In vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the GP33-43 epitope by H-2Db but not H-2Kb

CD8 T cells drive the protective immune response to lymphocytic choriomeningitis virus (LCMV) infection and are thus a determining force in the selection of viral variants. To examine how escape mutations affect the presentation and recognition of overlapping T-cell epitopes, we isolated an LCMV variant that is not recognized by T-cell receptor (TCR)-transgenic H-2Db-restricted LCMV GP33-41-specific cytotoxic T lymphocytes (CTL). The variant virus carried a single-amino-acid substitution (valine to alanine) at position 35 of the viral glycoprotein. This region of the LCMV glycoprotein encodes both the Db-restricted GP33-43 epitope and a second epitope (GP34-42) presented by the Kb molecule. We determined that the V-to-A CTL escape mutant failed to induce a Db GP33-43-specific CTL response and that Db-restricted GP33-43-specific CTL induced by the wild-type LCMV strain were unable to kill target cells infected with the variant LCMV strain. In contrast, the Kb-restricted response was much less affected. We found that the V-to-A substitution severely impaired peptide binding to Db but not to Kb molecules. Strikingly, the V-to-A mutation did not change any of the anchor residues, and the dramatic effect on binding was therefore unexpected. The strong decrease in Db binding explains why the variant virus escapes the Db GP33-43-specific response but still elicits the Kb-restricted response. These findings also illustrate that mutations within regions encoding overlapping T-cell epitopes can differentially affect the presentation and recognition of individual epitopes.     

Pleiotropic effects of post-translational modifications on the fate of viral glycopeptides as cytotoxic T cell epitopes

The fate of viral glycopeptides as cytotoxic T lymphocyte (CTL) epitopes is unclear. We have dissected the mechanisms of antigen presentation and CTL recognition of the peptide GP392-400 (WLVTNGSYL) from the lymphocytic choriomeningitis virus (LCMV) and compared them with those of the previously reported GP92-101 antigen (CSANNSHHYI). Both GP392-400 and GP92-101 bear a glycosylation motif, are naturally N-glycosylated in the mature viral glycoproteins, bind to major histocompatibility complex H-2D(b) molecules, and are immunogenic. However, post-translational modifications differentially affected GP92-101 and GP392-400. Upon N-glycosylation or de-N-glycosylation, a marked decrease in major histocompatibility complex binding was observed for GP392-400 but not for GP92-101. Further, under its N-glycosylated or de-N-glycosylated form, GP392-400 then lost its initial ability to generate a CTL response in mice, whereas GP92-101 was still immunogenic under the same conditions. The genetically encoded form of GP392-400, which on the basis of its immunogenicity could still be presented with H-2D(b) during the course of LCMV infection, does not in fact appear at the surface of LCMV-infected cells. Our results show that post-translational modifications of viral glycopeptides can have pleiotropic effects on their presentation to and recognition by CTL that contribute to either creation of neo-epitopes or destruction of potential epitopes.     

Diversity of T-cell receptors in virus-specific cytotoxic T lymphocytes recognizing three distinct viral epitopes restricted by a single major histocompatibility complex molecule.

Cytotoxic T lymphocytes (CTL) recognize virus peptide fragments complexed with class I major histocompatibility complex (MHC) molecules on the surface of virus-infected cells. Recognition is mediated by a membrane-bound T-cell receptor (TCR) composed of alpha and beta chains. Studies of the CTL response to lymphocytic choriomeningitis virus (LCMV) in H-2b mice have revealed that three distinct viral epitopes are recognized by CTL of the H-2b haplotype and that all of the three epitopes are restricted by the Db MHC molecule. The immunodominant Db-restricted CTL epitope, located at LCMV glycoprotein amino acids 278 to 286, was earlier noted to be recognized by TCRs that consistently contained V alpha 4 segments but had heterogeneous V beta segments. Here we show that CTL clones recognizing the other two H-2Db-restricted epitopes, LCMV glycoprotein amino acids 34 to 40 and nucleoprotein amino acids 397 to 407 (defined in this study), utilize TCR alpha chains which do not belong to the V alpha 4 subfamily. Hence, usage of V alpha and V beta in the TCRs recognizing peptide fragments from one virus restricted by a single MHC molecule is not sufficiently homogeneous to allow manipulation of the anti-viral CTL response at the level of TCRs. The diversity of anti-viral CTL likely provides the host with a wider option for attacking virus-infected cells and prevents the emergence of virus escape mutants that might arise if TCRs specific for the virus were homogeneous.     

