 expressing T cells to reject M-MuLV-infected tumor cells (17), congenic B6.V^a mice, in which the dominant V5 chain is not expressed, use alternative V chains to recognize the same immunodominant epitope (18). However, previous observations speak against a class I-restricted TCR repertoire flexibility because the elimination of T cells bearing the specific V region for virus- or chemically induced tumor Ag(s), through endogenous superantigen expression, abrogates tumor immunosurveillance (19, 20).

The present study investigates the flexibility of MHC class I-restricted TCR usage after injection of Moloney-murine sarcoma virus (M-MSV)/M-MuLV complex (M-MSV/MuLV) in mice in which the preferential V responses were either naturally or intentionally abrogated. The injection of transforming M-MSV and its natural MuLV helper gives rise to sarcomas that rapidly undergo spontaneous regression within a few days, mostly due to CD8 response (21). Different MHC class II and I Ag presentation pathways are used in generating virus-specific CTL (22), and a preferential use of specific V chain rearrangements in the H-2^d- and H-2^b-restricted CD8 recognition of M-MuLV-induced Ags has been shown (17). To date, only peptides recognized in the H-2^b context have been identified: the immunodominant CCLCLTVFL, epitope shared by gag-encoded protein of Friend/Moloney/Rauscher (FMR) retrovirus group (23), recognized by CD8⁺V5⁺V3.2⁺ (17), and a subdominant env-encoded SSWDFITV epitope (24) that is less efficient in inducing a protective immune response against M-MuLV tumor cell challenge (25).

The results presented in this paper show that in recognizing M-MuLV-induced tumor Ags, alternative TCRV chain usage can hierarchically substitute for the lacking dominant TCRV elements. Moreover, we observed that H-2D^b-restricted virus-specific CTL, which express dominant V5 or any alternative V chain, recognize the same immunodominant gag-encoded epitope. However, while CD8⁺V5⁺ effector cells confer full protection against a M-MuLV leukemic tumor cell challenge, the alternative V usage cannot supply in vivo the same long-lasting functional activity.

	Materials and Methods

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Mice

BALB/c and C57BL/6 (B6) mice originally obtained from Harlan Italy were bred in our specific pathogen-free facility for one to two generations and then crossed to obtain (BALB/c x B6)F₁ hybrids. Procedures involving animals and their care conformed to institutional guidelines that comply with all national and international regulatory laws and policies.

Virus-immune and carrier mice

Adult (7- to 8-wk-old) mice were inoculated i.m. in the thigh region with 0.1 ml of a cell-free preparation of M-MSV/MuLV complex that had an in vitro M-MSV titer of 1.2 x 10⁵ focus-forming U/ml on 3T3/FL cells, and M-MuLV titer of 2 x 10⁶ PFU/ml on SC-1 XC cells. These mice developed sarcomas at the site of injection that, in all instances, regressed within 14 days and were thus used as immune mice. To obtain virus carriers, mice were injected s.c., within 48 h after birth, with 0.05 ml of 0.1 gEq of a primary leukemia cell-free M-MuLV extract which had a titer of 5 x 10⁸ PFU/ml on SC-1 XC cells. When these mice were 8–10 wk old, they served as virus carrier spleen cell donors.

Tumor cell lines

M-MuLV-infected MBL-2 (H-2^b), LSTRA (H-2^d), A6ATL (H-2^t1) (26), and chemically induced thymoma EL-4 (H-2^b) cell lines were maintained in vitro or by in vivo i.p. passage in syngeneic recipients. Experimental groups of mice received 2 x 10⁵ MBL-2 or 1 x 10⁶ LSTRA viable leukemic cells s.c. in the flank region and were then examined daily for any sign of tumor growth. Previous experiments showed this minimal cell dose induced tumor growth in >95% of naive mice (data not shown). Tumor-bearing mice were sacrificed within 2 wk from tumor onset.

In vivo mAb treatments

BALB/c mice were depleted of lymphocytes expressing specific TCRV (>95% cell depletion as evidenced by indirect immunofluorescence PBL staining) by i.p. injection of 0.5 mg of anti-V4 (KT4) and anti-V8 (F23.1) 3 and 2 days before, and then weekly, after virus immunization, until sacrifice. Only relative increases in the percentages of remaining TCRV families, to compensate the specific V deletion, were detected by direct PBL flow cytometry analysis.

Cell cultures and CTL lines

Virus-specific CTL were generated in vitro in a 5-day mixed leukocyte tumor cell culture (MLTC) (27) or mixed leukocyte peptide culture (MLPC) (25). Usually, 25 x 10⁶ responder spleen or 20–25 x 10⁶ lymph node cells from virus-immune mice and 5 x 10⁶ irradiated leukemic (MBL-2/LSTRA) or 2 x 10⁷ stimulator spleen cells from carrier mice were cocultured in MLTC in 15 ml of DMEM (Invitrogen Life Technologies) supplemented with 2 mM L-glutamine, 10 mM HEPES, 20 µM 2-ME, antibiotics, and 10% heat-inactivated FBS (Invitrogen Life Technologies). In the MLPC, responder cells were stimulated in the presence of the peptide corresponding to aa 85–93 of the FMR gag-encoded protein (CCLCLTVFL) at a final concentration of 1 µM.

