Molecular and functional dissection of the H-2Db-restricted subdominant cytotoxic T-cell response to lymphocytic choriomeningitis virus

Infection of H-2b mice with lymphocytic choriomeningitis virus (LCMV) generates an H-2Db-restricted cytotoxic T-lymphocyte (CTL) response whose subdominant component is directed against the GP92-101 (CSANNSHHYI) epitope. The aim of this study was to identify the functional parameters accounting for this subdominance. We found that the two naturally occurring (genetically encoded and posttranslationally modified) forms of LCMV GP92-101 were immunogenic, did not act as T-cell antagonists, and bound efficiently to but were unable to form stable complexes with H-2Db, a crucial factor for immunodominance. Thus, the H-2Db-restricted subdominant CTL response to LCMV resulted not from altered T-cell activation but from impaired major histocompatibility complex presentation properties.     

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.     

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.     

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.     

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)     

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.     

Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance

The primary CD8(+) T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8(+) T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking H-2K(b), resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F(1) hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8(+) T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33-41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the immunodominance hierarchy of CD8(+) T cell responses.     

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.     

Comparative analysis of core amino acid residues of H-2D(b)-restricted cytotoxic T-lymphocyte recognition epitopes in simian virus 40 T antigen.

Simian virus 40 (SV40) tumor (T) antigen expressed in H-2b SV40-transformed cells induces the generation of Lyt-2+ (CD8+) cytotoxic T lymphocytes (CTL), which are involved in tumor rejection, in syngeneic mice. Five CTL recognition sites on T antigen have been described by using mutant T antigens. Four of the sites (I, II, III, and V) are H-2Db restricted and have been broadly mapped with synthetic peptides of 15 amino acids in length overlapping by 5 residues at the amino and carboxy termini. The goal of this study was to define the minimal and optimal amino acid sequences of T antigen which would serve as recognition elements for the H-2Db-restricted CTL clones Y-1, Y-2, Y-3, and Y-5, which recognizes sites I, II, III, and V, respectively. The minimal and optimal residues of T antigen recognized by the four CTL clones were determined by using synthetic peptides truncated at the amino or carboxy terminus and an H-2Db peptide-binding motif. The minimal site recognized by CTL clone Y-1 was defined as amino acids 207 to 215 of SV40 T antigen. However, the optimal sequence recognized by CTL clone Y-1 spanned T-antigen amino acids 205 to 215. The T-antigen peptide sequence LT223-231 was the optimal and minimal sequence recognized by both CTL clones Y-2 and Y-3. Site V was determined to be contained within amino acids 489 to 497 of T antigen. The lytic activities of CTL clones Y-2 and Y-3, which recognize a single nonamer peptide, LT223-231, were affected differently by anti-Lyt-2 antibody, suggesting that the T-cell receptors of these two CTL clones differ in their avidities. As the minimal and optimal H-2Db-restricted CTL recognition sites have been defined by nonamer synthetic peptides, it is now possible to search for naturally processed H-2Db-restricted epitopes of T antigen and identify critical residues involved in processing, presentation, and recognition by SV40-specific CTL.     

Protection against CTL escape and clinical disease in a murine model of virus persistence

CTL escape mutations have been identified in several chronic infections, including mice infected with mouse hepatitis virus strain JHM. One outstanding question in understanding CTL escape is whether a CD8 T cell response to two or more immunodominant CTL epitopes would prevent CTL escape. Although CTL escape at multiple epitopes seems intuitively unlikely, CTL escape at multiple CD8 T cell epitopes has been documented in some chronically infected individual animals. To resolve this apparent contradiction, we engineered a recombinant variant of JHM that expressed the well-characterized gp33 epitope of lymphocytic choriomeningitis virus, an epitope with high functional avidity. The results show that the presence of a host response to this second epitope protected mice against CTL escape at the immunodominant JHM-specific CD8 T cell epitope, the persistence of infectious virus, and the development of clinical disease.     

Cytotoxic T lymphocyte responses to minor H-43 alloantigens in H-43a and H-43b mice. Both anti-H-43b and anti-H-43a CTL activities are generated exclusively in the context of the same H-2Kb restriction element

We have previously demonstrated that anti-H-43a CTL response of H-43b responder mice was exclusively restricted by self H-2Kb (Kb) but not by the other nine self MHC class I alleles from independent origins, i.e., Kbml,d,k,s and Db,d,k,q,s. In the present study, we verified that Kf,q,r and Df,r alleles could also not serve as restricting class I elements in the CTL response to H-43a alloantigen. Another notable observation made in the earlier study was the fact that, in H-43 incompatibility of the alternative combination, H-43a mice were incapable of generating CTL activity against H-43b alloantigen. However, by means of employing new in vivo immunization procedures, we discovered that some but not all genetically identical H-43a responder mice could mount anti-H-43b CTL response restricted by self Kb. Again, no anti-H-43b CTL activity could be generated in the context of self Kk, Kj, Db or Dk molecules. Although the number of class I alleles we examined is still limited, these results indicate that antigenic fragments derived from the processed H-43a and H-43b alloantigens possess an indistinguishable epitope (agretope), and that such agretope either interacts only with the privileged Kb molecules or allows to bestow the immunogenic conformation of allodeterminants on the fragments solely in the context of the restricting Kb element.     