Uncovering subdominant cytotoxic T-lymphocyte responses in lymphocytic choriomeningitis virus-infected BALB/c mice.

The cytotoxic T-lymphocyte response against lymphocytic choriomeningitis virus (LCMV) in BALB/c mice is predominantly directed against a single, Ld-restricted epitope in the viral nucleoprotein (residues 118 to 126). To investigate whether any Kd/Dd-restricted responses were activated but did not expand during the primary response, we used a BALB/c mutant, BALB/c-H-2dm2, which does not express the Ld molecule. Splenocytes from LCMV-infected BALB/c mice were transferred into irradiated BALB/c-H-2dm2 mice and rechallenged with LCMV. Thus, they were exposed to an antigenic stimulus without the involvement of the immunodominant Ld-restricted epitope. In this adoptive transfer model, the donor splenocytes protected the recipient mice against chronic LCMV infection by mounting a potent Kd- and/or Dd-restricted secondary antiviral response. Analysis of a panel of Kd binding LCMV peptides revealed that residues 283 to 291 from the viral glycoprotein (GP(283-291)) comprise a major new epitope in the adoptive transfer model. Because the donor splenocytes were first activated during the primary infection in BALB/c mice, the GP(283-291) epitope is a subdominant epitope in BALB/c mice that becomes dominant after rechallenge in BALB/c-H-2dm2 mice. This study makes two points. First, it shows that subdominant CTL responses can be protective, and second, it provides a general experimental approach for uncovering subdominant CTL responses in vivo. This strategy can be used to identify subdominant T-cell responses in other systems.     

Genetically encoded and post-translationally modified forms of a major histocompatibility complex class I-restricted antigen bearing a glycosylation motif are independently processed and co-presented to cytotoxic T lymphocytes

The mechanisms by which antigenic peptides bearing a glycosylation site may be processed from viral glycoproteins, post-translationally modified, and presented by major histocompatibility complex class I molecules remain poorly understood. With the aim of exploring these processes, we have dissected the structural and functional properties of the MHC-restricted peptide GP92-101 (CSANNSHHYI) generated from the lymphocytic choriomeningitis virus (LCMV) GP1 glycoprotein. LCMV GP92-101 bears a glycosylation motif -NXS- that is naturally N-glycosylated in the mature viral glycoprotein, displays high affinity for H-2D(b) molecules, and elicits a CD8(+) cytotoxic T lymphocyte response. By analyzing the functional properties of natural and synthetic peptides and by identifying the viral sequence(s) from the pool of naturally occurring peptides, we demonstrated that multiple forms of LCMV GP92-101 were generated from the viral glycoprotein and co-presented at the surface of LCMV-infected cells. They corresponded to non-glycosylated and post-translationally modified sequences (conversion of Asn-95 to Asp or alteration of Cys-92). The glycosylated form, despite its potential immunogenicity, was not detected. These data illustrate that distinct, non-mutually exclusive antigen presentation pathways may occur simultaneously within a cell to generate structurally and functionally different peptides from a single genetically encoded sequence, thus contributing to increasing the diversity of the T cell repertoire.     