In some experiments, responder B6 spleen cells were partially depleted of V5⁺ T cells (>95% as evidenced by flow cytometry analysis) by panning on anti-mouse plus anti-V5 (MR9-4) mAb precoated petri dishes (28) before culture. Cells recovered from mixed cultures were washed and maintained for an additional 3 days in culture (2 x 10⁶ cells/ml) in complete medium supplemented with 10 U/ml rIL-2 (a generous gift of Sandoz) to allow for flow cytometry analysis of TCRV expression (17). In some experiments, spleen cells from virus-immune mice were stimulated repeatedly for multiple cycles (MLTC-n) in the presence of syngeneic irradiated leukemic cells and spleen cells as a source of APC in 10 U/ml rIL-2-supplemented complete medium.

Cytotoxic assays

Cytotoxic activity of CTL generated in MLTC was determined by a 4-h incubation in ⁵¹Cr release assay in which 2 x 10³ labeled target cells and different numbers of effector cells were plated in 96-well microplates. Target cells were either M-MuLV infected LSTRA (H-2^d), MBL-2(H-2^b), A6ATL(H-2^t1), or EL-4 (FMR-uninfected H-2^b thymoma) cells. Cytotoxic activity was expressed as the percentage of specific lysis or in terms of lytic units (LU) where 1 LU was the number of cells producing 30% specific lysis of 5 x 10³ targets (27).

For peptide pulsing, 10^{6 51}Cr-labeled EL-4 cells per milliliter were incubated for 30 min at 37°C with 10 µM final concentration of the FMR gag (CCLCLTVFL)- or env (SSWDFITV)-encoded peptide (25) and then washed three times before use. In some experiments, effector cells were preincubated for 20 min at 37°C in microplates with variable concentrations of anti-CD8 (53-6.72) mAb or with an anti-K^b, K^d cross-reacting (20-8-4s) mAb (10 µg/ml) before target cells addition for Ab blocking. Finally, to evaluate the peptide/MHC requirements of effector cells, EL-4 target cells were pulsed with variable concentrations of CCLCLTVFL peptide in the presence or absence of blocking anti-CD8 mAb (10 µg/ml).

Flow cytometry

Cells recovered from in vitro cultures were double stained with anti-CD8 PE-conjugated (CT-CD8A; Caltag Laborartories) and a panel of anti-V FITC-conjugated mAbs including V2 (B20.6), V4 (KT4), V5 (MR9-4), V6 (RR4-7), V7 (TR310), V8.3 (1B3.3), V9 (MR10-2), V10 (B21.5), V13 (MR12-3), V14 (14-2) (BD Pharmingen), and V8.1,2 (KJ16; Caltag Laboratories). In some experiments, 50-µl aliquots of PBL from retro-orbital sinus, collected from virus-immune mice after viable MBL-2 leukemic cell challenge, were triple stained with anti-CD8 PE, anti-V FITC, and biotin-conjugated anti-CD62L (Mel-14; BD Pharmingen) mAbs, followed by streptavidin SpectralRed (SPRD)-conjugated (Southern Biotechnology Associates). The erythrocytes were lysed by incubation with isotonic ammonium chloride buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA). To examine TCRV expression in CD8⁺-gated tumor-infiltrating lymphocytes (TIL), the MBL-2 tumor mass was removed and forced trough a multilayer nylon sieve to obtain cell suspension before labeling. All the samples were gated on viable cells on the basis of forward and side scatter parameters and run on EPICS ELITE (Coulter) or FACSCalibur (BD Biosciences).

	Results

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Identification of the CD8TCRV repertoire specific for the H-2^d-restricted anti-M-MuLV immune response

In the immune response to M-MuLV-induced Ags, BALB/c (H-2^d) mice preferentially use CTL expressing the TCRV4 region as shown in vivo by PBL analysis and, more markedly, in vitro after secondary stimulation of their spleen cells in MLTC (17). Cytometric analysis of the TCRV repertoire expressed by the CD8⁺ cells using a panel of anti-V mAbs revealed an increase in the TCRV4 cell percentage (V4⁺CD8⁺/CD8⁺ = 35.1–39.8%) in five MLTC as compared with the baseline levels in immune (13.2 ± 1.3 SD) and in six naive spleen cells (8.0 ± 03 SD).

To investigate whether the expansion of V4⁺ T cells can mask the involvement of other V families in the M-MuLV-specific H-2^d-restricted response, BALB/c mice were treated with a V4⁺ cell-depleting mAb before virus priming. These mice, like mAb-untreated control mice, developed sarcomas that regressed in a few days, suggesting the involvement of V families different from V4. To identify the vicarious V specificity used by the virus-specific effectors, spleen cells from anti-V4 mAb treated mice were used as responders in syngeneic MLTC 2 wk after M-MSV/MuLV injection. The ⁵¹Cr release assay performed against syngeneic leukemic target cells (LSTRA) evidenced the generation of a strong cytotoxic activity (Fig. 1A) although lower than that generated in control cultures conducted with spleen cells from mAb-untreated immune mice (149 ± 82 vs 286 ± 91 LU/culture, mean ± SD in 12 MLTC). The cytotoxic activity was virus-specific and H-2-restricted, as CTLs were unable to kill either BALB/c Con A blast spleen cells, antigenically unrelated L1210 (H-2^d) or the H-2^b M-MuLV-induced MBL-2 leukemic target cells (data not shown). Cytometric analysis of the TCRV repertoire in five MLTC always revealed a substantial increase in the V8.3 cell percentage (V8.3⁺CD8⁺/CD8⁺ = 23–32% from 6.8 ± 0.7% SD of immune spleen cells) with an additional increase in the V 6% (16%) in only one MLTC. Representative results of ⁵¹Cr release assay (Fig 1A) and V expression (Fig. 1B) of above described experiments are reported in Fig. 1.

