Murine IgE and IgG responses to melittin and its analogs.

Melittin, a bee-venom peptide of 26 amino acids, induces IgE and IgG responses in man and animals. The antibody response was shown previously to be specific primarily for the C-terminal 6 residues and its T cell epitope in H-2d restricted mice was shown to be in residue 11-19 of melittin. To study the relationship of peptide structure and immunogenicity in mice, we have prepared a series of melittin analogs varied in length and composition at the C-terminus. Immunogenicity of the analogs for IgG and IgE responses was found to correlate with two factors: a peptide length of more than 24 residues and the presence of a hydrophilic C-terminal region preferably with two to four cationic groups. These factors result in the ability of peptide to bind to cell membranes. Analogs that possess these features are good immunogens whereas those lacking any of these features are weak immunogens.     

Functionally distinct agretopic and epitopic sites. Analysis of the dominant T cell determinant of moth and pigeon cytochromes c with the use of synthetic peptide antigens

The dominant T cell determinant on moth and pigeon cytochromes c in B10.A (E beta k:E alpha k) mice is located in the C-terminal portion of the protein, contained within residues 93-103 or 93-104. Thirty-seven antigen analogs, containing single amino acid substitutions at positions 98, 99, 101, 102, 103, and 104, were synthesized. The effects of the substitutions on in vitro antigenicity and in vivo immunogenicity were determined. Functional assays with T cell clones identified residues 99, 101, 102, and 103 as critical, based on their effect on antigenic potency. Peptides containing substitutions at residues 99, 101, and 102 were capable of eliciting unique clones upon immunization of B10.A mice. This was consistent with the identification of these residues as part of the epitope, the site on the antigen that interacts with the T cell receptor. Immunization with peptides substituted at residue 103, however, failed to elicit clones with unique specificity for the immunogen. When these peptides were tested for their ability to stimulate the T cell clones with antigen-presenting cells from B10.A(5R) mice expressing the E beta b:E alpha k Ia molecule, a consistent change in the relative antigenic potency was observed with 50% of the peptides. The effect of the Ia molecule on the antigenic potency ruled out the possibility that residue 103 nonspecifically affected antigen uptake or processing and identified residue 103 as part of the agretope, the site that interacts with the Ia molecule. The locations of the agretope and the epitope on this antigenic determinant appear to be fixed, even in the presence of large numbers of amino acid substitutions. However, some substitutions were found to affect both the agretope and the epitope, placing limits on the functional independence of the two sites. The results are discussed in terms of the trimolecular complex model of T cell activation and the implications of these data for antigen-Ia molecule interactions.     

Polymorphic residues on the I-A beta chain modulate the stimulation of T cell clones specific for the N-terminal peptide of the autoantigen myelin basic protein

The effect of polymorphic residues on the A alpha A beta molecule on T cell recognition of the N-terminal nonapeptide of myelin basic protein (R1-9) was determined. Ak-restricted T cell clones recognizing R1-9 were isolated. The peptide-Ia specificities of these clones were determined by testing the response to 1) a panel of peptide analogs of R1-11, 2) splenic APC from mice expressing MHC molecules from serologically distinct haplotypes, and 3) L cell transfectants expressing mutant/recombinant A beta cDNA containing combinations of polymorphic nucleotide sequences from the k and u alleles. Comparisons were made between the Ak-restricted clones and a previously characterized panel of Au-restricted clones. Certain Ak-restricted clones were able to recognize MBP peptide analogs that were not recognized by any of the Au-restricted clones. The Au-restricted T cell clones did not cross-react with R1-9 presented in the context of Ak, whereas the majority of the Ak-restricted clones responded to R1-9 presented in the context of Au. This nonreciprocal cross-reactivity was also reflected in the relative responses of the two sets of T cell clones to the interchange of u- and k-derived residues in the A beta chain. Residues in regions corresponding both the alpha-helical or beta-sheet portions of the hypothetical Ia three-dimensional structure were involved. The results suggest that overall specificity of the T cell clones is the summation of numerous distinct subspecificities for different regions of the peptide-Ia ligand. These results indicate that there can be striking differences in T cell specificity for an autoantigenic epitope, even in the context of A alpha A beta molecules from very closely related haplotypes.     

