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Characterization of the Fas Ligand/Fas-Dependent Apoptosis of Antiretroviral, Class I MHC Tetramer-Defined, CD8⁺ CTL by In Vivo Retrovirus-Infected Cells¹

Department of Microbiology and Immunology and The Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, NH 03756

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
C57BL/6 (B6; H-2^b) mice mount strong AKR/Gross murine leukemia virus (MuLV)-specific CD8⁺ CTL responses to the immunodominant K^b-restricted epitope, KSPWFTTL, of endogenous AKR/Gross MuLV. In sharp contrast, spontaneous virus-expressing AKR.H-2^b congenic mice are low/nonresponders for the generation of AKR/Gross MuLV-specific CTL. Furthermore, when viable AKR.H-2^b spleen cells are cocultured with primed responder B6 antiviral precursor CTL, the AKR.H-2^b cells function as "veto" cells that actively mediate the inhibition of antiviral CTL generation. AKR.H-2^b veto cell inhibition is virus specific, MHC restricted, contact dependent, and mediated through veto cell Fas ligand/responder T cell Fas interactions. In this study, following specific priming and secondary in vitro restimulation, antiretroviral CD8⁺ CTL were identified by a labeled K^b/KSPWFTTL tetramer and flow cytometry, enabling direct visualization of AKR.H-2^b veto cell-mediated depletion of these CTL. A 65–93% reduction in the number of B6 K^b/KSPWFTTL tetramer⁺ CTL correlated with a similar reduction in antiviral CTL cytotoxicity. Addition on sequential days to the antiviral CTL restimulation cultures of either 1) AKR.H-2^b veto cells or 2) a blocking Fas-Ig fusion protein (to cultures also containing AKR.H-2^b veto cells) to block inhibition demonstrated that AKR.H-2^b veto cells begin to inhibit B6 precursor CTL/CTL expansion during days 2 and 3 of the 6-day culture. Shortly thereafter, a high percentage of B6 tetramer⁺ CTL cocultured with AKR.H-2^b veto cells was annexin V positive and Fas^high, indicating apoptosis as the mechanism of veto cell inhibition. Experiments using the irreversible inhibitor emetine demonstrated that AKR.H-2^b cells had to be metabolically active and capable of protein synthesis to function as veto cells. Of the tetramer-positive CTL that survived veto cell-mediated apoptosis, there was no marked skewing from the preferential usage of V4, 8.1/8.2, and 11 TCR normally observed. These findings provide further insight into the complexity of host/virus interactions and suggest a fail-safe escape mechanism by virus-infected cells for epitopes residing in critical areas of viral proteins that cannot accommodate variations of amino acid sequence.

	Introduction

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There is general acceptance that neutralizing Abs and antiviral T cells, especially CTL, are particularly important in providing protection against viral disease. Neutralizing Abs are effective against free virions, specifically in reinfections where preexisting Abs may protect against initial infection and/or memory Ab responses may be elicited with sufficiently rapid kinetics to limit the infection after the first few rounds of viral replication. In a complementary fashion, antiviral CTL, via their ability to lyse virus-infected cells and to secrete antiviral cytokines such as IFN-, are very efficient in defending against viruses that are transmitted by infected cells and cell:cell contact. CTL responses may also be critical in resolving a primary encounter with virus before sufficient cycles of infection have occurred to spread the virus systemically.

To evade recognition of virus-infected cells by CD8⁺ CTL, viruses have developed a number of clever escape strategies (1, 2, 3, 4, 5, 6, 7). These evasion mechanisms fall into two classical categories: 1) variation in viral amino acid sequences responsible for epitope production, and 2) viral genome encoding of proteins that actively interfere with the production and presentation of unmutated viral epitopes. For variations directly affecting viral epitope presentation, evidence has accumulated not only for changes within the epitopes that inhibit either binding to MHC class I alleles or by the TCR, as expected, but also processing (8). In addition, viral variations include various alterations in the amino acid flanking sequences, resulting in impaired processing of the epitopes from their precursor proteins/larger peptides or their transport to the endoplasmic reticulum by TAP 1/2 (9, 10, 11, 12, 13, 14, 15, 16, 17). Such viral evasion by amino acid variation within or adjacent to the CTL epitope requires that the amino acid sequence of this region of the viral protein is not critical to its function such that the alteration can be tolerated by the virus. Alternatively, for viral proteins that inhibit epitope processing or presentation it is conceptually possible that every step of the endogenous class I pathway may provide an opportunity for a virus counter measure and escape from antiviral T cells. Indeed, there has been a growing number of descriptions of a variety of viral proteins encoded particularly by the large-genome DNA viruses, such as the herpes family, that interfere not only with transport of MHC/peptide complexes from the endoplasmic reticulum but also other steps of the presentation pathway, such as the TAP 1/2 transporter (reviewed in Ref. 18). However, most retroviruses have relatively small genomes, particularly the simple murine leukemia viruses (MuLV)³ that are discussed herein. There are only three genes—gag, pol, and env—encoding highly conserved polyproteins that collectively perform all of the functions necessary for viral replication, spread, and pathogenesis.