View larger version (48K): [in this window] [in a new window]   FIGURE 1. a, Analysis of the H-2d-restricted virus-specific T cell repertoire in BALB/c mice. Spleen cells from BALB/c mice were used as responders in syngeneic MLTC 2 wk after M-MSV/MuLV injection. Evaluation of cytotoxic activity generated in MLTC and TCRV usage in CD8 cells of one representative experiment are reported in A and B, respectively. b, BALB/c mice were depleted of cells expressing specific TCRV by anti-V4 or anti-V4 plus anti-V8 mAb treatments before virus immunization. c, LSTRA were used as target cells in the 51Cr release assay at different E:T cell ratios and LU calculated per culture are reported. R = percentage of TCRV+CD8+ among CD8+ cells.

Even though the M-MuLV dominant epitope recognized by V4⁺CD8⁺ cells in H-2^d mice remains unknown, it has long been established that the CTL response in these mice is restricted by K^d class I molecules (29). Therefore, we studied whether the effectors that emerge after mAb treatments use the same H-2^d class I restriction molecule for viral epitope/s recognition. As shown in Table I, regardless of V expression, all CTL generated in MLTC (following one or multiple cycles of stimulation with LSTRA) recognized M-MuLV-induced Ags associated with the K^d molecule, because they were neither able to lyse the M-MuLV induced A6.ATL (K^sD^d) leukemic cell line nor recognize LSTRA leukemic cells in the presence of anti-K^d cross-reacting (20-8-4s) mAb.

View this table: [in this window] [in a new window]   Table I. Kd-restricted recognition of M-MuLV-induced Ags by anti-V mAb-treated BALB/c mice

Identification of the CD8TCRV repertoire specific for the H-2^b-restricted anti-M-MuLV immune response

Previous studies have shown the preferential usage of the TCRV5 rearrangements in the H-2^b-restricted response of B6 mice to M-MuLV-induced Ags (17). To investigate the possible involvement of different TCRV families in the H-2^b-restricted virus recognition, we used (BALB/c x B6)F₁ (H-2^d/b) mice in which the BALB/c background leads to the clonal deletion of V5 T cells due to Mls Ag tolerance. Two weeks after M-MSV/MuLV injection, spleen cells were stimulated in different MLTC with parental H-2^d (LSTRA) or H-2^b (MBL-2) leukemic cells, or spleen cells from H-2^d/b syngeneic M-MuLV carrier mice, or a mixture of LSTRA and MBL-2.

As shown in the results of one representative experiment reported in Table II, the (BALB/c x B6)F₁ effectors generated in response to parental H-2^d M-MuLV leukemic cells exerted a strong H-2-restricted cytotoxic activity against LSTRA target cells. As expected, analysis of the CD8V repertoire disclosed that CD8⁺V4⁺ cells were overrepresented in these cultures (data not shown). Furthermore, high H-2^d-restricted CTL generation was seen following stimulation in MLTC with syngeneic M-MuLV infected H-2^d/b cells (Table II, line 3; 188 ± 82 SD LU/culture in nine MLTCs). In contrast, a low H-2^b-restricted cytotoxic activity (18 ± 14 SD LU/culture) was generated in the same MLTCs in response to M-MuLV-infected H-2^d/b target cells. However, the same spleen cells could recognize the M-MuLV-induced Ags in the H-2^b context because they were able to generate H-2^b-restricted effectors (126 ± 83 LU/culture in nine MLTCs) when stimulated in MLTC by MBL-2 cells (Table II, line 2). Moreover, in four different experiments, a substantial H-2^d (119 ± 50 SD)- plus H-2^b-restricted (116 ± 51 SD) response was detected in response to a mixture of LSTRA and MBL-2 leukemic cells (Table II, line 4).

Previous studies in B6 mice have show that M-MuLV-specific H-2^b-restricted CD8⁺V5⁺ cells recognize the gag-encoded peptide CCLCLTVFL (23). We then evaluated whether (BALB/c x B6)F₁ effector cells recovered from MLTC after repeated cycles of restimulation with MBL-2 leukemic cells were able to recognize the same gag-encoded peptide. The results of one representative experiment show that CTL from three different MLTC (Fig. 2), in which the respective increase of either V7, V8.1,2, or V13⁺ among CD8⁺ cells are reported, were indifferently able to specifically lyse EL-4 lymphoma cells pulsed with the CCLCLTVFL peptide. In addition, analysis during consecutive MLTC restimulation showed that cytotoxic activity expressed in terms of LU/recovered cells paralleled the increase in the percent of V bias, and in one MLTC-2, selected for a simultaneous increase of V7, 8.1,2, and 13 CD8⁺ subsets, these effectors almost exclusively mediated the lytic activity. In fact, the depletion by cytometric cell sorting of these V subsets, but not of a unrelated V5, 6, 8.3, 14 expressing counterparts, produced a quite complete loss of specific cytotoxic activity measured at a E:T cell ratio of 60:1 against either MBL-2 or EL-4 gag-peptide pulsed target cells (data not shown).

View larger version (34K): [in this window] [in a new window]   FIGURE 2. M-MuLV peptide specificity of H-2b-restricted (BALB/c x B6)F1 CTL. Spleen cells from (BALB/c x B6)F1 immune mice were repeatedly stimulated for three to six cycles with irradiated MBL-2 plus syngeneic APC in MLTC. TCRV usage (A), and the results of a 51Cr release assay against MBL-2, EL-4 (H-2b, FMR uninfected), and gag- (CCLCLTVFL) or env- (SSWDFITV) encoded peptide pulsed EL-4 (B) are shown.