Cytotoxic T-cell antagonism in HIV-1

The cytotoxic T-cell (CTL) response to human immunodeficiency virus Type 1 (HIV-1) is vigorous and sustained, but despite this, the virus persists. Natural variation arising within CTL epitopes may affect CTL recognition of infected targets and allow viral escape. Some of these variant epitopes appear to engage T-cell receptors but fail to activate the CTL normally. This can interfere with recognition of the unmutated epitope — a phenomenon known as T-cell antagonism. We discuss the evidence for this in HIV-1 using CTL and epitope variants derived from infected donors, and discuss its possible relevancein vivo.     

Recognition of H-2Kb mutant target cells by Moloney virus-specific cytotoxic T lymphocytes from bm13 (H-2Db mutant) mice. I. Full recognition of Kbm11 by Kb-restricted CTL

In C57BL/6 (B6, H-2b) mice, the secondary in vitro CTL response against Moloney leukemia virus is restricted and regulated by the H-2Db locus. B6.C-H- 2bm13 ( bm13 ) mice, however, carrying a mutation at the Db locus, show an increased H-2Kb-restricted CTL response without a demonstrable CTL component restricted by the mutant Dbm13 molecule (D----K shift). These purely Kb-restricted bm13 virus-specific CTL were incubated with a series of Kb mutant virus-infected target cells to study the effect of the mutations at the target cell level. Of six Kb-mutant virus-infected target cells tested, bm1 cells were not recognized and bm8 cells were recognized only marginally by bm13 virus-specific CTL, whereas bm3 , bm5 , bm6 , and bm11 cells were fully recognized. Thus, the bm3 , bm5 , bm6 , and bm11 Kb mutants fully share the relevant H-2K restriction specificities with H-2Kb, whereas the bm1 mutant totally and the bm8 mutant almost completely lack these specificities. This result differs markedly from the restriction site relationships among B6 and these Kb mutants in other antigenic systems. The most striking example concerns the bm11 mutant, which is fully recognized by Moloney-specific CTL, but not at all by Sendai, minor H (H-3.1, H-4.2), and sulfhydryl hapten-specific CTL. Monoclonal anti-H-2Kb antibody B8-3-24 inhibited virus-specific lysis by bm13 CTL of all Kb virus-infected mutant target cells to which this antibody binds. Lysis of bm5 and bm11 but not of bm3 target cells was inhibited, in line with the fact that B8-3-24 antibody does not bind bm3 . On the other hand, not only bm5 and bm11 but also bm3 virus-infected target cells blocked virus-specific lysis to the same extent as syngeneic bm13 target cells. Therefore, bm13 virus-specific CTL populations do not recognize the discrete cluster alteration in the Kbm3 molecule, as identified by antibody B8-3-24. The bm1 and the bm8 mutations, which have structural alterations in completely different sites of the Kb molecule, show complete or almost complete loss, respectively, of Kb-Moloney restriction sites. This finding supports the notion that these virus-specific CTL recognize conformational determinants rather than linear amino acid sequences.     

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.     

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.     

Simian virus 40 T antigen as a carrier for the expression of cytotoxic T-lymphocyte recognition epitopes.

Simian virus 40 (SV40) large T antigen can immortalize a wide variety of mammalian cells in culture. We have taken advantage of this property of T antigen to use it as a carrier for the expression of cytotoxic T-lymphocyte (CTL) recognition epitopes. DNA sequences corresponding to an H-2Db-restricted SV40 T-antigen site I (amino acids 205 to 215) were translocated into SV40 T-antigen DNA at codon positions 350 and 650 containing EcoRI linkers. An H-2Kb-restricted herpes simplex virus glycoprotein B epitope (amino acids 498 to 505) was also expressed in SV40 T antigen at positions 350 and 650. Primary C57BL/6 mouse kidney cells were immortalized by transfection with the recombinant and wild-type T-antigen DNA. Clonal isolates of cells expressing chimeric T antigens were shown to be specifically susceptible to lysis by CTL clones directed to SV40 T-antigen site I and herpes simplex virus glycoprotein B epitopes, indicating that CTL epitopes restricted by two different elements can be processed, presented, and recognized by the epitope-specific CTL clones. Our results suggest that SV40 T antigen can be used as a carrier protein to express a wide variety of CTL epitopes.     