Regulation of polyclonal T cell responses by an MHC anchor-substituted variant of myelin oligodendrocyte glycoprotein 35-55

Analogs of immunogenic peptides containing substitutions at TCR contact residues (altered peptide ligands (APLs)) have been used to manipulate Ag-specific T cell responses in models of autoimmunity, including experimental autoimmune encephalomyelitis. However, recent clinical trials with APL of a myelin basic protein epitope revealed limitations of this therapy. In this study, we demonstrate that individual myelin oligodendrocyte glycoprotein (MOG) 35-55-specific T cell clones responded differentially to a MOG 35-55 APL, raising questions about the ability of peptide analogs containing amino acid substitutions at TCR contact residues to control polyclonal populations of T cells. In contrast, we found that a variant peptide containing a substitution at an MHC anchor residue uniformly affected multiple MOG 35-55-specific clones and polyclonal lines. Stimulation of polyclonal MOG 35-55-specific T cells with an MHC variant peptide resulted in the induction of anergy, as defined by a dramatic reduction in proliferation and IL-2 production upon challenge with wild-type peptide. Furthermore, treatment of T cell lines with this peptide in vitro resulted in a significant reduction in their encephalitogenicity upon adoptive transfer. These results indicate that the use of MHC anchor-substituted peptides may be efficacious in the regulation of polyclonal T cell responses such as those found in EAE.     

Human CD4+ T cells induced by synthetic peptide malaria vaccine are comparable to cells elicited by attenuated Plasmodium falciparum sporozoites

Peptide vaccines containing minimal epitopes of protective Ags provide the advantages of low cost, safety, and stability while focusing host responses on relevant targets of protective immunity. However, the limited complexity of malaria peptide vaccines raises questions regarding their equivalence to immune responses elicited by the irradiated sporozoite vaccine, the "gold standard" for protective immunity. A panel of CD4+ T cell clones was derived from volunteers immunized with a peptide vaccine containing minimal T and B cell epitopes of the Plasmodium falciparum circumsporozoite protein to compare these with previously defined CD4+ T cell clones from volunteers immunized with irradiated P. falciparum sporozoites. As found following sporozoite immunization, the majority of clones from the peptide-immunized volunteers recognized the T* epitope, a predicted universal T cell epitope, in the context of multiple HLA DR and DQ molecules. Peptide-induced T cell clones were of the Th0 subset, secreting high levels of IFN-gamma as well as variable levels of Th2-type cytokines (IL-4, IL-6). The T* epitope overlaps a polymorphic region of the circumsporozoite protein and strain cross-reactivity of the peptide-induced clones correlated with recognition of core epitopes overlapping the conserved regions of the T* epitope. Importantly, as found following sporozoite immunization, long-lived CD4+ memory cells specific for the T* epitope were detectable 10 mo after peptide immunization. These studies demonstrate that malaria peptides containing minimal epitopes can elicit human CD4+ T cells with fine specificity and potential effector function comparable to those elicited by attenuated P. falciparum sporozoites.     

Identification and characterization of a T cell-inducing epitope of bovine ribonuclease that can be restricted by multiple class II molecules.

An immunodominant epitope of bovine RNase restricted by I-Ek molecules was identified using a T cell hybridoma recognizing RNase. This epitope was localized to the peptide RNase(90-105). Single conservative amino acid substitutions were made at each of the positions 94 through 105. It was found that only at one position, Asn-103, were conservative substitutions not allowed. This residue was shown to be the critical residue in determining T cell specificity. The ability of RNase(90-105) and the well-defined T cell epitope, HEL(46-61) to stimulate mouse strains expressing different independent H-2 haplotypes was examined using a T cell proliferation assay. The response to HEL(46-61) was completely restricted to mice expressing an I-Ak molecule. In striking contrast, 6 of 10 different mouse strains, H-2b,f,k,q,s,u, mounted vigorous T cell responses to RNase(90-105). The response was restricted to both I-A and I-E molecules, including I-Ab, I-Af, I-Ek, I-Aq, and I-As. H-2d mice were nonresponders to RNase(90-105), which was shown to be due to the failure of RNase(90-105) to bind to I-Ad molecules. A variant RNase(90-105) peptide was generated, containing an I-Ad binding motif, that could bind to I-Ad molecules. Despite its ability to bind, this variant peptide was not able to stimulate a response in H-2d mice. This result demonstrates that the ability of a peptide to bind to an Ia molecule is necessary but not always sufficient for a response to occur. Thus, in contrast to the highly restricted HEL(46-61) determinant, the RNase(90-105) determinant is permissive in its binding to Ia molecules. These results show that in the universe of T cell inducing epitopes contains both highly restricted and broadly restricted epitopes are found.     