We have previously reported (19, 20) that C57BL/6 (B6; H-2^b) mice generate type-specific CTL responses to an immunodominant K^b-restricted epitope, KSPWFTTL, located in the membrane-spanning domain of p15E/TM of AKR/Gross MuLV. AKR.H-2^b congenic mice, though carrying the responder H-2^b haplotype, naturally express endogenous MuLV and are low/nonresponders for AKR/Gross MuLV-specific CTL (21, 22), apparently due to inhibitory AKR.H-2^b cells, as demonstrated both in vivo (23) and in vitro (24). For example, despite their expression of endogenous retroviral Ags and K^b (25), untreated viable AKR.H-2^b spleen cells cause dramatic inhibition of the B6 antiviral CTL response to in vitro restimulation with AKR/Gross MuLV-induced tumor cells (24). This inhibition is specific (AKR.H-2^b modulator spleen cells do not inhibit allogeneic MHC or minor histocompatibility-specific CTL production), MHC restricted, dependent on direct contact of AKR.H-2^b cells in a dose-dependent manner with the responder cell population, and not due to soluble factors. The mechanism of inhibition of the antiviral CTL response was shown to depend on Fas/Fas ligand (FasL) interactions (26). Although B6.gld (FasL^-) responders were as sensitive to inhibition by AKR.H-2^b modulator cells as B6, B6.lpr (Fas^-) responders were largely insensitive to inhibition, indicating that the responder cells needed to express Fas. A Fas-Ig fusion protein, when added to the in vitro CTL restimulation cultures, relieved the inhibition caused by the AKR.H-2^b cells if the primed responder cells were from either B6 or B6.gld mice, indicating that the inhibitory AKR.H-2^b cells express FasL. These results collectively implicate a veto cell (reviewed in Ref. 27)-mediated, activation-induced cell death (AICD) type of mechanism: viral Ag-positive AKR.H-2^b cells expressing FasL induce apoptosis of the antiviral T cells (or "veto" them) when the AKR.H-2^b cells are specifically recognized by responder T cells via their TCR. The CTL response in the presence of inhibitory cells could be restored by several cytokines/agents that have been shown to interfere with FasL/Fas induced cell death (e.g., IL-2, IL-15, TGF-, LPS, and 9-cis-retinoic acid) but not others, such as TNF- (26).

In this study we further define this type of escape mechanism by the use of labeled K^b/KSPWFTTL tetrameric analysis and multicolor flow cytometry to detect CD8⁺ CTL specific for this immunodominant retroviral epitope. The kinetics of expansion of such antiviral CTL and the effect of AKR.H-2^b veto cells on this expansion are determined. The mechanism of AKR.H-2^b veto cell-mediated inhibition of antiviral CTL production and the characteristics of the sensitive CD8⁺ CTL population are further defined. In this way, we provide a better understanding of how retroviruses may take advantage of the normal, physiological AICD mechanism to turn it into an ultimate virus escape mechanism.

	Materials and Methods

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Mice

B6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The AKR.H-2^b congenic mouse strain was maintained through breeding of brother/sister pairs in the Animal Health Resource facility of Dartmouth Medical School (Lebanon, NH). Breeding pairs were originally provided by Dr. D. Myers (Sloan-Kettering Memorial Institute, New York, NY).

Cell lines

The EG2, Gross virus-induced and Gross cell surface Ag (GCSA)⁺ tumors and EK1, AKR virus-induced but GCSA^- tumors are of the B6 (H-2^b) strain origin. AKR.H-2^b SL1 (SL1), a spontaneous GCSA⁺ tumor, was originally derived from the AKR.H-2^b congenic mouse strain. B.GV, a Gross virus-induced GCSA⁺ tumor, was derived from a BALB.B (H-2^b) mouse. These tumor cell lines have previously been described in detail (28). Cell lines were maintained by in vitro passage three times weekly in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 5% FBS, 5 x 10^-5 M 2-ME, L-glutamine, and antibiotics.

Polyclonal CTL, AKR.H-2^b veto cell-mediated inhibition, Fas-Ig blocking, and emetine treatment

