Murine T cell-stimulatory peptides from the 19-kDa antigen of Mycobacterium tuberculosis. Epitope-restricted homology with the 28-kDa protein of Mycobacterium leprae.

Fifteen overlapping synthetic peptides, spanning the entire amino acid sequence of the Mycobacterium tuberculosis 19-kDa protein, were used to identify epitopes recognized by murine T cells. Five of the 15 peptides tested were able to elicit in vitro lymph node T cell proliferative responses in C57BL/10 mice primed by footpad inoculation with homologous peptide. Analysis in congenic strains of mice revealed H-2 restriction in the response to four peptides. However, one peptide, 19.7 (residues 61 to 80), induced T cell responses in all four haplotypes tested. This peptide was also unique in being able to stimulate lymph node cells from C57BL/10 mice immunized with recombinant 19-kDa protein, killed M. tuberculosis, or live bacillus Calmette Guerin infection. T cell lines specific for peptide 19.7 were of the CD4 phenotype. Significantly, sequence analysis revealed that residues 61 to 80 of the 19-kDa protein exhibited considerable homology with a single 20-amino acid sequence (residues 120 to 140), but not with any other region of the 28-kDa protein expressed in Mycobacterium leprae. This finding is the first evidence of epitope-restricted homology between otherwise structurally unrelated microbial Ag.     

Genetic control of immune responses to the 18-kDa protein of Mycobacterium leprae. Different TH1 subsets may be involved in proliferative and delayed-type hypersensitivity responses.

In vitro and in vivo responses to the 18-kDa protein of Mycobacterium leprae have been analysed in different strains of mice. Lymphocytes from BALB/cJ (H-2d), BALB.B (H-2b), B10.BR (H-2k), and B10.M (H-2f) mice primed with 18-kDa protein yielded high T cell proliferative responses, while those from C57BL/10J (H-2b) mice yielded lower responses. Both H-2 and non-H-2 genes contributed to the magnitude of responsiveness. F1 mice from high and low responder strains showed high responsiveness to the 18-kDa protein. Supernatants from lymph node cell cultures prepared from 18-kDa protein-immunised BALB/cJ, B10.BR, and C57BL/10J mice contained IL-2 but no IL-4, indicating that activated T cells from both high and low responder mice were of a TH1 phenotype. Cell cultures from low responder C57BL/10J mice produced less IL-2 than those from high responders. The low responsiveness to the 18-kDa protein in proliferative assays might be due to a low frequency of antigen-specific T cells in the C57BL/10J mouse strain. BALB/cJ, C57BL/10J, and F1 (BALB/cJ x B10.BR) mouse strains were tested for in vivo DTH reactions to the 18-kDa protein. All strains, including C57BL/10J, were high DTH responders. Although DTH effector cells and 18-kDa protein-specific proliferative T cells belong to the TH1 subset, our data comparing high and low responder status indicate that distinct TH1 subpopulations are stimulated in response to the 18-kDa protein of M. leprae.     

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.     

Identification of T cell stimulatory peptides from the 38-kDa protein of Mycobacterium tuberculosis.

T cell specificity to individual antigenic epitopes could determine the distinction between protective and pathogenic host reactions in tuberculous infections. Therefore, T cell stimulatory epitopes of the Mycobacterium tuberculosis 38-kDa lipoprotein, of known structure and specificity and of prominent immunogenicity, have been examined. To identify potential T cell epitopes, eight peptides, seven of which were predicted to form amphiphatic helices, were used for immunization of various inbred mice and for elicitation of in vitro T cell proliferative responses. Three different response patterns were observed. 1) Lymph node cells from mice immunized with peptide, recombinant 38-kDa Ag, killed M. tuberculosis strain H37Ra, or live Mycobacterium bovis bacillus Calmette Guerin infection responded to peptide 38.G (residues 350 to 369). Responses were observed in mice of H-2b, H-2d, and H-2k haplotypes. 2) Peptide 38.C (residues 201 to 220) induced proliferation of lymph node cells from 38-kDa protein-, but not from peptide-immunized mice. 3) Peptide 38.F (residues 285 to 304) only elicited a response of the homologous peptide-primed cells. Analysis of CD4+ T cell lines confirmed the distinct specificities and stimulatory features of peptides 38.F and 38.G. The described attributes of peptide 38.C and 38.G could be of potential interest for diagnostic evaluation in tuberculous infections.     