Molecularly engineered vaccine which expresses an immunodominant T-cell epitope induces cytotoxic T lymphocytes that confer protection from lethal virus infection

Identification of a single viral T-cell epitope, associated with greater than 95% of the virus-specific cytotoxic T-lymphocyte (CTL) activity in BALB/c (H-2d) mice (J. L. Whitton, A. Tishon, H. Lewicki, J. Gebhard, T. Cook, M. Salvato, E. Joly, and M. B. A. Oldstone, J. Virol. 63:4303-4310, 1989), permitted us to design a CTL vaccine and test its ability to protect against a lethal virus challenge. Here we show that a single immunization with a recombinant vaccinia virus-lymphocytic choriomeningitis virus (LCMV) vaccine (VVNPaa1-201) expressing the immunodominant epitope completely protected H-2d mice from lethal infection with LCMV but did not protect H-2b mice. Furthermore, we show that the success or failure of immunization was determined entirely by the host class I major histocompatibility glycoproteins. The difference in outcome between mice of these two haplotypes was consistent with the presence or absence in the immunizing sequences of an epitope for CTL recognition and is correlated with the induction of LCMV-specific H-2-restricted CTL in H-2d mice. Protection is not conferred by a humoral immune response, since LCMV-specific antibodies were not detectable in sera from VVNPaa1-201-immunized mice. In addition, passive transfer of sera from vaccinated mice did not confer protection upon naive recipients challenged with LCMV. Hence, the molecular dissection of viral proteins can uncover immunodominant CTL epitope(s) that can be engineered into vaccines that elicit CTL. A single CTL epitope can protect against a lethal virus infection, but the efficacy of the vaccine varies in a major histocompatibility complex-dependent manner.     

Polymorphism of cytotoxic T-lymphocyte clones that recognize a defined nine-amino-acid immunodominant domain of lymphocytic choriomeningitis virus glycoprotein

To assess the heterogeneity of cytotoxic T lymphocytes (CTLs) directed against viral epitopes, we studied six class I major histocompatibility complex-restricted (H-2Db) CTL clones that recognize the same 9-amino-acid immunodominant epitope, amino acids 278 to 286 from envelope glycoprotein 2 (GP2) of lymphocytic choriomeningitis virus (LCMV). Using Southern blot analysis of beta-chain rearrangements, we found that each clone has a unique restriction pattern, providing evidence of the independent derivation of the clones and suggesting that the clones express different beta-chain sequences for their T-cell receptor. All these clones killed syngeneic target cells infected with strain Armstrong or WE of LCMV; however, two of the six clones failed to recognize target cells infected with the Pasteur strain of LCMV. Sequence analysis of LCMV Armstrong, WE, and Pasteur GP in the region of amino acids 272 to 293 demonstrated a single-amino-acid substitution at amino acid 278 in the region of the defined epitope in the Pasteur strain. Interestingly, one of the two CTL clones that failed to lyse LCMV Pasteur-infected target cells nevertheless efficiently and specifically killed uninfected target cells coated with the appropriate LCMV Pasteur peptide, while the other clone failed to do so. This indicated a dichotomy between processing of the synthesized protein initiated by infection and a peptide exogenously applied. Dose-response studies utilizing several peptides with substitutions in GP amino acid 278 indicate that CTL recognition occurs at the level of a single amino acid and suggest that this difference is likely recognized at the level of the T-cell receptor.     

Mapping and characterization of the primary and anamnestic H-2(d)-restricted cytotoxic T-lymphocyte response in mice against human metapneumovirus

Cytotoxic T lymphocytes (CTLs) are important for the control of virus replication during respiratory infections. For human metapneumovirus (hMPV), an H-2(d)-restricted CTL epitope in the M2-2 protein has been described. In this study, we screened the hMPV F, G, N, M, M2-1, and M2-2 proteins using three independent algorithms to predict H-2(d) CTL epitopes in BALB/c mice. A dominant epitope (GYIDDNQSI) in positions 81 to 89 of the antitermination factor M2-1 and a subdominant epitope (SPKAGLLSL) in N(307-315) were detected during the anti-hMPV CTL response. Passive transfer of CD8(+) T-cell lines against M2-1(81-89) and N(307-315) protected Rag1(-/-) mice against hMPV challenge. Interestingly, diversification of CTL targets to include multiple epitopes was observed after repetitive infections. A subdominant response against the previously described M2-2 epitope was detected after the third infection. An understanding of the CTL response against hMPV is important for developing preventive and therapeutic strategies against the virus.     