Briefly, AKR/Gross MuLV-specific CTL were generated through in vivo inoculation of B6 responder mice with 10⁶ nonsyngeneic, H-2^b-matched B.GV tumor cells. Eleven to 14 days postinoculation, 10⁷ immune spleen cells were cultured in mixed lymphocyte tumor cell cultures (MLTC) with 2 x 10⁵ irradiated (DMS irradiation facility) EG2 tumor stimulator cells, as previously described (29). MLTC conditions where responder cells were cultured alone, without additional tumor cell restimulation (e.g., Fig. 1), are referred to as "none." For experiments designed to measure veto cell-mediated inhibition, 2 x 10⁶ viable AKR.H-2^b spleen cells were included in MLTC wells either at the initiation of the cultures (day 0), or, for kinetic experiments, on consecutive days thereafter, in which case the results have been presented as averaged data of three to six experiments per kinetic time point. To block Fas/FasL interactions, a 2x concentrated supernatant of a blocking Fas-Ig fusion protein (30) was derived as a secreted product of the National Institutes of Health 3T3 Fas-Ig transfectant cell line, generously provided by Dr. P. Leder (Howard Hughes Medical Institute, Boston, MA). To verify the presence of Fas-Ig (human IgG1 tail) in each supernatant preparation used, indirect flow cytometric analysis was performed using RF33.7 T hybridoma cells (31), the kind gift of Dr. K. Rock (Dana-Farber Cancer Institute, Boston, MA). The FasL⁺ RF33.7 cells were incubated with each Fas-Ig-containing supernatant preparation, followed by incubation with FITC F(ab')₂ goat anti-human IgG H and L chains (Jackson ImmunoResearch Laboratories, West Grove, PA), and flow cytometric analysis was performed. As a negative control for both flow cytometric analysis and in vitro blocking of Fas/FasL interactions, a concentrated supernatant of cultured, nontransfected National Institutes of Health 3T3 cells was used in parallel. For emetine (Sigma-Aldrich, St. Louis, MO) treatment, AKR.H-2^b splenocytes were incubated in 2 µg/ml emetine for 17 h in a 37°C CO₂ incubator and then washed five times before use of the pretreated cells in experiments. Following 6 days of in vitro restimulation in medium containing RPMI 1640 supplemented with 5% FBS, L-glutamine, and antibiotics, ⁵¹Cr release assays were conducted as previously described (29) to measure CTL cytotoxicity from these bulk MLTC cultures. In short, 10⁴ radiolabeled tumor target cells were mixed with varying numbers of effector cells (i.e., several E:T ratios), centrifuged, and incubated for 4 h at 37°C. E:T ratios were established by counting all viable cells remaining at the end of the MLTC. Thus, in restimulation conditions including AKR.H-2^b veto cells (added as one-sixth of the total cells at day 0), the actual percentage of responder B6 effector cells (and thus the E:T ratio) may be diminished by up to 16%. At the end of this incubation, the cells were centrifuged again and an aliquot of cell-free supernatant was removed for gamma counting and data reduction. Percentage of specific lysis against tumor cells is defined according to the following formula: [(X - Y)/Z] x 100, in which X = cpm released by target cells incubated with effector cells, Y = cpm released by target cells incubated alone, and Z = cpm released by the freeze-thaw of target cells (80% of total cpm incorporated).

View larger version (47K): [in this window] [in a new window]   FIGURE 1. Concurrent reduction in the frequency of tetrameric MHC class I/peptide complex binding and inhibition of antiviral CTL cytotoxicity. B6 mice were inoculated with AKR/Gross MuLV+ tumor cells. Eleven days later, responder lymphocytes were cultured in vitro for 6 days with irradiated, viral Ag+ EG2 tumor cells, without or with viable AKR.H-2b veto cells. MLTC conditions where responder cells were cultured alone, without additional tumor cell restimulation, are referred to as "None." At the end time point of these MLTC cultures, responder cells were stained via flow cytometric analysis to identify and enumerate Kb/KSPWFTTL tetramer (PE-labeled) and anti-CD8a (FITC-labeled) binding to CTL generated without (A) or with (B) addition of AKR.H-2b veto cells. Concurrently, lymphocytes were tested for their ability to lyse 51Cr-labeled GCSA viral Ag-positive EG2 tumor target cells (C), which had a spontaneous release of 5.6%. In these and all other cytolytic assays GCSA viral Ag-negative EK1 tumor cells were always used as a negative control target and consistently were lysed <3%.

Purified KSPWFTTL peptide was synthesized by Research Genetics (Huntsville, AL) and then sent to the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility (Atlanta, GA) for the production of PE-labeled K^b/KSPWFTTL tetramer. For flow cytometric analysis, 2 x 10⁵ splenocytes were incubated at 37°C for 15 min in 20 µl of a 1/100 dilution of tetramer preparation (pretitrated optimal concentration determined using A610.G5 cloned CTL cells of K^b/KSPWFTTL specificity). Cells were then washed once in HBSS supplemented with 2 mg/ml BSA and HEPES buffer (flow medium), centrifuged, and further incubated (4°C for 30 min) with the following reagents (or their appropriate isotype control mAb) at 0.2 µg/2 x 10⁵ cells/reagent in a total volume of 50 µl: FITC-anti-CD8a (BD PharMingen, San Diego, CA) or allophycocyanin-anti-CD8a (Caltag Laboratories, Burlingame, CA); FITC-anti-CD95 (BD PharMingen); FITC anti-CD44 (BD PharMingen); or biotinylated annexin V (Caltag Laboratories). Following incubation of cells with biotinylated annexin V, two washes in flow medium were subsequently performed followed by incubation with allophycocyanin-streptavidin (Caltag Laboratories). PE-rat IgG2a and biotinylated P3 mAb (unknown specificity) were used as control Abs for tetramer and annexin V staining, respectively. Cells incubated in biotinylated preparations were further incubated in medium containing streptavidin-allophycocyanin.