Heterogeneity among human T cell clones recognizing an HLA-DR4,Dw4-restricted epitope from the 18-kDa antigen of Mycobacterium leprae defined by synthetic peptides.

Synthetic peptides have been used to exactly define a T cell epitope region from the immunogenic 18-kDa protein of Mycobacterium leprae. Four M. leprae reactive CD4+ T cell clones, isolated from two healthy individuals vaccinated with killed M. leprae, recognized a determinant initially defined by the peptide (38-50). However, fine mapping of the minimal sequence required for T cell recognition revealed heterogeneity among the T cell clones with regard to the N- and carboxyl-terminal boundaries of the epitopes recognized. MHC restriction analysis showed that the immunogenic peptides were presented to the T cells in an HLA-DR4,Dw4-restricted manner in all cases. The results suggest that a polyclonal T cell response representing different fine specificities is directed toward a possible immunodominant epitope from the M. leprae 18-kDa Ag in individuals carrying this MHC haplotype.     

Genetic control of murine T cell proliferative responses to Mycobacterium leprae. V. Evidence for cross-reactivity between host antigens and Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured by immunization of mice at the base of the tail with Ag and challenging lymphocytes from draining lymph nodes in culture with M. leprae. C57BL/10J and B10.BR mice were identified as low responder mice and the congenic strains B10.M, B10.Q, and B10.AKM as high responders whereas F1 (high x low) hybrid mice were found to be low responders. The cellular basis of low responsiveness did not appear to result from a defect in Ag-presenting cells or the activation of suppressor T cells by M. leprae. The influence of the environment in which T cells developed on responsiveness to M. leprae was analyzed in chimeric mice prepared by irradiating F1(C57BL/10J x B10.M) mice and reconstituting with bone marrow from C57BL/10J, B10.M, or F1 donors. Six weeks later, chimeric mice were immunized with M. leprae, lymph node cells were subsequently prepared, and H-2 phenotyped and challenged in culture with M. leprae Ag. T cell proliferative responses were found to be low in all cases, similar to those observed using lymph node cells from F1 hybrid mice. These results suggested that high responder B10.M lymphocytes developing in the irradiated F1 mice became tolerized to antigenic determinants found on M. leprae. This implied cross-reactive epitopes existed between some mouse strains and M. leprae. Low responsiveness to M. leprae in low responder and F1 hybrid mice may result from tolerance to H-2-encoded Ag that show cross-reactivity with M. leprae.     

Mapping of T helper cell epitopes by using peptides spanning the 19-kDa protein of Mycobacterium tuberculosis. Evidence for unique and shared epitopes in the stimulation of antibody and delayed-type hypersensitivity responses.

In vivo and in vitro T cell responses to overlapping 20-mer peptides that span the entire 19-kDa protein of Mycobacterium tuberculosis have been compared in three different strains of mice. Immunization of the mice with peptides and analysis of specific antibody production is an in vivo assay of Th cell activity. Peptides 1-20 and 61-80 elicited strong IgG1 responses in BALB/cJ, C57BL/10J, and B10.BR mice, indicating that these peptides could stimulate Th cells, possibly of a Th2 phenotype. T cells isolated from peptide-immunized mice were challenged in vitro with peptide, and their proliferative responses were analyzed. T cells from these three strains of mice immunized with peptides 1-20, 61-80, and 76-95 also responded to challenge with specific peptide in vitro. In addition, B10.BR mice and BALB/cJ mice showed antibody and T cell proliferative responses to peptides 136-155 and 145-159, respectively. Thus, in vitro proliferating T cells were found to possess specificities for peptide epitopes that were almost identical to those of the antibody-producing cells. Delayed-type hypersensitivity (DTH) responses to these peptides were also examined in the three strains. Interestingly, the T cells responding in the DTH assay had Ag specificities that were quite different from those identified in the antibody and proliferation assays. These results suggested that DTH Th cells form a separate population from antibody Th and proliferative T cells and these populations of cells were differentially activated, in an Ag-specific manner.     

Immune responses to the 18-kDa protein of Mycobacterium leprae. Similar B cell epitopes but different T cell epitopes seen by inbred strains of mice.