Multispecific CD4+ T cell response to a single 12-mer epitope of the immunodominant heat-shock protein 60 of Yersinia enterocolitica in Yersinia-triggered reactive arthritis: overlap with the B27-restricted CD8 epitope, functional properties, and epitope presentation by multiple DR alleles

Yersinia heat-shock protein 60 (Ye-hsp60) has recently been found to be a dominant CD4 and CD8 T cell Ag in Yersinia-triggered reactive arthritis. The nature of this response with respect to the epitopes recognized and functional characteristics of the T cells is largely unknown. CD4+ T cell clones specific for Ye-hsp60 were raised from synovial fluid mononuclear cells from a patient with Yersinia-triggered reactive arthritis. and their specificity was determined using three recombinant Ye-hsp60 fragments, overlapping 18-mer synthetic peptides as well as truncated peptides. Functional characteristics were assessed by cytokine secretion analysis in culture supernatants after specific antigenic stimulation. Amino acid positions relevant for T cell activation were detected by single alanine substitutions within the epitopes. Fragment II comprising amino acid sequence 182-371 was recognized by the majority of clones. All these clones were specific for peptide 319-342. Th1 clones and IL-10-secreting clones occurred in parallel, sometimes with the same fine specificity. The 12-mer core epitope 322-333 is a degenerate MHC binder and is presented to some T cell clones in a "promiscuous" manner. This epitope is almost identical with a B27-restricted CTL epitope of Ye-hsp60. Cross-reactivity of Ye-hsp60-specific T cell clones with self-hsp60 was not observed. In conclusion, an interesting Ye-hsp60 T cell epitope has been identified and characterized. It remains to be determined whether this epitope is also relevant in other reactive arthritis patients.     

Determinant selection in murine experimental autoimmune myasthenia gravis. Effect of the bm12 mutation on T cell recognition of acetylcholine receptor epitopes.

C57BL/6 (B6) mice respond to immunization with acetylcholine receptor (AChR) from Torpedo californica as measured by T cell proliferation, antibody production, and the development of muscle weakness resembling human myasthenia gravis. The congenic strain B6.C-H-2bm12 (bm12), which differs from B6 by three amino acid substitutions in the beta-chain of the MHC class II molecule I-A, develops a T cell proliferative response but does not produce antibody or develop muscle weakness. By examining the fine specificity of the B6 and bm12 T cell responses to AChR by using T cell clones and synthetic AChR peptides, we found key differences between the two strains in T cell epitope recognition. B6 T cells responded predominantly to the peptide representing alpha-subunit residues 146-162; this response was cross-reactive at the clonal level to peptide 111-126. Based on the sequence homology between these peptides and the T cell response to a set of truncated peptides, the major B6 T cell epitope was determined to be residues 148-152. The cross-reactivity of peptides 146-162 and 111-126 could also be demonstrated in vivo. Immunization of B6 mice with either peptide primed for T cell responses to both peptides. In contrast, immunization of bm12 mice with peptide 111-126 primed for an anti-peptide response, which did not cross-react with 146-162. Peptide-reactive T cells were not elicited after immunization of bm12 mice with 146-162. These results define a major T cell fine specificity in experimental autoimmune myasthenia gravis-susceptible B6 mice to be directed at alpha-subunit residues 148-152. T cells from disease-resistant bm12 mice fail to recognize this epitope but do recognize other portions of AChR. We postulate that alpha-148-152 is a disease-related epitope in murine experimental autoimmune myasthenia gravis. In this informative strain combination, MHC class II-associated determinant selection, rather than Ag responsiveness per se, may play a major role in determining disease susceptibility.     