For V typing of K^b/KSPWFTTL tetramer-positive CTL, FITC-labeled mAbs to V2, V3, V4, V5.1/5.2, V6, V7, V8.1/8.2, V8.3, V9, V10^b, V11, V12, V13, V14, or V17a (BD PharMingen) were used. Flow cytometric analysis was performed on a FACSCalibur (BD Biosciences, Mountain View, CA) using CellQuest software (BD Biosciences at the Herbert C. Englert Flow Cytometry Facility).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Quantification of K^b/KSPWFTTL tetramer-positive CTL and deletion mediated by viable AKR.H-2^b veto cells

Following priming and in vitro restimulation with AKR/Gross MuLV⁺ tumor cells to generate anti-AKR/Gross MuLV CTL primarily directed to the KSPWFTTL immunodominant viral epitope (19, 20), viable B6 splenic lymphocytes were selected through forward and side scatter profiling for two-color flow cytometric analysis (Fig. 1, A and B). Fluorochrome-labeled anti-CD8a (Fig. 1, x-axis) and K^b/KSPWFTTL tetramer (Fig. 1, y-axis) reagents were used to identify and quantitate double positive lymphocytes at the end time point of in vitro restimulation cultures (day 7). In the representative experiment shown (Fig. 1A), following priming and restimulation with viral Ag⁺ tumor cells, 12% of all gated lymphocytes (36.4% of CD8a-positive cells) proved to be CD8a/KSPWFTTL tetramer double-positive (Fig. 1A, upper right quadrant). Importantly, and in contrast, the addition of viable, viral Ag-positive AKR.H-2^b veto cells at the beginning time point to parallel CTL restimulation cultures mediated a dramatic (86%) decrease in CD8a/KSPWFTTL tetramer double-positive T cells to only 1.7% of all lymphocytes (6.6% of CD8a-positive T cells, representing an 82% decrease). To test whether the veto cell-mediated inhibition of the number of CD8a/KSPWFTTL tetramer double-positive cells correlated with a decrease in antiviral CTL cytotoxicity, parallel ⁵¹Cr release assays were performed (Fig. 1C). Following tumor cell restimulation (in the absence of veto cells), high levels of lysis of specific tumor target cells were observed. Addition of AKR.H-2^b veto cells reduced the level of cytotoxicity by 70%, a decrease in the generation of functional B6 antiviral CTL roughly proportional to the reduced number of CD8a/KSPWFTTL tetramer double-positive cells. This experiment was repeated seven times with the same result, although the capacity of AKR.H-2^b veto cells to inhibit the number of tetramer⁺ CTL and corresponding antiviral lysis varied among experiments, ranging from 65 to 93%. Similarly, if in place of irradiated tumor stimulator cells KSPWFTTL peptide was used as a source of specific Ag for in vitro restimulation, both the numbers of tetramer⁺ cells and the development of cytotoxic K^b/KSPWFTTL-specific CD8⁺ T cells was similarly inhibited (data not shown). Taking advantage of the alternatively expressed Thy1 alleles of the B6 (Thy 1.2⁺) responder, vs AKR.H-2^b (Thy 1.1⁺) veto, T cell populations, four-color flow cytometric analysis was performed to rule out the possibility that antiviral CTL nonresponder AKR.H-2^b veto CD8⁺ T cells contributed to the detectable double-positive K^b/KSPWFTTL tetramer-binding subset (data not shown). Similarly, naive B6 spleen cell populations were determined to be below the level of detection for K^b/KSPWFTTL tetramer positivity (data not shown).

The kinetics of K^b/KSPWFTTL-specific CTL expansion

Using the same two-color flow cytometric protocol described above, a study of the kinetic expansion of CD8a/KSPWFTTL-specific CTL was performed by staining aliquots of cells harvested on varied days of the MLTC. As shown in the representative experiment of Fig. 2, CD8a/KSPWFTTL double-positive lymphocytes first become clearly discernable following 4 days of specific tumor cell restimulation: 11% of CD8a-positive cells were tetramer positive. The number of CD8a/KSPWFTTL double-positive lymphocytes expanded to 21% of CD8a⁺ T cells by the termination of the MLTC at day 7. Conversely, all tetramer-positive cells were shown to be CD8a⁺, consistent with the KSPWFTTL epitope being the immunodominant peptide recognized by AKR/Gross MuLV-specific CD8⁺ CTL. Over a course of four experiments, we verified that the observable expansion of Ag-specific CD8a⁺ CTL occurred starting at day 3 or 4 of the restimulation cultures, with a range of 2- to 5-fold further increase (in percentage of CD8⁺ cells) by day 7.