Immunobiology of cytotoxic T-cell escape mutants of lymphocytic choriomeningitis virus.

Infection with virus variants exhibiting changes in the peptide sequences defining immunodominant determinants that abolish recognition by antiviral cytotoxic T cells (CTL) presents a considerable challenge to the antiviral T-cell immune system and may enable some viruses to persist in hosts. The potential importance of such variants with respect to mechanisms of viral persistence and disease pathogenesis was assessed by infecting adult mice with variants of lymphocytic choriomeningitis virus (LCMV) strain WE. These variants were selected in vivo or in vitro for resistance to lysis by CD8+ H-2b-restricted antiviral CTL. The majority of anti-LCMV CTL in infected H-2b mice recognize epitopes defined by residues 32 to 42 and 275 to 289 (epitopes 32-42 and 275-289) of the LCMV glycoprotein or 397 to 407 of the viral nucleoprotein. The 8.7 variant exhibits a change in the epitope 32-42 (Val-35-->Leu), and variant CL1.2 exhibits a change in the epitope 275-289 (Asn-280-->Asp) of the wild-type LCMV-WE. The double-mutated 8.7-B23 variant had the variation of 8.7 and an additional change located in the epitope 275-289 (Asn-280-->Ser). The 8.7 variant peptide with unchanged anchor positions bound efficiently to H-2Db and H-2Kb molecules but induced only a very weak CTL response. CTL epitope 275-289 of CL1.2 and 8.7-B23 altered at predicted anchor residues were unable to bind Db molecules and were also not recognized by antiviral CTL. Infection of C57BL/6 mice (H-2b) with the variants exhibiting mutations of one of the CTL epitopes, i.e., 8.7 or CL1.2, induced CTL responses specific for the unmutated epitopes comparable with those induced by infection with WE, and these responses were sufficient to eliminate virus from the host. In contrast, infection with the double-mutated variant 8.7-B23 induced CTL activity that was reduced by a factor of about 50-fold compared with wild-type LCMV. Consequently, high doses (10(7) PFU intravenously) of this virus were eliminated slowly and only by about day 100 after infection. 8.7-B23 failed to cause lethal lymphocytic choriomeningitis after intracerebral infection with a dose of > 10(4) PFU in C57BL/6 mice (but not in mice of nonselecting H-2d haplotype); with the other variants or wild-type LCMV, doses greater than 10(6) to 10(7) PFU were necessary to avoid lethal choriomeningitis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Capsid-specific cytotoxic T lymphocytes recognize three distinct H-2D(b)-restricted regions of the BeAn strain of Theiler's virus and exhibit different cytokine profiles

The role of virus-specific cytotoxic T lymphocytes (CTL) in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a viral model for multiple sclerosis, is not yet clear. To investigate the specificity and function of CTL generated in response to TMEV infection, we generated a panel of overlapping 20-mer peptides encompassing the entire capsid and leader protein region of the BeAn strain of TMEV. Binding of these peptides to H-2K(b) and H-2D(b) class I molecules of resistant mice was assessed using RMA-S cells. Several peptides displayed significant binding to H-2K(b), H-2D(b), or both. However, infiltrating cytotoxic T cells in the central nervous system of virus-infected mice preferentially lysed target cells pulsed with VP2(111-130/121-140) or VP2(121-130), a previously defined CTL epitope shared by the DA strain of TMEV and other closely related cardioviruses. In addition, at a high effector-to-target cell ratio, two additional peptides (VP2(161-180) and VP3(101-120)) sensitized target cells for cytolysis by infiltrating T cells or splenic T cells from virus-infected mice. The minimal epitopes within these peptides were defined as VP2(165-173) and VP3(110-120). Based on cytokine profiles, CTL specific for these subdominant epitopes are Tc2, in contrast to CTL for the immunodominant epitope, which are of the Tc1 type. Interestingly, CTL function towards both of these subdominant epitopes is restricted by the H-2D molecule, despite the fact that these epitopes bind both H-2K and H-2D molecules. This skewing toward an H-2D(b)-restricted response may confer resistance to TMEV-induced demyelinating disease, which is known to be associated with the H-2D genetic locus.     