Immunodominance of the TCRV repertoire in the anti-M-MuLV immune response

Further studies were addressed to determine why the alternative M-MuLV-specific TCRV effectors do not expand, either in vivo or in vitro, in the presence of the main TCRV responses. We reasoned that, at least for the H-2^b-restricted response, the predominance of one V cannot be due to differences in the immunogenicity of the recognized epitope, nor likely due to the V pool size of M-MuLV-specific CTL precursors, because mice lacking the main V are competent in generating a substantial cytotoxic T cell response in MLTC.

We first ascertained whether the priming of the alternative M-MuLV-specific TCRV repertoire had occurred in the presence of V5⁺ CTL precursors. Spleen cells from immune B6 mice, after partial (>95%) TCRV5 cell depletion by panning on anti-mouse plus anti-V5 mAbs, were stimulated in MLTC to evaluate the possible alternative M-MuLV-specific TCRV repertoire expansion. Flow cytometry analysis of seven different primary MLTC (MLTC-1) evidenced that among CD8⁺, V13⁺ cells were enriched by about 5-fold (30%) in one, or 3-fold (23%) together with a slight increase of V5 in another MLTC. In the other three MLTC, an expansion of V5 alone was observed, while in the remaining two V expansion was not evidenced using the mAb we used despite a moderate, virus-specific CTL generation was detected. The cells recovered from these latter two MLTC-1 were then repeatedly stimulated in vitro until a bias in V13 (33%) or V7 (28%) was clearly observed in MLTC-4.

The results provide evidence that a wide potentially M-MuLV-reactive repertoire was primed by in vivo sensitization. However, the fact that detectable alternative V expansion is not easily reached in B6 mice, even after the elimination of V5⁺ cells, possibly indicates that M-MuLV-specific precursors different from V5 are numerically limited and/or their differentiation into the memory pool is shaped by the numerical expansion of V5⁺ cells.

It is possible that the hierarchal emergence of V usage could reflect differences in TCRV affinity/avidity for the MHC/Ag complex. In this regard, it was recently observed that the wide range of TCR affinities among different EBV-reactive clonotypes segregates with V usage, and it was proposed that the variable contribution of the CD8 coreceptor to their functional activity may govern in vivo clonotype persistence (30). Pioneering studies reported that the virus-specific activity of CTL generated in MLTC by spleen cells from M-MSV/MuLV primed B6 mice were not significantly inhibited by anti-CD8 mAb (31); therefore, we evaluated the effect of anti-CD8 mAb on H-2^b-restricted cytotoxic activity of CTL generated in MLTC by V5^– (BALB/c x B6)F₁ mice. The results of three experiments comparing the effect of different anti-CD8 mAb dilutions on the activity of CTL generated in MLTC by (BALB/c x B6)F₁, and B6 immune mice against MBL-2 cells are reported in Fig. 3A. They show that the cytotoxic activity of effectors from V5⁺ B6 strain was not significantly inhibited in the presence of high concentrations (10 µg/ml) of anti-CD8 mAb, while the activity from V5^– (BALB/c x B6)F₁ mice was reduced in a dose-dependent fashion. Furthermore, we observed a comparable inhibition of (BALB/c x B6)F₁ CTL activity independently of the TCRV7, 8.1,2, 13 expression (data not shown).

View larger version (18K): [in this window] [in a new window]   FIGURE 3. CD8 dependency of the anti-MuLV H-2b-restricted immune response of B6 and (BALB/c x B6)F1 mice. A, Cells recovered from MLTC-1 were preincubated for 20 min at 37°C with a 5-fold dilution of anti-CD8 mAb, and assayed for cytotoxic activity at 60:1 E:T cell ratio against 2 x 103 51Cr-labeled MBL-2 cells. Data as mean ± SD (three experiments) of percent of lysis inhibition vs controls in the absence of mAb. B, Cells from MLTC-1 were assayed at 60:1 E:T cell ratio against 2 x 103 51Cr-labeled EL-4 loaded with variable concentrations of CCLCLTVFL peptide in the absence (•, ) or presence (, ) of 10 µg/ml blocking anti-CD8 mAb.

Lack of anti-M-MuLV protection by alternative V repertoire

Other studies were undertaken to ascertain whether the alternative V usage can provide in vivo a functional anti-M-MuLV immune response in (BALB/c x B6)F₁ mice. The presence of alternative V bias was evaluated among activated CD62L^–CD8⁺ cells in the PBL of mice challenged with 2 x 10⁵ MBL-2 cells 4 wk after M-MSV/MuLV injection, as previously described to detect the dominant V5 bias in B6 mice (17).

The results of one representative experiment reported in Fig. 4A show the expansion of the alternative V repertoire in the CD62L^–CD8⁺ PBL subset detected 10 days after tumor cell boosting. PBL, triple stained with mAbs against CD8, CD62L, and VTCR, show a strong down-regulation of CD8 coreceptor, a common feature of Ag-specific activation of CD8⁺ cells in vivo, and a high bias of V8.1,2, but not V8.3, in the CD62L^– when compared with the CD62L⁺ subset, immune and normal counterparts.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Alternative V repertoire expansion in M-MuLV-immune (BALB/c x B6)F1 mice at different days after MBL-2 challenge. Cells from PBL, lymph node, and TIL were triple stained with mAbs against CD8, CD62L, and TCRV. Representative results of cytometric analysis: A, PBL from normal or M-MuLV-immune mice 10 days after challenge. In the first line, the cytograms show CD8 vs CD62L expression and the histogram the CD8 fluorescence gated on CD62L– or CD62L+ subsets of CD8+ cells. Histograms below represent V expression in the indicated subsets. B, V7, V8.1,2, and V13 expression in the PBL of three (BALB/c x B6)F1 mice during tumor progression 16, 20, and 18 days and V5 in one B6 mouse 20 days after MBL-2 challenge, respectively. C, V13 expression in PBL, lymph node, and TIL of (BALB/c x B6)F1 and V5 in lymph node of B6 mice.