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Kinetics of Kb/KSPWFTTL-specific CTL expansion. B6 splenocytes were primed in vivo and restimulated in vitro with viral Ag+ tumor cells in MLTC cultures (see Fig. 1). An aliquot of cells was stained for Kb/KSPWFTTL tetramer/CD8a dual positivity on each of the indicated sequential days of the MLTC, fixed in 2% paraformaldehyde, and subsequently analyzed at the same time by flow cytometry.

As shown in Fig. 1, addition of viable AKR.H-2^b veto cells at the initiation of the restimulation cultures leads to a dramatic decrease in the generation of K^b/KSPWFTTL-specific CTL. To define when the veto cells actually mediated inhibition of CTL generation, two different series of kinetic experimental studies were conducted. In the first, equal numbers of viable AKR.H-2^b veto cells, obtained from sex-matched sibling mice, were added on consecutive days to parallel culture wells already containing primed B6 responder splenocytes and viral Ag-positive tumor stimulator cells. As can clearly be seen in Fig. 3A, viable AKR.H-2^b veto cells substantially inhibited antiviral CTL generation when added to restimulation cultures through day 2, with a much lesser degree of inhibition when added thereafter. To assess whether other factors could influence the boundaries of this kinetic window of precursor CTL (pCTL)/CTL susceptibility, we conducted additional experiments. In one such experiment we took advantage of an AKR.H-2^b cell population that proved to be exceptionally efficient, causing nearly complete inhibition of antiviral CTL generation. In this case substantial inhibition was observed when veto cells were added through day 3 of the MLTC. In another experiment, adding 2-fold more AKR.H-2^b cells, the maximal allowable number of veto cells (see Materials and Methods), also extended the capacity of the veto cells to inhibit CTL responsiveness by 1 day. However, inclusion of veto cells during the last 24–48 h of the MLTC did not cause significant inhibition. In keeping with these kinetics for optimal pCTL/CTL inhibition, irradiated AKR.H-2^b cells, like their viable counterparts, were able to inhibit antiviral CTL generation, albeit with lower efficiency, only when added to cultures on day 1 or 2 (data not shown).

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Kinetics of AKR.H-2b veto cell inhibition and abrogation of inhibition with blocking Fas-Ig fusion protein. A, Fresh viable AKR.H-2b veto cells were added on sequential days to in vitro MLTC cultures containing primed B6 responder cells and irradiated EG2 tumor stimulator cells to determine the kinetics of veto cell inhibition. Values shown represent averaged data of three to six experiments/kinetic time point. The values for spontaneous 51Cr release by EG2 tumor target cells ranged from 4.9 to 10.7%. B, MLTC cultures were set up as in A above but with AKR.H-2b splenic veto cells included at the initiation of in vitro cultures in all cases. Then, a Fas-Ig fusion protein (2x concentrate supernatant) was added on sequential days to parallel wells to determine the kinetic time frame of FasL/Fas-mediated inhibition via restoration of the generation of CTL that lyse viral Ag+ SL1 target cells, assessed by testing the time points in a single 51Cr release assay. SL1 target cells had a spontaneous 51Cr release of 9.2%.

To test whether inhibition of antiviral CTL expansion by AKR.H-2^b veto cells is dependent on their ability to synthesize proteins, perhaps including FasL, the irreversible protein synthesis inhibitor emetine was used (Fig. 4). AKR.H-2^b veto cells were either untreated or emetine-pretreated (followed by extensive washing) before their inclusion at equal numbers of viable cells to CTL restimulation cultures at day 2, a time point at which there is still maximal sensitivity of the pCTL/CTL but which minimizes the time the veto cells may need to persist to mediate inhibition. As previously shown in Fig. 3, the high levels of cytotoxicity observed following tumor cell restimulation were inhibited substantially when viable, untreated AKR.H-2^b veto cells were added at day 2. In contrast, AKR.H-2^b veto cells pretreated with emetine had essentially no effect on the generation of antiviral CTL. Emetine-treated AKR.H-2^b cells cultured alone for up to 4 days were checked for viability by erythrosin B staining and were found to be as viable as their untreated counterparts. This experiment was repeated three times with essentially the same result, suggesting that protein synthesis by the veto cells is required for veto cell-mediated inhibition of antiviral CTL generation.

View larger version (37K): [in this window] [in a new window]   FIGURE 4. Emetine treatment of AKR.H-2b veto cells. To determine whether AKR.H-2b cells must be metabolically active and have the ability of de novo protein synthesis to function as veto cells, the irreversible protein synthesis inhibitor emetine was used. Primed B6 responder cells were restimulated in vitro with EG2 tumor stimulator cells (at day 0) either alone or with the addition (at day 2) of viable, vs emetine-treated (17 h), AKR.H-2b spleen cells. Erythrosin B viability staining was performed and equal numbers of viable, untreated vs emetine-treated, AKR.H-2b cells were added to cultures at a responder:AKR.H-2b cell ratio of 5:1. The value for spontaneous 51Cr release by SL1 target cells was 5.8%.