Antibody responses to the 18-kDa protein of Mycobacterium leprae have been analyzed in different strains of mice. High, intermediate, and low responder strains have been identified and these response patterns show clear linkage to genes encoded in the H-2 complex. Three peptides, residues 1-50, 51-100, and 101-148 have been synthesized, as well as a series of 20-mer peptides, which span the entire 18-kDa protein. Repeated immunization of different strains of mice with the 18-kDa protein resulted in IgG responses to epitopes found on all three synthetic peptides. Immunization of BALB/cJ and B10.BR mice, two high responder strains, with 18-kDa protein resulted in high levels of IgG antibody to epitopes found on peptides 1-20, 16-35, 31-50, 46-65, and 76-95. B10.BR mice also contained IgG that bound peptide 61-80 and BALB/cJ mice produced IgG that bound peptide 91-110. Although B10.BR mice produced IgG that bound the 50-mer peptide 101-148, this IgG was not detected by binding to peptides 91-110, 106-125, 121-140, and 131-148. Immunization of B10.BR mice with individual overlapping 20-mer peptides as Ag revealed that peptides 1-20, 16-35, 31-50, and 76-95 elicited high titers of IgG that bound both the immunizing peptide as well as 18-kDa protein. As these peptides induce antibody synthesis they must contain both B cell and T cell epitopes. By contrast, immunization of BALB/cJ mice with the same 20-mer peptides, all of which contain B cell epitopes for this strain, failed to elicit IgG responses with one exception. Peptide 91-110 induced IgG that bound peptide 91-110, but not the intact 18-kDa protein. We conclude that peptides 1-20, 16-35, 31-50, and 76-95 either lack T cell epitopes for BALB/cJ mice, or activate different T cell subpopulations in the two strains. We suggest that the induction of IgG responses to small peptide Ag is an in vivo assay of the activity of Th2 cell subpopulations.     

Human T cell recognition of the Mycobacterium leprae LSR antigen: epitopes and HLA restriction

We have in this work mapped epitopes and HLA molecules used in human T cell recognition of the Mycobacterium leprae LSR protein antigen. HLA typed healthy subjects immunized with heat killed M. leprae were used as donors to establish antigen reactive CD4+ T cell lines which were screened for proliferative responses against overlapping synthetic peptides covering the C-terminal part of the antigen sequence. By using this approach we were able to identify two epitope regions represented by peptide 2 (aa 29-40) and peptide 6 (aa 49-60), of which the former was mapped in detail by defining the N- and C-terminal amino acid positions necessary for T cell recognition of the core epitope. MHC restriction analysis showed that peptide 2 was presented to T cells by allogeneic cells coexpressing HLA-DR4 and DRw53 or DR7 and DRw53. In contrast, peptide 6 was presented to T cells only in the context of HLA-DR5 molecules. In conclusion, the M. leprae LSR protein antigen can be recognized by human T cells in the context of multiple HLA-DR molecules, of which none are reported to be associated with the susceptibility to develop leprosy. The results obtained are in support of using the LSR antigen in subunit vaccine design.     

Identification of a T-cell epitope adjacent to neutralization antigenic site 1 of poliovirus type 1.

Proliferative T-cell responses to poliovirus in various strains of mice have been analyzed by using either killed purified virus or capsid protein VP1 synthetic peptides. Following immunization of mice with inactivated poliovirus type 1 (PV1), a specific proliferative response of their lymph node CD4+ T cells was obtained after in vitro stimulation with purified virus. In mice immunized with PV1, PV2, or PV3, a strong cross-reactivity of the T-cell responses was observed after in vitro stimulation with heterologous viruses. By using various strategies, a dominant T-cell epitope was identified in the amino acid 103 to 115 region of capsid polypeptide VP1, close by the C3 neutralization epitope. The T-cell response to VP1 amino acids 103 to 115 is H-2 restricted: H-2d mice are responders, whereas H-2k and H-2b mice do not respond to this T-cell epitope. Immunization of BALB/c (H-2d) mice with the uncoupled p86-115 peptide, which represents VP1 amino acids 86 to 115 and contains both the T-cell epitope and the C3 neutralization epitope, induced poliovirus-specific B- and T-cell responses. Moreover, these mice developed poliovirus neutralizing antibodies.     