View this table: [in this window] [in a new window]   Table III. The dominant V repertoire is required for full protection against M-MuLV tumor growth

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Kinetics of dominant and alternative anti-M-MuLV H-2b-restricted immune response of B6 and (BALB/c x B6)F1 mice. Spleen and lymph node cells were cultured in MLTC with MBL-2 cells 2, 4, and 7 wk after M-MSV/MuLV injection and used as effectors in 51Cr release assay against 2 x 103 MBL-2 cells at different E:T cell ratios. Each value represents the mean ± SD of percent of 51Cr release (four different MLTC) in two experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

To this end, we used mice in which the dominant V responses were either intentionally abrogated before Ag priming by depleting mAb treatment or clonally deleted by natural tolerance. In both instances, anti-V4 mAb-treated BALB/c or V5^– (BALB/c x B6)F₁ mice were fully capable of regressing sarcomas induced by the injection of the M-MSV/MuLV complex. In addition, spleen cells from the same mice were able to generate a substantial H-2-restricted cytotoxic activity when stimulated in MLTC with LSTRA (H-2^d) or MBL-2 (H-2^b) leukemic cells, respectively. Flow cytometry analysis of CTL thus generated suggests that different V gene segments (V8.3 and 6 for H-2^d, and V7, 8.2, and 13 for H-2^b) can be used for the recognition of M-MuLV-induced Ags. Thus, in both instances a shift of TCR gene usage appears to compensate for the lack of the dominant V response.

However, it is important to note that sarcoma regression in (BALB/c x B6)F₁ mice could mainly be due to the H-2^d-restricted recognition of M-MuLV-induced Ags. In fact, spleen cells from these mice give rise almost exclusively to an H-2^d-restricted CTL response, following in vitro stimulation with syngeneic M-MuLV-infected H-2^d/b cells. The low H-2^b-restricted CTL generation cannot be due to a lack of epitope recognition because a substantial CTL generation was induced in the same responder cells following MBL-2 stimulation. Moreover, suppressive effects possibly exerted by H-2^d- on the H-2^b-restricted CTL differentiation can be ruled out because responses against both the parental haplotypes were elicited by the simultaneous stimulation with LSTRA and MBL-2 leukemic cells, i.e., by class I/epitope presentation on separate cells.

On the whole, the results indicate that the "immunodomination" by CTL sharing defined V gene segments is a central feature of the response to M-MuLV-induced Ags. The term immunodomination has mostly been used to describe the inhibition of a specific immune response to a subdominant epitope during a concomitant response to a dominant epitope of the same molecule (33), thus indicating that the determinants can be ordered in a hierarchy of domination. Interestingly, for the H-2^b-restricted response to M-MuLV-induced Ags, we show that the same immunodominant gag-encoded peptide (23) is recognized by different TCRV specificities: the dominant V5 and alternative V. This situation provides a unique opportunity to demonstrate that hierarchical response is dominated by the expansion of lymphocytes that use preferential V chain rearrangements; indeed, in both anti-M-MuLV H-2^d- and H-2^b-restricted responses, only the deletion of dominant V rescues the nondominant V response.

In this light, if the V hierarchy of domination equally governs the concomitant Ag recognition on different class I molecules, then the immunodomination of V4 (H-2^d-restricted) on the nondominant H-2^b-restricted (V7, 8.1,2, 13) CTL counterpart would be expected following in vitro stimulation with syngeneic M-MuLV-infected H-2^d/b cells. The H-2^d-restricted immunodomination detected in (BALB/c x B6)F₁ mice parallels previous results on in vitro CTL generation against minor histocompatibility Ags showing that subdominant epitope recognition is inhibited by the response to the dominant epitope when presented on the same APC, but not when subdominant and dominant moieties are recognized on separate cells (34). Therefore, the preferential clonal V expansion, that indifferently occurred following the recognition of epitope(s) associated with the same or different class I allomorph(s), very likely could be explained by a phenomenon of T cell competition around a same APC (1, 35).

When questioning whether priming of the alternative V repertoire also occurs in the presence of the V dominant counterpart, we reasoned that the subdominant H-2^b-restricted repertoire in (BALB/c x B6)F₁ mice must be sensitized by virus injection. This because, despite the presence of H-2^d-restricted dominant V4 CTL precursors, the H-2^b-restricted CTL would not be as easily evidenced in MLTC in secondary response to MBL-2 (21). The demonstration that spleen cells from M-MSV/MuLV immune B6 mice, after depletion of the V5⁺ subset, were able to differentiate the virus-specific alternative V repertoire in MLTC directly demonstrated that the priming of all the potentially M-MuLV-reactive lymphocytes had occurred. However, despite alternative V T cell priming, the immune response to M-MuLV-induced Ags is dominated by the almost exclusive expansion of T cell clones sharing V5 gene segments (17). These findings confirm the observations that the massive CD8⁺ T cell expansion during viral infection (1, 2) in many cases is accompanied by the selection of a V restricted T cell repertoire (7, 8, 9, 10, 11).

It has been proposed that the emergence of a preferential V usage in the immune response to virus-induced Ags could be a related requirement in narrowing the memory T cell pool during infectious recall (36). However, the rules and mechanisms that govern the shaping (expansion and/or narrowing) of the activated Ag-specific T cells remain to be investigated, even to a better understanding as to how and to what extent the naive and memory T cell repertoires differ (37).