An altered T cell repertoire in MECL-1-deficient mice

Immunoproteasome subunits low-molecular mass polypeptide (LMP)2 and LMP7 affect Ag presentation by MHC class I molecules. In the present study, we investigated the function of the third immunosubunit LMP10/multicatalytic endopeptidase complex-like (MECL)-1 (beta2i) in MECL-1 gene-targeted mice. The number of CD8+ splenocytes in MECL-1-/- mice was 20% lower than in wild-type mice. Infection with lymphocytic choriomeningitis virus (LCMV) elicited a markedly reduced cytotoxic T cell (CTL) response to the LCMV epitopes GP276-286/Db and NP205-212/Kb in MECL-1-/- mice. The weak CTL response to GP276-286/Db was not due to an impaired generation of this epitope but was attributed to a decreased precursor frequency of GP276-286/Db-specific T cells. The expansion of TCR-Vbeta10+ T cells, which contain GP276-286/Db-specific cells, was reduced in LCMV-infected MECL-1-/- mice. Taken together, our data reveal an in vivo function of MECL-1 in codetermining the T cell repertoire for an antiviral CTL response.     

Virus specificity of cytotoxic T lymphocytes generated during acute lymphocytic choriomeningitis virus infection: role of the H-2 region in determining cross-reactivity for different lymphocytic choriomeningitis virus strains

We have compared the relatedness of five different strains of lymphocytic choriomeningitis virus (LCMV) as assessed by LCMV-specific cytotoxic T lymphocytes (CTL). Several different mouse strains were injected with each of the five LCMV strains, and the cross-reactivity of virus-specific CTL generated during the acute infection was tested by killing on a panel of target cells infected with the various LCMV strains. We found that the cross-reactivity pattern of LCMV-specific CTL generated in mice of H-2d haplotype (BALB/c WEHI and DBA/2) was strikingly different from that in mice of H-2b haplotype (C57BL/6 and C3H.Sw/Sn), suggesting that the fine specificity of LCMV-specific CTL is a function of the H-2 region. The characteristic cross-reactivity patterns were also observed in (C57BL/6 X DBA/2)F1 mice, demonstrating that the repertoire of the H-2b- and H-2d-restricted LCMV-specific CTL is not changed as a result of complementation by gene products of the other major histocompatibility haplotype. Studies with congenic BALB.B10 and (BALB.B10 X BALB/c)F1 mice firmly established that the characteristic cross-reactivity patterns of LCMV-specific CTL map to the H-2 region and are not influenced by background genes outside the major histocompatibility locus. These results suggest that LCMV determinants seen in the context of H-2d-restricting elements are different from those seen in the context of H-2b-restricting elements. Moreover, our studies show that CTL can be used as probes for dissecting differences among various LCMV strains, but the degree of relatedness between the different LCMV strains is not absolute when measured by CTL recognition. Since the H-2 region regulates the fine specificity of CTL generated during LCMV infection in its natural host, the degree of cross-protective immunity developed during a viral infection apparently depends on the major histocompatibility haplotype. The importance of these findings lies in understanding susceptibility or resistance of various host populations to viral infections and in designing vaccination programs to provide immunity.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. I. Generation and recognition of virus strains and H-2b mutants