To directly test whether AKR.H-2^b veto cells were mediating apoptosis of B6 antiviral CTL, following restimulation for 4 days, without (Fig. 5, A–C) or with (Fig. 5, D–F) addition of AKR.H-2^b veto cells, CD8a/KSPWFTTL tetramer dual-positive CTL were analyzed for their ability to bind allophycocyanin-labeled annexin V. Annexin V staining of cells occurs via binding to exposed membrane phospholipid phosphatidylserine moieties characteristic of the early stages of apoptosis (32). Day 4 of restimulation cultures was selected as the earliest time providing consistently adequate numbers of tetramer⁺ CTL to analyze (Fig. 2), and taking into account the kinetics of functional inhibition by AKR.H-2^b veto cells (Fig. 3). In the representative experiment shown, following tumor cell restimulation, 23% of all CD8⁺ T cells were K^b/KSPWFTTL tetramer-specific (Fig. 5A). Such antiviral CTL were shown to bind annexin V at relatively low levels (31%, total mean fluorescence intensity (TMFI) = 17) (Fig. 5B), and were highly cytotoxic when parallel cultures were assayed following an additional 2 days of MLTC culture (Fig. 5C). In contrast, adding AKR.H-2^b veto cells at the initiation of the CTL restimulation cultures decreased K^b/KSPWFTTL-specific CTL to 10% of CD8⁺ T cells (Fig. 5D). In addition to this substantial veto cell-dependent inhibition, these remaining antiviral CTL were found to be highly annexin V positive (85% positive, TMFI = 182) (Fig. 5E), suggesting that a high proportion of residual antiviral CTL were also beginning to undergo apoptosis at day 4. Indeed, when assayed for cytotoxic function at day 6, there was a 71% veto cell-dependent reduction in lysis of specific tumor target cells (Fig. 5F), in keeping with the likelihood that the annexin V-positive, tetramer⁺ antiviral CTL, caught in the act of undergoing apoptosis due to veto cells at day 4, were depleted by the time cytotoxicity assays were performed 2 days later. As an internal control, consistent with the exquisite specificity of AKR.H-2^b veto cell inhibition (24, 26), the CD8a⁺ T cell subpopulation that was clearly negative for K^b/KSPWFTTL tetramer binding (Fig. 5, A and D, below dotted lines) bound annexin V with only low intensity (e.g., Fig. 5D, 41% positive, TMFI = 20). In contrast, those CD8a⁺ T cells that were scored as tetramer negative but showed a +/- degree of tetramer staining, especially those cocultured with AKR.H-2^b veto cells (Fig. 5D, between dotted line and solid line), showed intermediate levels of annexin V binding (67% positive, TMFI = 72). This observation is perhaps indicative of bona fide tetramer⁺ CTL whose immediately previous interaction with AKR.H-2^b veto cells lead to a down regulation of tetramer binding ability in conjunction with the initiation of apoptosis (33). These data, which were repeated with the same pattern of results in two additional experiments, directly demonstrated that adding AKR.H-2^b veto cells to restimulation cultures caused a dramatic reduction in the generation of antiviral CTL by mediating the apoptosis specifically of K^b/KSPWFTTL tetramer-positive CD8a⁺ CTL.

View larger version (64K): [in this window] [in a new window]   FIGURE 5. AKR.H-2b veto cell-dependent apoptosis of Kb/KSPWFTTL-specific CTL. Responder Ag-specific B6 antiviral CTL (CD8a/tetramer double-positive), expanded in MLTC culture without or with AKR.H-2b veto cells (added at day 0), were stained for annexin V binding at day 4 via three-color flow cytometry. B and E, Control, Biotinylated P3 mAb (unknown binding specificity), which was used as a negative control for experimental binding by biotinylated annexin V. A and D, The observed differential intensity of the binding of Kb/KSPWFTTL tetramer to CD8a-positive cells is indicated as follows: below dotted lines, clearly tetramer negative; above the dotted line and below the solid line, intermediate tetramer binding; above the solid line, strong tetramer binding. Corresponding cytotoxicity data obtained at the end time point of the MLTC (day 7) is also shown. The value for spontaneous 51Cr release by SL1 target cells was 12.3%.

To test whether K^b/KSPWFTTL-specific CTL displayed an activated phenotype that correlated with susceptibility to inhibition by AKR.H-2^b veto cells, CD8a/KSPWFTTL tetramer double-positive CTL were also stained for CD44 and Fas (CD95) expression at day 4. Such double-positive CTL generated without or with AKR.H-2^b veto cells proved to be CD44^high vs the expression found on naive B6 CD8⁺ T cells, indicating their activation status (data not shown). Similarly, the majority of the tetramer⁺ CTL from all day 4 cultures were also Fas⁺ (vs naive CD8⁺ T cells). However, those CTL from MLTC wells including AKR.H-2^b veto cells clearly showed increased Fas expression vs CTL from cultures that did not include veto cells (Table I). These data are consistent with a possible Fas up-regulation through interaction with AKR.H-2^b veto cells and were coordinate with increased annexin V binding at day 4 (Fig. 5) as an indicator of susceptible K^b/KSPWFTTL-specific CTL undergoing FasL/Fas-mediated apoptosis. This differential expression of Fas was reproduced in a second experiment.