The selective V immunodomination observed in our study cannot be due exclusively to a different frequency of virus-specific CTL precursors, because a conspicuous expansion of alternative V elements was detected, in the absence of the V immunodominant precursors, in both in vitro and in vivo response to M-MuLV. It is, instead, more likely that the V-driven expansion could result from a proliferative advantage due to differences in the TCR affinity for the recognized MHC/peptide complex. In agreement, by evaluating the CD8 dependency of H-2^b-restricted CTL activity, as an indirect estimate of TCR affinity, we observed that the cytotoxic activity of alternative V5^–, but not dominant V5⁺ CTL, against MBL-2 and EL-4 pulsed with appropriate amounts of peptide was strongly inhibited by anti-CD8 mAb. In the opposite situation when no single TCRV dominated the others as in the response to HBV, it was observed that T cell clones, expressing different TCRV, recognize the same dominant epitope with similar affinity (38).

By analyzing the functional activity of the H-2^b-restricted T cell repertoire involved in the virus-specific response, we observed that in (BALB/c x B6)F₁ mice the alternative V T cell repertoire was unable to confer full immune protection against the MBL-2 tumor cell challenge even if it was expanded, retained the activated phenotype during tumor progression and infiltrated growing tumors. Therefore, despite the evident TCR flexibility in the recognition of M-MuLV Ags, in these mice the TCR repertoire capable of counteracting tumor growth appears in great part negatively selected. This does not necessarily exclude that different genetic-encoded backgrounds favor the expression of a protective H-2^b-retricted TCR flexibility, like that observed in the congenic B6.V^a mice (18).

Our in vivo findings are in line with previous observations that activated virus-specific CD8 T cells can persist without effector function in LCMV chronically infected mice (39) and circulating CD8⁺ T cells specific for melanoma Ags are unable to contain tumor progression in humans (40, 41, 42).

When the ability of (BALB/c x B6)F₁ mice to generate an anti-M-MuLV immune response was investigated, we evidenced in their spleen a strong decrease in the H-2^b-restricted CTL generation in vitro few weeks postimmunization. In some viral systems it was suggested that functional immune response declines rapidly because T cell help, required to sustain and expand an initial Th-independent response, is not available (43). This does not to apply to our study as the H-2^b-restricted anti-M-MuLV response is strictly CD4-dependent in B6 (27) as well as in (BALB/c x B6)F₁ mice (data not shown). The declining of CTL generation we detected in (BALB/c x B6)F₁ mice is likely due to the absence of T_CM that has the intrinsic capacity to proliferate following Ag recall. In this line, in the lymph node, where T_CM subset persists long-term (32), the alternative TCRV repertoire was not expanded at any time after virus immunization.

Whether T_CM and effector memory cells represent separate or related lineages is still unclear (32, 44). In line with the hypothesis that T_CM represents a separate lineage, the lack of CTL generation we described in (BALB/c x B6)F₁ mice might simply derived from the fact that the virus-specific alternative TCRV repertoire represent a subset incapable to generate T_CM and so, functionally different from the dominant TCRV5 subset. Conversely, whether effector memory cells and T_CM are part of a continuum in a linear differentiation pathway (32), then in B6 mice the dominant TCRV5 repertoire would rapidly seed and convert in the lymph node as T_CM in <2 wk postimmunization. Even though it is unclear as to why (BALB/c x B6)F₁ mice become unresponsive, on the whole our findings indicate that a high proportion of CD8⁺ T cells is involved in Ag-specific recognition, but only a restricted subset is able to generate T_CM and mediate long lasting functions. Because T cells have the potential to recognize self Ags as well (45), the narrowing of the immune response, as previously suggested (6), could indeed be advantageous to limit cross-reactivity and autoimmunity. However, in some circumstances the selected T cell repertoire would be unable to recognize immunodominant peptides or, even with recognition, as in our study, may not be fully efficient in counteracting tumor growth.

	Acknowledgments

 
We are grateful to Dr. Vincenzo Bronte for critical reading of this manuscript and Dr. Valeria Tosello for cell sorting.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	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 This work was supported in part by MURST and Fondazione Carivezona "Integrazione tra tecnologia e sviluppo d’settore" Biomedical Research Program 2004.

² Address correspondence and reprint requests to Dr. Giovanni Biasi, c/o Department of Oncology and Surgical Sciences, via Gattamelata 64, 35128 Padova, Italy. E-mail address: g.biasi{at}univpm.it

³ Abbreviations used in this paper: M-MuLV, Moloney-murine leukemia virus; M-MSV, Moloney-murine sarcoma virus; MLTC, mixed leukocyte tumor cell culture; MLPC, mixed leukocyte peptide culture; TIL, tumor-infiltrating lymphocyte; LU, lytic unit; FMR, Friend/Moloney/Rauscher; T_CM, T central memory.