Cytotoxic T lymphocytes (CTL) were induced in C57BL/6 and (C57BL/6 X DBA/2)F1 mice after immunization with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV-Arm) and were cloned by limiting dilution in vitro. The cytotoxic activity of these clones was LCMV specific and H-2 restricted. All clones induced in C57BL/6 (H-2b) mice with LCMV-Arm lysed target cells infected with each of five distinct strains of LCMV (Arm, Traub , WE, Pasteur, and UBC ), suggesting recognition of common regions of viral proteins in association with H-2b molecules. In contrast, one clone obtained from (B6 X D2)F1 mice and restricted to the H-2d haplotype only lysed cells infected with one of three strains of virus (Arm, Traub , WE) but not two others (Pasteur, UBC ), suggesting recognition of variable regions of viral proteins in the context of H-2d molecules. To assess the fine specificity for H-2 molecules, we tested H-2Kb-restricted CTL clones for their ability to kill LCMV-infected target cells bearing mutations in their H-2Kb, and we tested clones presumed to be restricted to the H-2Db region for their ability to all LCMV targets cells bearing a mutation in the H-2Db region. Several different patterns of killing of the mutant targets were observed, indicating that a number of different epitopes on the H-2b molecules were used as restricting determinants for LCMV antigen recognition by CTL. Thus, cross-reactive viral determinants were recognized in the context of several different restricting determinants. Mutations in the N or C1 domains of the H-2 molecule affected recognition by a single LCMV specific CTL clone. One implication of this result is that CTL recognize a conformational determinant on the H-2 molecule formed by the association of virus antigen(s) with H-2. An alternate explanation is that one site on the H-2 molecule is involved in the interaction of viral antigens with H-2, whereas another may serve as a binding site for the CTL receptor.     

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.     

Molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic T-lymphocyte response and establishment of persistence.

Isolates of lymphocytic choriomeningitis virus (LCMV) that elicit a cytotoxic T-lymphocyte response (CTL+) have been compared with isolates that suppress the CTL response (CTL-) in an effort to map this phenotype. A single amino acid change in the glycoprotein of the LCMV Armstrong (ARM) strain is consistently associated with the CTL- trait and the ability of the virus to persist (P+). The CTL+ P- parental strain spontaneously gives rise to CTL- P+ variants within lymphoid tissues of mice persistently infected from birth. To map the structural basis of the phenotype, the complete RNA sequence of LCMV ARM 53b (CTL+) was compared with that of its variant ARM clone 13 (CTL-). Differences in 5 of 10,600 nucleotides were found. Three changes are noted in the large L RNA segment, and two are noted in the small S RNA segment. Only two of the changes distinguishing CTL+ from CTL- isolates affect amino acid coding: lysine to glutamine at amino acid 1079 of the polymerase protein, and phenylalanine to leucine at amino acid 260 of the envelope glycoprotein (GP). We also analyzed two additional CTL- variants and four spontaneous CTL+ revertants. All three CTL- variants differ from the original CTL+ parental strain at GP amino acid 260, indicating that this amino acid change is consistently associated with the CTL- phenotype. By contrast the other four mutations in LCMV are not associated with the CTL- phenotype. Sequence analysis of the coding regions of four CTL+ revertants of ARM clone 13 did not reveal back mutations at the GP 260 locus. This finding indicates that the GP 260 mutation is necessary but not sufficient for a CTL- P+ phenotype and that the reversion to CTL+ P- is likely either due to secondary mutations in other regions of the viral genome or to quasispecies within the revertant population that make significant contributions to the phenotype.     