Identification of an I-Ad restricted peptide on the 65-kilodalton heat shock protein of Mycobacterium avium

The 65 kilodalton heat shock protein (Hsp65) from mycobacterial species elicits immune responses and in some cases protective immunity. Here we have used a DNA sublibrary approach to identify antigenic fragments of Mycobacterium avium Hsp65 and a synthetic peptide approach to delineate CD4+ T cell determinants. A panel of Hsp65 reactive CD4+ T cell clones was established from lymph node cells obtained from BALB/c mice immunized with recombinant Hsp65. The clones were tested for proliferative reactivity against the products of the DNA sublibrary of the hsp65 gene. A T cell epitope, restricted by the I-Ad molecule, was identified within the C-terminal region of Hsp65 and the minimal epitope (amino acid residues 489-503) delineated using overlapping peptides spanning the C-terminal fragment. Additionally, the CD4+ T cell clone recognizing this epitope also responded to native Hsp65 present in M. avium lysates by both proliferation and cytokine production, indicating that the epitope was present and processed similarly both in the native and the recombinant forms of Hsp65. This sequence identified in BALB/c mice (Hsp65 489-503) is identical in other mycobacteria, notably M. tuberculosis, M. bovis and M. leprae, suggesting the epitope may have wider application in murine models of other mycobacterial infections.     

The immunodominant CD8 T cell response to the human cytomegalovirus tegument phosphoprotein pp65(495-503) epitope critically depends on CD4 T cell help in vaccinated HLA-A*0201 transgenic mice

Immunodominance hierarchies operating in immune responses to viral Ags limit the diversity of the elicited CD8 T cell responses. We evaluated in I-A(b+)/A2-HHD-II and HLA-DR1(+)/A2-DR1 mice the HLA-A*0201-restricted, multispecific CD8 T cell responses to the human CMV tegument phosphoprotein pp65 (pp65) Ag. Vaccination of mice with pp65-encoding DNA elicited high IFN-γ(+) CD8 T cell frequencies to the pp65(495-503)/(e6) epitope and low responses to the pp65(320-328)/(e3) and pp65(522-530)/(e8) epitopes. Abrogation of the e6-specific immunity efficiently enhanced e3- and e8-specific T cell responses by a pp65(Δ501-503) DNA vaccine. The immunodominant e6-specific (but not the e3- and e8-specific) CD8 T cell response critically depends on CD4 T cell help. Injection of monospecific DNA- or peptide-based vaccines encoding the e3 or e8 (but not the e6) epitope into mice elicited CD8 T cells. Codelivering the antigenic peptides with different heterologous CD4 T cell helper epitopes enhanced e6-specific (but not e3- or e8-specific) CD8 T cell responses. Similarly, homologous CD4 T cell help, located within an overlapping (nested) pp65(487-503) domain, facilitated induction of e6-specific CD8 T cell responses by peptide-based vaccination. The position of the e6 epitope within this nested domain is not critical to induce the immunodominant, e6-specific CD8 T cell response to the pp65 Ag. Distant CD4 T cell epitope(s) can thus provide efficient help for establishing pp65-e6 immunodominance in vaccinated mice. These results have practical implications for the design of new T cell-stimulating vaccines.     

A SmD peptide induces better antibody responses to other proteins within the small nuclear ribonucleoprotein complex than to SmD protein via intermolecular epitope spreading

Autoantibody response against the small nuclear ribonucleoprotein (snRNP) complex is a characteristic feature of systemic lupus erythematosus. The current investigation was undertaken to determine whether activation of SmD-reactive T cells by synthetic peptides harboring T cell epitopes can initiate a B cell epitope spreading cascade within the snRNP complex. T cell epitopes on SmD were mapped in A/J mice and were localized to three regions on SmD, within aa 26-55, 52-69, and 86-115. Immunization with synthetic peptides SmD(31-45), SmD(52-66), and SmD(91-110) induced T and B cell responses to the peptides, with SmD(31-45) inducing the strongest response. However, only SmD(52-66) immunization induced T cells capable of reacting with SmD. Analysis of sera by immunoprecipitation assays showed that intermolecular B cell epitope spreading to U1RNA-associated A ribonucleoprotein and SmB was consistently observed only in the SmD(52-66)-immunized mice. Surprisingly, in these mice, Ab responses to SmD were at low levels and transient. In addition, the sera did not react with other regions on SmD, indicating a lack of intramolecular B cell epitope spreading within SmD. Our study demonstrates that T cell responses to dominant epitope on a protein within a multiantigenic complex are capable of inducing B cell responses to other proteins within the complex. This effect can happen without generating a good Ab response to the protein from which the T epitope was derived. Thus caution must be taken in the identification of Ags responsible for initiating autoimmune responses based solely on serological analysis of patients and animals with systemic autoimmune disorders.     