Received for publication August 9, 2004. Accepted for publication February 25, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Butz, E. A., M. J. Bevan. 1998. Massive expansion of antigen-specific CD8⁺ T cells during an acute virus infection. Immunity 8:167.[Medline]
2. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177.[Medline]
3. Townsend, A. R., F. M. Gotch, J. Davey. 1985. Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell 42:457.[Medline]
4. Gotch, F., A. McMichael, G. Smith, B. Moss. 1987. Identification of viral molecules recognized by influenza-specific human cytotoxic T lymphocytes. J. Exp. Med. 165:408.[Abstract/Free Full Text]
5. Spencer, J. V., T. J. Braciale. 2000. Incomplete CD8⁺ T lymphocyte differentiation as a mechanism for subdominant cytotoxic T lymphocyte responses to a viral antigen. J. Exp. Med. 191:1687.[Abstract/Free Full Text]
6. Deng, Y., J. W. Yewdell, L. C. Eisenlohr, J. R. Bennink. 1997. MHC affinity, peptide liberation, T cell repertoire, and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL. J. Immunol. 158:1507.[Abstract]
7. Chen, Z. W., H. Yamamoto, D. I. Watkins, G. Levinson, N. L. Letvin. 1992. Predominant use of a T-cell receptor V gene family in simian immunodeficiency virus Gag-specific cytotoxic T lymphocytes in a rhesus monkey. J. Virol. 66:3913.[Abstract/Free Full Text]
8. Deckhut, A. M., W. Allan, A. McMickle, M. Eichelberger, M. A. Blackman, P. C. Doherty, D. L. Woodland. 1993. Prominent usage of V 8.3 T cells in the H-2D^b-restricted response to an influenza A virus nucleoprotein epitope. J. Immunol. 151:2658.[Abstract]
9. Pantaleo, G., J. F. Demarest, H. Soudeyns, C. Graziosi, F. Denis, J. W. Adelsberger, P. Borrow, M. S. Saag, G. M. Shaw, R. P. Sekaly, et al 1994. Major expansion of CD8⁺ T cells with a predominant V usage during the primary immune response to HIV. Nature 370:463.[Medline]
10. Callan, M. F., N. Steven, P. Krausa, J. D. Wilson, P. A. Moss, G. M. Gillespie, J. I. Bell, A. B. Rickinson, A. J. McMichael. 1996. Large clonal expansions of CD8⁺ T cells in acute infectious mononucleosis. Nat. Med. 2:906.[Medline]
11. Lim, A., L. Trautmann, M. A. Peyrat, C. Couedel, F. Davodeau, F. Romagne, P. Kourilsky, M. Bonneville. 2000. Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire. J. Immunol. 165:2001.[Abstract/Free Full Text]
12. Zamvil, S. S., D. J. Mitchell, N. E. Lee, A. C. Moore, M. K. Waldor, K. Sakai, J. B. Rothbard, H. O. McDevitt, L. Steinman, H. Acha-Orbea. 1988. Predominant expression of a T cell receptor V gene subfamily in autoimmune encephalomyelitis. J. Exp. Med. 167:1586.[Abstract/Free Full Text]
13. Sakai, K., A. A. Sinha, D. J. Mitchell, S. S. Zamvil, J. B. Rothbard, H. O. McDevitt, L. Steinman. 1988. Involvement of distinct murine T-cell receptors in the autoimmune encephalitogenic response to nested epitopes of myelin basic protein. Proc. Natl. Acad. Sci. USA 85:8608.[Abstract/Free Full Text]
14. Frangoulis, B., M. Pla, H. G. Rammensee. 1989. Alternative T cell receptor gene usage induced by self tolerance. Eur. J. Immunol. 19:553.[Medline]
15. Falcioni, F., Z. Dembic, S. Muller, P. V. Lehmann, Z. A. Nagy. 1990. Flexibility of the T cell repertoire. Self tolerance causes a shift of T cell receptor gene usage in response to insulin. J. Exp. Med. 171:1665.[Abstract/Free Full Text]
16. Liang, H. E., C. C. Chen, D. L. Chou, M. Z. Lai. 1994. Flexibility of the T cell receptor repertoire. Eur. J. Immunol. 24:1604.[Medline]
17. Brawand, P., G. Biasi, C. Horvath, J. C. Cerottini, H. R. MacDonald. 1998. Flow-microfluorometric monitoring of oligoclonal CD8⁺ T cell responses to an immunodominant Moloney leukemia virus-encoded epitope in vivo. J. Immunol. 160:1659.[Abstract/Free Full Text]
18. Brawand, P., J. C. Cerottini, H. R. MacDonald. 1999. Hierarchal utilization of different T-cell receptor V gene segments in the CD8⁺-T-cell response to an immunodominant Moloney leukemia virus-encoded epitope in vivo. J. Virol. 73:9161.[Abstract/Free Full Text]
19. Lukacher, A. E., Y. Ma, J. P. Carroll, S. R. Abromson-Leeman, J. C. Laning, M. E. Dorf, T. L. Benjamin. 1995. Susceptibility to tumors induced by polyoma virus is conferred by an endogenous mouse mammary tumor virus superantigen. J. Exp. Med. 181:1683.[Abstract/Free Full Text]
20. Schirrmacher, V., U. Beutner, M. Bucur, V. Umansky, M. Rocha, P. von Hoegen. 1998. Loss of endogenous mouse mammary tumor virus superantigen increases tumor resistance. J. Immunol. 161:563.[Abstract/Free Full Text]
21. Chieco-Bianchi, L., D. Collavo, G. Biasi. 1988. Immunologic unresponsiveness to murine leukemia virus antigens: mechanisms and role in tumor development. Adv. Cancer Res. 51:277.[Medline]
22. Biasi, G., D. Collavo, L. Chieco-Bianchi. 1983. Selective failure of accessory cell function in Moloney murine leukemia virus-tolerant mice. J. Immunol. 131:16.[Abstract]
23. Chen, W., H. Qin, B. Chesebro, M. A. Cheever. 1996. Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors. J. Virol. 70:7773.[Abstract]