V TCR usage by KSPWFTTL-specific CTL

Because AKR.H-2^b veto cell inhibition, though substantial, was never complete as measured by either the frequency of tetramer⁺ CD8 T cells or cytolytic activity, it was of interest to determine whether the resistant antiviral CTL comprised a distinct phenotypic subset. To address this possibility, responder cells were restimulated in culture for 7 days to allow for optimal in vitro expansion of specific CTL. Then, CD8a/KSPWFTTL double-positive cells were detected as above, and their TCR V usage was further determined via three-color flow cytometric analysis with a broad panel of anti-V mAbs (see Materials and Methods for details). In the representative experiment shown, in the absence of AKR.H-2^b veto cells the antiviral CTL response displayed a highly skewed TCR repertoire (Fig. 6, left panels). Thus, V 4, 11, and 8.1/8.2 accounted for nearly all TCR V used by tumor cell-restimulated, K^b/KSPWFTTL-specific CTL. Similarly, the residual K^b/KSPWFTTL-specific CTL generated in parallel cultures that included AKR.H-2^b veto cells (Fig. 6, right panels) were shown to use roughly the same percentages of each of these three TCR V elements. This experiment was repeated two times with the same pattern of results. Therefore, the possibility that CTL resistance to apoptosis might correlate with differential V usage, perhaps due to varied CTL TCR avidity for K^b/KSPWFTTL complexes on the veto cell, was not supported. Similarly, although Fas expression on K^b/KSPWFTTL-specific CTL was increased due to the inclusion of AKR.H-2^b veto cells in restimulation cultures when assessed at day 4 (Table I), at day 7 residual CTL (which were not vetoed) displayed levels of Fas (and CD44) which were indistinguishable from the levels expressed on CTL generated in the absence of AKR.H-2^b veto cells.

View larger version (47K): [in this window] [in a new window]   FIGURE 6. TCR V usage by Kb/KSPWFTTL-specific antiviral CTL. Primed B6 responder splenocytes were cultured with irradiated tumor stimulator cells without or with AKR.H-2b veto cells for 7 days as shown. CD8a/KSPWFTTL tetramer double-positive cells (68 and 27% of all gated CD8+ T cells generated without or with AKR.H-2b veto cells, respectively) were further phenotyped for TCR V usage (see Materials and Methods). The appropriate rat IgG2b isotype control mAb for the anti-V4 and V11 mAbs is shown. The data for mouse IgG2a isotype control mAb, appropriate for the anti-V8.1/8.2 mAb, also demonstrated very low background staining and was omitted for the sake of brevity.

The results of these studies, taken together, suggest that the basis for the AKR/Gross MuLV-specific CTL nonresponder phenotype of AKR.H-2^b mice is the inhibition of clonal expansion of antiviral CTL directed against the immunodominant K^b-presented viral epitope, KSPWFTTL (Fig. 1). Such inhibition is caused by normal AKR.H-2^b veto cells spontaneously expressing viral epitopes as a result of infection in vivo by endogenous MuLV. By use of K^b/KSPWFTTL tetramers direct evidence was presented for veto cell-dependent apoptosis of specific pCTL/CTL via analysis with annexin V staining (Fig. 5). These findings were consistent with our previous data demonstrating that veto cell inhibition of B6 antiviral CTL generation was dependent on both Fas expression by responder T cells and FasL expression by the AKR.H-2^b veto cells (26).

Several additional observations were not only in keeping with this model but further suggested that such veto cell-dependent apoptosis is a dynamic process involving prior activation of both the responder pCTL/CTL and the AKR.H-2^b veto cells. First, the veto cells must be capable of de novo protein synthesis to exert their inhibitory effect (Fig. 4). Whether this requirement for protein synthesis simply reflects a need to induce and/or up-regulate the level of veto cell FasL, or alternatively, or in addition, relates to the expression of other activation, adhesion, or costimulatory molecules is at present unclear. Cell division of veto cells was not required, however, as lethally gamma-irradiated AKR.H-2^b cells, provided they were added at the correct kinetic window of responder T cell susceptibility, were still able to substantially veto the CTL response. Second, the kinetics of responder T cell susceptibility to veto cell inhibition was somewhat delayed (Figs. 3 and 4), also consistent with required activational events. We interpret the experiments in which viable AKR.H-2^b veto cells were added on sequential days of the in vitro restimulation MLTC as indicating that day 2 was the latest time point at which veto cells could set in motion the series of events culminating in the apoptosis of antiviral CTL. In contrast, the experiments varying the time of addition of blocking Fas-Ig fusion protein defined 24 h after the establishment of the restimulation cultures as the initiation of irreversible FasL/Fas interactions leading to apoptosis. This distinction is valid because this blocking approach is essentially a competition between soluble Fas-Ig and responder T cell surface Fas for binding to veto cell FasL. Consistent with an activation-dependent delayed kinetic window of susceptibility of pCTL/CTL to veto cell action, their expression of the activation markers CD44 and Fas (Table I) was up-regulated. Also by day 4, a high proportion of the antiviral CTL which had not already lysed were annexin V positive (Fig. 5). Indeed, by tetramer staining and functional assessment at day 7, it was apparent that these annexin V-expressing cells were subsequently lysed. Collectively, these results implied a necessary, and potentially reciprocal, activation of responder T cell and AKR.H-2^b veto cell following TCR-mediated recognition of the veto cells before the commitment to terminal FasL/Fas-mediated apoptotic events. This interpretation of an AICD/veto cell type mechanism was in keeping with our earlier observation that pCTL frequencies in naive AKR.H-2^b nonresponder mice were similar to those of responder B6 mice (34). Why some K^b/KSPWFTTL-specific pCTL/CTL survive veto cell-mediated apoptosis was unclear; discordant use of V TCR subunits did not seem to be the explanation (Fig. 6), but we could not rule out differential expression of other required molecules or other features impacting overall TCR system affinity/avidity.