Diverse fine specificity and receptor repertoire of T cells reactive to the major VP1 epitope (VP1230-250) of Theiler's virus: V beta restriction correlates with T cell recognition of the c-terminal residue.

Theiler's murine encephalomyelitis virus induces chronic demyelinating disease in genetically susceptible mice. The histopathological and immunological manifestation of the disease closely resembles human multiple sclerosis, and, thus, this system serves as a relevant infectious model for multiple sclerosis. The pathogenesis of demyelination appears to be mediated by the inflammatory Th1 response to viral epitopes. In this study, T cell repertoire reactive to the major pathogenic VP1 epitope region (VP1233-250) was analyzed. Diverse minimal T cell epitopes were found within this region, and yet close to 50% of the VP1-reactive T cell hybridomas used V beta 16. The majority (8/11) of the V beta 16+ T cells required the C-terminal amino acid residue on the epitope, valine at position 245, and every T cell hybridoma recognizing this C-terminal residue expressed V beta 16. However, the complementarity-determining region 3 sequences of the V beta 16+ T cell hybridomas were markedly heterogeneous. In contrast, such a restriction was not found in the V alpha usage. Only restricted residues at this C-terminal position allowed for T cell activation, suggesting that V beta 16 may recognize this terminal residue. Further functional competition analysis for TCR and MHC class II-contacting residues indicate that many different residues can be involved in the class II and/or TCR binding depending on the T cell population, even if they recognize the identical minimal epitope region. Thus, recognition of the C-terminal residue of a minimal T cell epitope may associate with a particular V beta (but not V alpha) subfamily-specific sequence, resulting in a highly restricted V beta repertoire of the epitope-specific T cells.     

Identification of two T-cell epitopes on the candidate Epstein-Barr virus vaccine glycoprotein gp340 recognized by CD4+ T-cell clones.

Current efforts to develop an Epstein-Barr virus subunit vaccine are based on the major envelope glycoprotein gp340. Given the central role of CD4+ T cells in regulating immune responses to subunit vaccine antigens, the present study has begun the work of identifying linear epitopes which are recognized by human CD4+ T cells within the 907-amino-acid sequence of gp340. A panel of gp340-specific CD4+ T-cell clones from an Epstein-Barr virus-immune donor were first assayed for their proliferative responses to a series of truncated gp340 molecules expressed from recombinant DNA vectors in rat GH3 cells, by using an autologous B lymphoblastoid cell line as a source of antigen-presenting cells. The first four T-cell clones analyzed all responded to a truncated form of gp340 which contained only the first 260 N-terminal amino acids. These clones were subsequently screened for responses to each of a panel of overlapping synthetic peptides (15-mers) corresponding to the primary amino acid sequence of the first 260 N-terminal amino acids of gp340. One clone (CG2.7) responded specifically to peptides from the region spanning amino acids 61 to 81, while three other clones (CG5.15, CG5.24, and CG5.36) responded specifically to peptides from the region spanning amino acids 163 to 183. Work with individual peptides from these regions allowed finer mapping of the T-cell epitopes and also revealed the highly dose-dependent nature of peptide-induced responses, with inhibitory effects apparent when the most antigenic peptides were present at supraoptimal concentrations. Experiments using homozygous typing B lymphoblastoid cell lines as antigen-presenting cells showed that the T-cell clones with different epitope specificities were restricted through different HLA class II antigens; clone CG2.7 recognized epitope 61-81 in the context of HLA DRw15, whereas clones CG5.15, CG5.24, and CG5.36 recognized epitope 163-183 in the context of HLA DRw11. The present protocol therefore makes a systematic analysis of CD4+ T-cell epitopes within gp340 possible; it will be necessary to screen gp340-specific T-cell clones from a variety of donors to assess the wider influence of HLA class II polymorphism upon epitope choice.     