The CD8+ T-cell response to lymphocytic choriomeningitis virus involves the L antigen: uncovering new tricks for an old virus

CD8(+) T-cell responses control lymphocytic choriomeningitis virus (LCMV) infection in H-2(b) mice. Although antigen-specific responses against LCMV infection are well studied, we found that a significant fraction of the CD8(+) CD44(hi) T-cell response to LCMV in H-2(b) mice was not accounted for by known epitopes. We screened peptides predicted to bind major histocompatibility complex class I and overlapping 15-mer peptides spanning the complete LCMV proteome for gamma interferon (IFN-gamma) induction from CD8(+) T cells derived from LCMV-infected H-2(b) mice. We identified 19 novel epitopes. Together with the 9 previously known, these epitopes account for the total CD8(+) CD44(hi) response. Thus, bystander T-cell activation does not contribute appreciably to the CD8(+) CD44(hi) pool. Strikingly, 15 of the 19 new epitopes were derived from the viral L polymerase, which, until now, was not recognized as a target of the cellular response induced by LCMV infection. The L epitopes induced significant levels of in vivo cytotoxicity and conferred protection against LCMV challenge. Interestingly, protection from viral challenge was best correlated with the cytolytic potential of CD8(+) T cells, whereas IFN-gamma production and peptide avidity appear to play a lesser role. Taken together, these findings illustrate that the LCMV-specific CD8(+) T-cell response is more complex than previously appreciated.     

Restricted V-segment usage in T-cell receptors from cytotoxic T lymphocytes specific for a major epitope of lymphocytic choriomeningitis virus.

Cytotoxic T lymphocytes (CTL) play an important role in recovery from a number of viral infections. They are also implicated in virus-induced immunopathology as best demonstrated in lymphocytic choriomeningitis virus (LCMV) infection of adult immunocompetent mice. In the present study, the structure of the T-cell receptor (TCR) in LCMV-specific CTL in C57BL/6 (B6) mice was investigated. Spleen T cells obtained from LCMV-infected mice were cultured in vitro with virus-infected stimulator cells and then stained with anti-TCR V beta antibodies. A skewing of V beta usage was noticeable in T cells enriched for their reactivity to LCMV, suggesting that particular V segments are important for the recognition of LCMV T-cell epitopes in B6 mice. To gain more detailed information on the structure of the TCR specific for LCMV epitopes, we studied CTL clones. It has been shown that approximately 90% of LCMV-reactive CTL clones generated in H-2b mice are specific for a short peptide fragment of the LCMV glycoprotein, residues 278 to 286, recognized in the context of the class I major histocompatibility complex molecule, Db. Four CTL clones possessing the specificity were randomly selected from a collection of clones, and their TCR genes were isolated by cDNA cloning or by the anchored polymerase chain reaction. All four clones were found to use V alpha gene segments belonging to the V alpha 4 subfamily. By RNA blot analysis, two more clones with the same specificity were also shown to express the V alpha 4 mRNA. In contrast, three different V beta gene segments were used among the four clones examined. J beta 2.1 was used by three of the clones. Although amino acid sequences in the V(D)J junctional regions were dissimilar, aspartic acid was found in the V alpha J alpha and/or V beta D beta J beta junctions of all four of these clones, suggesting that this residue is involved in binding the LCMV fragment. Restricted usage of V alpha and possibly J beta segments in the CTL response to a major T-cell epitope of LCMV raises the possibility that immunopathology in LCMV infection can be treated with antibodies directed against such TCR segments. Thus, similar analysis of the TCR in other virus infections is warranted and may lead to therapeutic strategies for immunopathology due to virus infections.     

Immunoregulation by mouse T-cell clones. I. Suppression and amplification of cytotoxic responses by cloned H-Y-specific cytolytic T lymphocytes