24. Sijts, A. J., M. L. De Bruijn, M. E. Ressing, J. D. Nieland, E. A. Mengede, C. J. Boog, F. Ossendorp, W. M. Kast, C. J. Melief. 1994. Identification of an H-2K^b-presented Moloney murine leukemia virus cytotoxic T-lymphocyte epitope that displays enhanced recognition in H-2D^b mutant bm13 mice. J. Virol. 68:6038.[Abstract/Free Full Text]
25. Milan, G., A. Zambon, M. Cavinato, P. Zanovello, A. Rosato, D. Collavo. 1999. Dissecting the immune response to moloney murine sarcoma/leukemia virus- induced tumors by means of a DNA vaccination approach. J. Virol. 73:2280.[Abstract/Free Full Text]
26. Biasi, G., D. Collavo, P. Zanovello, L. Chieco-Bianchi. 1981. Leukemia-cell rejection due to T-region encoded antigens. Immunogenetics 12:433.[Medline]
27. Biasi, G., A. Facchinetti, M. Panozzo, P. Zanovello, L. Chieco-Bianchi, D. Collavo. 1991. Moloney murine leukemia virus tolerance in anti-CD4 monoclonal antibody-treated adult mice. J. Immunol. 147:2284.[Abstract]
28. Wysocki, L. J., V. L. Sato. 1978. "Panning" for lymphocytes: a method for cell selection. Proc. Natl. Acad. Sci. USA 75:2844.[Abstract/Free Full Text]
29. Biasi, G., D. Collavo, P. Zanovello, A. Colombatti, L. Chieco-Bianchi. 1979. Lack of T-cell mediated cytotoxicity in M-MSV system depending on H-2 haplotypes. J Immunogenet. 6:341.[Medline]
30. Couedel, C., M. Bodinier, M. A. Peyrat, M. Bonneville, F. Davodeau, F. Lang. 1999. Selection and long-term persistence of reactive CTL clones during an EBV chronic response are determined by avidity, CD8 variable contribution compensating for differences in TCR affinities. J. Immunol. 162:6351.[Abstract/Free Full Text]
31. MacDonald, H. R., A. L. Glasebrook, J. C. Cerottini. 1982. Clonal heterogeneity in the functional requirement for Lyt-2/3 molecules on cytolytic T lymphocytes: analysis by antibody blocking and selective trypsinization. J. Exp. Med. 156:1711.[Abstract/Free Full Text]
32. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H. von Andrian, R. Ahmed. 2003. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4:225.[Medline]
33. Yewdell, J. W., J. R. Bennink. 1999. Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu. Rev. Immunol. 17:51.[Medline]
34. Wolpert, E. Z., P. Grufman, J. K. Sandberg, A. Tegnesjo, K. Karre. 1998. Immunodominance in the CTL response against minor histocompatibility antigens: interference between responding T cells, rather than with presentation of epitopes. J. Immunol. 161:4499.[Abstract/Free Full Text]
35. Grufman, P., E. Z. Wolpert, J. K. Sandberg, K. Karre. 1999. T cell competition for the antigen-presenting cell as a model for immunodominance in the cytotoxic T lymphocyte response against minor histocompatibility antigens. Eur. J. Immunol. 29:2197.[Medline]
36. Sourdive, D. J., K. Murali-Krishna, J. D. Altman, A. J. Zajac, J. K. Whitmire, C. Pannetier, P. Kourilsky, B. Evavold, A. Sette, R. Ahmed. 1998. Conserved T cell receptor repertoire in primary and memory CD8 T cell responses to an acute viral infection. J. Exp. Med. 188:71.[Abstract/Free Full Text]
37. Bousso, P., F. Lemaitre, J. Bilsborough, P. Kourilsky. 2000. Facing two T cell epitopes: a degree of randomness in the primary response is lost upon secondary immunization. J. Immunol. 165:760.[Abstract/Free Full Text]
38. Ishikawa, T., D. Kono, J. Chung, P. Fowler, A. Theofilopoulos, S. Kakumu, F. V. Chisari. 1998. Polyclonality and multispecificity of the CTL response to a single viral epitope. J. Immunol. 161:5842.[Abstract/Free Full Text]
39. Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh, J. D. Altman, R. Ahmed. 1998. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188:2205.[Abstract/Free Full Text]
40. Lee, P. P., C. Yee, P. A. Savage, L. Fong, D. Brockstedt, J. S. Weber, D. Johnson, S. Swetter, J. Thompson, P. D. Greenberg, M. Roederer, M. M. Davis. 1999. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat. Med. 5:677.[Medline]
41. Pittet, M. J., D. Valmori, P. R. Dunbar, D. E. Speiser, D. Lienard, F. Lejeune, K. Fleischhauer, V. Cerundolo, J. C. Cerottini, P. Romero. 1999. High frequencies of naive Melan-A/MART-1-specific CD8⁺ T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals. J. Exp. Med. 190:705.[Abstract/Free Full Text]
42. Anichini, A., A. Molla, R. Mortarini, G. Tragni, I. Bersani, M. Di Nicola, A. M. Gianni, S. Pilotti, R. Dunbar, V. Cerundolo, G. Parmiani. 1999. An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions. J. Exp. Med. 190:651.[Abstract/Free Full Text]
43. Deeths, M. J., R. M. Kedl, M. F. Mescher. 1999. CD8⁺ T cells become nonresponsive (anergic) following activation in the presence of costimulation. J. Immunol. 163:102.[Abstract/Free Full Text]
44. Sallusto, F., A. Lanzavecchia. 2001. Exploring pathways for memory T cell generation. J. Clin. Invest. 108:805.[Medline]
45. Facchinetti, A., P. Gallo, P. Perini, S. Mezzalira, F. Ronchese, G. Biasi. 2004. The MBP-reactive repertoire is shaped by recognition of minor histocompatibility antigens. J. Neuroimmunol. 148:154.[Medline]

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