The LCMV gp33-specific memory T cell repertoire narrows with age

ABSTRACT: BACKGROUND: The memory response to LCMV in mice persists for months to years with only a small decrease in the number of epitope specific CD8 T cells. This long persistence is associated with resistance to lethal LCMV disease. In contrast to studies focused on the number and surface phenotype of the memory cells, relatively little attention has been paid to the diversity of TCR usage in these cells. CD8+ T cell responses with only a few clones of identical specificity are believed to be relatively ineffective, presumably due to the relative ease of virus escape. Thus, a broad polyclonal response is associated with an effective anti-viral CD8+ T cell response. RESULTS: In this paper we show that the primary CD8+ T cell response to the LCMV gp33-41 epitope is extremely diverse. Over time while the response remains robust in terms of the number of gp33-tetramer+ T cells, the diversity of the response becomes less so. Strikingly, by 26 months after infection the response is dominated by a small number TCRbeta sequences. In addition, it is of note the gp33 specific CD8+ T cells sorted by high and low tetramer binding populations 15 and 22 months after infection. High and low tetramer binding cells had equivalent diversity and were dominated by a small number of clones regardless of the time tested. A similar restricted distribution was seen in NP396 specific CD8+ T cells 26 months after infection. The identical TCRVbeta sequences were found in both the tetramerhi and tetramerlo binding populations. Finally, we saw no evidence of public clones in the gp33-specific response. No CDR3 sequences were found in more than one mouse. CONCLUSIONS: These data show that following LCMV infection the CD8+ gp33-specific CD8 T cell response becomes highly restricted with enormous narrowing of the diversity. This narrowing of the repertoire could contribute to the progressively ineffective immune response seen in aging.     

CD4+ T-cell responses to Epstein-Barr virus (EBV) latent-cycle antigens and the recognition of EBV-transformed lymphoblastoid cell lines

There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4+ T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4+ T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4+ epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4+ T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4+ clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4+ T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells.     

Endogenous H/K ATPase beta-subunit promotes T cell tolerance to the immunodominant gastritogenic determinant

A CD4(+) T cell response to the gastric H/K ATPase beta-subunit (H/Kbeta) is required for the onset of experimental autoimmune gastritis in BALB/c mice. The extent to which endogenous H/Kbeta contributes toward the tolerance of the H/Kbeta-specific T cell repertoire in normal individuals is not known. By comparison of T cell responses in H/Kbeta-deficient (o/o) and H/Kbeta-expressing BALB/c mice, in this work we show that the endogenous H/Kbeta autoantigen plays a major role in the tolerance of pathogenic H/Kbeta-specific T cells. First, T cell-dependent Ab responses to the H/Kbeta Ag were enhanced in H/K ATPase-immunized H/Kbeta-deficient mice compared with wild-type mice. Second, peptide immunization experiments indicated that immune responses to the major gastritogenic epitope of the H/K ATPase, namely H/Kbeta(253-277), were significantly more vigorous in H/Kbeta-deficient mice compared with wild-type mice. Third, unfractionated splenocytes from H/Kbeta-deficient mice, but not H/Kbeta-expressing mice, induced autoimmune gastritis after adoptive transfer to BALB/c nude mice. The enhanced responses to H/Kbeta in H/Kbeta-deficient mice were shown to be intrinsic to CD4(+)CD25(-) T cells rather than a change in status of CD4(+)CD25(+) regulatory T cells. We conclude from these studies that the H/Kbeta-specific T cells in wild-type mice represent the residue of a T cell repertoire, directed toward a single determinant, that has been subjected to partial tolerance induction.     

A dominant Th epitope in influenza nucleoprotein. Analysis of the fine specificity and functional repertoire of T cells recognizing a single determinant.