H-Y-specific and H-2Db-restricted, Lyt-1-2+ T-cell clones ( CTLL ) with graded specific cytotoxic activities on male C57BL/6 (B6) target cells ( 1E3 , ; 2C5 , ++; 2A5 , +, 3E6 , +/-) were tested for their capacity to inhibit the generation of H-Y-specific cytotoxic T lymphocytes (CTL) in vitro. Addition of irradiated lymphocytes of CTLL 1E3 and CTLL 3E6 but not those of CTLL 2A5 or CTLL 2C5 abolished the generation of CTL from in vivo primed H-Y-specific precursor cells (CTLP) when added to fresh mixed-lymphocyte cultures (MLC). Exogenous sources of T-cell growth factors (TCGF) did not overcome suppression. Rather the presence of TCGF resulted in a further enhancement of suppressive activities in CTLL 1E3 and 3E6 and the induction of similar activities in cells from CTLL 2A5 and 2C5 , which by themselves were not inhibitory. Moreover when added to similar MLC on Day 1 instead of Day 0, only irradiated cells of CTLL 3E6 but not those of the other three CTLL were suppressive. Induction of suppressive activities in H-Y-specific CTLL was independent of the appropriate male stimulator cells since it was also observed in MLC induced by irrelevant antigens (H-2, trinitrophenol). Furthermore at low cell numbers, irradiated lymphocytes from any of the CTLL consistently enhanced CTL activities generated from H-Y-specific CTLP. This augmenting activity, which was not TCGF, could be transferred by soluble mediators present in antigen-sensitized CTLL cultures. Thus, these data indicate (i) that cytotoxic effector cells can function as suppressor cells in the generation of CTL, (ii) that the cytotoxic activity of cloned CTL does not correlate with their capacity to suppress CTL responses, (iii) that the inhibition of CTL responses by CTLL is not due to simple consumption of T-cell growth factors produced in MLC, and (iv) that different CTL clones may interfere with the generation of CTL at different stages of their maturation. Moreover, the experiments suggest an antigen-independent enhancement of suppression by the interaction of CTL with lymphokines. Together with the augmenting activity evoked by cloned CTL the data provide strong evidence for the expression of multiple immunological functions by one particular subset of T cells and suggest that cytotoxic effector cells can differentially regulate the maturation and/or clonal expression of their precursor cells.     

Molecular Anatomy and Number of Antigen Specific CD8 T Cells Required to Cause Type 1 Diabetes

We quantified CD8 T cells needed to cause type 1 diabetes and studied the anatomy of the CD8 T cell/beta (β) cell interaction at the immunologic synapse. We used a transgenic model, in situ tetramer staining to distinguish antigen specific CD8 T cells from total T cells infiltrating islets and a variety of viral mutants selected for functional deletion(s) of various CD8 T cell epitopes. Twenty percent of CD8 T cells in the spleen were specific for all immunodominant and subdominant viral glycoprotein (GP) epitopes. CTLs to the immunodominant LCMV GP33-41 epitope accounted for 63% of the total (12.5% of tetramers). In situ hybridization analysis demonstrated only 1 to 2% of total infiltrating CD8 T cells were specific for GP33 CD8 T cell epitope, yet diabetes occurred in 94% of mice. The immunologic synapse between GP33 CD8 CTL and β cell contained LFA-1 and perforin. Silencing both immunodominant epitopes (GP33, GP276–286) in the infecting virus led to a four-fold reduction in viral specific CD8 CTL responses, negligible lymphocyte infiltration into islets and absence of diabetes.     

H-2Kb mutations limit the CTL response to SV40 TASA

The cytotoxic T lymphocyte (CTL) responses directed towards SV40 tumor-associated specific antigen (TASA) in nine strains of spontaneously arising Kb mutant mice were analyzed. All nine mutants generated normal levels of H-2Db-restricted response, but the K-end-restricted CTL response varied. B6.C-H-2bm1 (bm1) did not produce K-end-restricted SV40 TASA-specific CTL upon immunization, and SV40-transformed bm1 cells were not lysed by intra-H-2 recombinant Kb [B10.A(5R)] CTL. Nonreciprocal cross-reactive lysis was seen between B6-H-2bm8 (bm8) and B10.A(5R). Strain B6-H-2bm8 mice produce highly specific Kbm8-restricted CTL that lyse SV40-transformed bm8 cells (Kbm8SV) but not B10.A(5R) target cells (K5RSV), although Kbm8SV targets can be partially lysed by B10.A(5R) CTL. The other seven Kb mutants cross-react with B10.A(5R). These experiments definitively show that genes mapping to the K and/or D region directly control the H-2-restricted CTL response to SV40 TASA.