In this report we have explored this system as a model of an alternative general mechanism by which viruses, specifically as virus-infected cells, may escape clearance by antiviral T cell-mediated immunity—the ability of virus-infected cells to serve as veto cells that inactivate activated antiviral T cells. The principal differences between the classic virus escape mechanisms affecting epitope integrity or processing, as discussed in the introduction, and the veto cell strategy are that in veto cell inhibition 1) viral epitopes recognized by the T cells are not modified, 2) viral epitope processing and presentation are not disturbed, and 3) TCR recognition and the initiation of the T cell response, and perhaps initial clonal expansion of antiviral T cells, are not inhibited. Rather, the veto cell mechanism embraces and takes advantage of normal Ag processing and presentation and MHC-restricted TCR recognition. In short, the virus-infected veto cell is a bona fide APC, but one which, subsequent to its specific recognition and binding by the antiviral T cell, functionally inactivates, or causes the apoptotic lysis of, that antiviral T cell.

In this context it is perhaps instructive to consider that virus-infected veto cells may represent an example of a "fail-safe" ultimate escape mechanism obtained by co-opting a normal immunological process. Thus, the needed down-regulation of the large number of effector T cells, once an infection has been successfully cleared, is generally considered to be accomplished by AICD. In AICD, activated T effector cells expressing FasL and/or Fas undergo apoptotic lysis by either "suicide" or "fratricide" upon engagement of FasL and Fas in an Ag-nonspecific manner, although the involvement of other TNF/TNFR family members has also been described. However, providing that the infected cells can express FasL, virus infection leading to presentation of viral peptides by MHC class I and/or II would overlay TCR recognition onto the system to substantially increase the efficiency of cell:cell interactions and render them antigenically specific. Such a veto cell would serve as a back-up escape device if the various viral strategies to mutate T cell epitopes or interfere with epitope processing have failed or are not available. Our studies presented herein using the AKR.H-2^b mouse strain, which exhibits spontaneous endogenous viral Ag expression triggering antiviral CTL nonresponsiveness, thus serve to emphasize the veto cell mechanism as a natural, physiologic virus escape mechanism. However, as has been recently suggested (35), FasL-expressing, specific epitope-presenting APCs could also be prospectively constructed to target and kill corresponding autoreactive T cells to potentially ameliorate autoimmune diseases.

	Acknowledgments

 
We thank Dr. John Altman and the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility for synthesis of the of PE-labeled K^b/KSPWFTTL tetramer. We also thank Dr. James Gorham, Dr. Hillary White, Kathy Green, On Ho, Jack Lin, Darshan Sappal, Amy Campopiano, and Shannon Baker for helpful scientific discussions. Jack Lin also contributed by performing preliminary experiments to determine the optimal tetramer staining conditions. We also thank Dr. Alice Givan, Gary Ward, and Kenneth Orndorff for assistance in performing flow cytometric analysis.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants CA69525 and CA82755. The DMS irradiation facilities and the Herbert C. Englert Flow Cytometer Facility, established by a grant from the Fannie E. Rippel Foundation, are partially supported by National Institutes of Health Core Grant CA-23108 of the Norris Cotton Cancer Center.

² Address correspondence and reprint requests to Dr. William R. Green, Department of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, Borwell 628 West, Lebanon, NH 03756. E-mail address: william.r.green{at}dartmouth.edu

³ Abbreviations used in this paper: MuLV, murine leukemia virus; FasL, Fas ligand; pCTL, precursor CTL; AICD, activation-induced cell death; GCSA, Gross cell surface Ag; MLTC, mixed lymphocyte tumor cell culture; TMFI, total mean fluorescence intensity.

Received for publication July 24, 2001. Accepted for publication January 14, 2002.

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