The sequence 260-283 of the nucleoprotein (NP) of influenza A virus is an epitope recognized by virus-immune lymph node cells from CBA (H-2k), B6 (H-2b), and B10.S (H-2s) mice. Further analysis shows that there are at least two Th epitopes within this sequence: the one close to the N-terminal (p260-273) is recognized by T cells from CBA and B6 mice while that close to the carboxyl-terminal (p270-283) is a dominant Th determinant in B10.S mice. The fine specificity of the recognition of this epitope by NP-specific T cell clones is also studied. When B10.S mice were infected intranasally or i.v. with live influenza virus, or immunized by different ways with various Ag preparations, P270-283 persistently emerged as a dominant T cell epitope. Immunization of B10.S mice with peptide p270-283 induces T cells with different in vivo functions including class II-restricted cytotoxicity, cognate help for Ag-specific antibody synthesis and delayed type hypersensitivity. This may have important implications for the understanding of the differentiation and classification of subsets of CD4+ T cells. The corresponding sequence of the NP of an equine influenza virus, A/Eq/Prague/56, which has a substitution (leucine to proline) at position 283, was not recognized by the lymph node cells from mice primed with either A/Okuda or A/Eq/Prague. However, the peptide, p270-283(E), representing this sequence induced T cell responses to both human and equine viruses. The data are discussed with respect to the development of viral vaccines.     

Recognition of homo- and heterosubtypic variants of influenza A viruses by human CD8+ T lymphocytes

In the present study, the recognition of epitope variants of influenza A viruses by human CTL was investigated. To this end, human CD8(+) CTL clones, specific for natural variants of the HLA-B*3501-restricted epitope in the nucleoprotein (NP(418-426)), were generated. As determined in (51)Cr release assays and by flow cytometry with HLA-B*3501-peptide tetrameric complexes, CTL clones were found to be specific for epitopes within one subtype or cross-reactive with heterosubtypic variants of the epitope. Using eight natural variants of the epitope, positions in the 9-mer important for T cell recognition and involved in escape from CTL immunity were identified and visualized using multidimensional scaling. It was shown that positions 4 and 5 in the 9-mer epitope were important determinants of T cell specificity. The in vivo existence of CD8(+) cells cross-reactive with homo- and heterosubtypic variants of the epitope was further confirmed using polyclonal T cell populations obtained after stimulation of PBMC with different influenza A viruses. Based on the observed recognition patterns of the clonal and polyclonal T cell populations and serology, it is hypothesized that consecutive infections with influenza viruses containing different variants of the epitope select for cross-reactive T cells in vivo.     

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.     

Functional analysis of birch pollen allergen Bet v 1-specific regulatory T cells

Allergen-specific immunotherapy using peptides is an efficient treatment for allergic diseases. Recent studies suggest that the induction of CD4+ regulatory T (Treg) cells might be associated with the suppression of allergic responses in patients after allergen-specific immunotherapy. Our aim was to identify MHC class II promiscuous T cell epitopes for the birch pollen allergen Bet v 1 capable of stimulating Treg cells with the purpose of inhibiting allergic responses. Ag-reactive CD4+ T cell clones were generated from patients with birch pollen allergy and healthy volunteers by in vitro vaccination of PBMC using Bet v 1 synthetic peptides. Several CD4+ T cell clones were induced by using 2 synthetic peptides (Bet v 1(141-156) and Bet v 1(51-68)). Peptide-reactive CD4+ T cells recognized recombinant Bet v 1 protein, indicating that these peptides are produced by the MHC class II Ag processing pathway. Peptide Bet v 1(141-156) appears to be a highly MHC promiscuous epitope since T cell responses restricted by numerous MHC class II molecules (DR4, DR9, DR11, DR15, and DR53) were observed. Two of these clones functioned as typical Treg cells (expressed CD25, GITR, and Foxp3 and suppressed the proliferation and IL-2 secretion of other CD4+ T cells). Notably, the suppressive activity of these Treg cells required cell-cell contact and was not mediated through soluble IL-10 or TGF-beta. The identified promiscuous MHC class II epitope capable of inducing suppressive Treg responses may have important implication for the development of peptide-based Ag-specific immunotherapy to birch pollen allergy.