Immunoproteasomes down-regulate presentation of a subdominant T cell epitope from lymphocytic choriomeningitis virus

The cytotoxic T cell response to pathogens is usually directed against a few immunodominant epitopes, while other potential epitopes are either subdominant or not used at all. In C57BL/6 mice, the acute cytotoxic T cell response against lymphocytic choriomeningitis virus is directed against immunodominant epitopes derived from the glycoprotein (gp33-41) and the nucleoprotein (NP396-404), while the gp276-286 epitope remains subdominant. Despite extensive investigations, the reason for this hierarchy between epitopes is not clear. In this study, we show that the treatment of cells with IFN-gamma enhanced the presentation of gp33-41, whereas presentation of the gp276-286 epitope from the same glycoprotein was markedly reduced. Because proteasomes are crucially involved in epitope generation and because IFN-gamma treatment in vitro and lymphocytic choriomeningitis virus infection in vivo lead to a gradual replacement of constitutive proteasomes by immunoproteasomes, we investigated the role of proteasome composition on epitope hierarchy. Overexpression of the active site subunits of immunoproteasomes LMP2, LMP7, and MECL-1 as well as overexpression of LMP2 alone suppressed the presentation of the gp276-286 epitope. The ability to generate gp276-286-specific CTLs was enhanced in LMP2- and LMP7-deficient mice, and macrophages from these mice showed an elevated presentation of this epitope. In vitro digests demonstrated that fragmentation by immunoproteasomes, but not constitutive proteasomes led to a preferential destruction of the gp276 epitope. Taken together, we show that LMP2 and LMP7 can at least in part determine subdominance and shape the epitope hierarchy of CTL responses in vivo.     

Molecular definition of a major cytotoxic T-lymphocyte epitope in the glycoprotein of lymphocytic choriomeningitis virus.

Analyses with segmental reassortants of lymphocytic choriomeningitis virus (LCMV) RNA have shown that cytotoxic T lymphocytes (CTL) are induced by and recognize proteins encoded by the viral short segment, which specifies two virus structural proteins, glycoprotein (GP) and nucleoprotein (NP). Expression of cDNA copies of these genes in vaccinia virus vectors demonstrates that C57BL/6 (H2bb) mice mount significant CTL responses to both GP and NP. We have used LCMV-specific H2bb-restricted CTL clones and a family of serial C-terminal truncations of the LCMV GP expressed in vaccinia virus to map the precise specificities of the anti-GP clones. Of the 18 CTL clones studied, 1 recognizes NP and the other 17 recognize GP. The reactivities of 14 of the 17 anti-GP CTL clones against the deleted GP molecules have been fully characterized, and two clear patterns of anti-GP activity have emerged, defining at least two CTL epitopes. The first epitope, recognized by only two of the clones, lies within GP residues 1 to 218. The second is recognized by all 12 of the remaining clones and was mapped, by using the GP deletions, to a 22-amino-acid region comprising GP residues 272 to 293. A synthetic peptide representing this area sensitized uninfected syngeneic target cells to lysis both by bulk CTL obtained from the spleen after a primary immunization and by appropriate CTL clones. Two sets of criteria are available which are said to identify potential T-cell epitopes, one based on primary amino acid sequence and the second based on protein secondary structure. Neither of these predictive schemes would have identified region 272 to 293 as a CTL recognition motif, indicating that such programs are of limited usefulness as presently conceived. Analysis of the CTL clones shows clearly that all three families (anti-NP and anti-GP 1 to 218 and 272 to 293) direct efficient cross-reactive killing against a variety of serologically distinct strains of LCMV.     

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.     

De novo generation of escape variant-specific CD8+ T-cell responses following cytotoxic T-lymphocyte escape in chronic human immunodeficiency virus type 1 infection

Human immunodeficiency virus type 1 (HIV-1) evades CD8(+) T-cell responses through mutations within targeted epitopes, but little is known regarding its ability to generate de novo CD8(+) T-cell responses to such mutants. Here we examined gamma interferon-positive, HIV-1-specific CD8(+) T-cell responses and autologous viral sequences in an HIV-1-infected individual for more than 6 years following acute infection. Fourteen optimal HIV-1 T-cell epitopes were targeted by CD8(+) T cells, four of which underwent mutation associated with dramatic loss of the original CD8(+) response. However, following the G(357)S escape in the HLA-A11-restricted Gag(349-359) epitope and the decline of wild-type-specific CD8(+) T-cell responses, a novel CD8(+) T-cell response equal in magnitude to the original response was generated against the variant epitope. CD8(+) T cells targeting the variant epitope did not exhibit cross-reactivity against the wild-type epitope but rather utilized a distinct T-cell receptor Vbeta repertoire. Additional studies of chronically HIV-1-infected individuals expressing HLA-A11 demonstrated that the majority of the subjects targeted the G(357)S escape variant of the Gag(349-359) epitope, while the wild-type consensus sequence was significantly less frequently recognized. These data demonstrate that de novo responses against escape variants of CD8(+) T-cell epitopes can be generated in chronic HIV-1 infection and provide the rationale for developing vaccines to induce CD8(+) T-cell responses directed against both the wild-type and variant forms of CD8 epitopes to prevent the emergence of cytotoxic T-lymphocyte escape variants.