Epitopes of the Mycobacterium tuberculosis 65-kilodalton protein antigen as recognized by human T cells

A synthetic peptide approach has been used to identify the epitopes recognized by clonal and polyclonal human T cells reactive to the recombinant mycobacterial 65-kDa protein Ag. Three of the four epitopes identified were recognized as cross-reactive between Mycobacterium tuberculosis and Mycobacterium leprae, although their amino acid sequence in two of three cases was not identical. The peptide (231-245) defining an epitope recognized as specific to the M. tuberculosis complex contains two substitutions compared with the homologous M. leprae region of which one or both are critical to T cell recognition. The reactive T cell clones showed helper/inducer phenotype (CD4+, CD8-), and secrete IL-2, granulocyte-macrophage-CSF, and IFN-gamma upon Ag stimulation. The same clones display cytotoxicity against macrophages pulsed with the relevant peptides or mycobacteria.     

Mapping an antibody-binding site and a T-cell-stimulating site on the 1A protein of respiratory syncytial virus.

A synthetic peptide modeled on residues 45 to 60 of the 1A protein of respiratory syncytial (RS) virus [1A(45-60)] was constructed and used for immunization of mice and rabbits. The immunoglobulin G fraction of the resulting rabbit antibody, purified on protein A-Sepharose, immunoprecipitated from RS-infected HEp-2 cells a protein with a molecular size of approximately 9.5 kilodaltons, which corresponds to the previously published molecular size of the 1A protein (Y. T. Huang, P. L. Collins, and G. W. Wertz, Virus Res. 2:157-173, 1985). To investigate the T-cell-inducing properties of 1A(45-60), six strains of mice were immunized and their popliteal lymph node cells were tested for proliferation upon restimulation with peptide in vitro. The lymph node cells of all six strains of mice were responsive to restimulation with 1A(45-60) and showed high- and low-responder strain variation. These peptide-primed lymph node cells also proliferated upon in vitro restimulation with RS virus-infected cells. Correlation of proliferation with interleukin 2 production suggested that the responding lymphocytes were T-helper cells. The antibody-binding and T-cell-stimulating sites of 1A were mapped by constructing a series of overlapping synthetic peptides and testing each for ability to react with antiserum prepared by immunization of BALB/C mice with free peptide 1A(45-60) or for ability to restimulate proliferation in 1A(45-60)-primed lymph node cells of BALB/C mice. Human antibody, obtained during confirmed RS virus infection, was similarly tested with the truncated peptides. Antibody-binding activity was reduced after truncation from the carboxy terminus, and a binding site was mapped to residues 51 through 60, the smallest peptide tested. T-cell-stimulating activity in mice was relatively resistant to truncation from the carboxy terminus and sensitive to truncation from the amino terminus. The smallest region which retained significant T-cell-stimulating activity mapped to residues 46 through 56. However, addition of the naturally occurring Cys at residue 45 and extension of the C terminus to residue 62 resulted in maximum T-cell-stimulating activity of the peptide. These data define both a T-cell epitope and a B-cell epitope of the 1A protein of RS virus and suggest that the carboxy terminus of 1A contains a B-cell epitope, involving residues 51 through 60, which is recognized during natural human infection.     

Exact definition of species-specific and cross-reactive epitopes of the 65-kilodalton protein of Mycobacterium leprae using synthetic peptides

With the use of solid phase synthesis of peptides corresponding to major and minor peaks in a Hopp-Woods hydrophilicity plot, the epitopes for 10 of 14 known different mAb to the Mycobacterium leprae 65-kDa protein, a prominent T and B cell Ag of this bacillus, have been located in the primary structure. Five epitopes have been precisely mapped by using the synthetic peptides in inhibition ELISA experiments, and five others have been located on peptides of 22 amino acids or less in length. The epitope of an important species-specific antibody, IIIE9, which may be useful for seriodiagnosis of leprosy, appears to be distinguished from the epitope of the antibody IVD2, widely cross-reactive among mycobacteria, not by its sequence, but only by its critical residues. All epitopes studied appear continuous insofar as can be determined by this approach.     

Sequence and immunogenicity of the 70-kDa heat shock protein of Mycobacterium leprae

The gene encoding the Mycobacterium leprae 70-kDa heat shock protein has been isolated from a cosmid library using a fragment of the clone JKL2. Southern blot analysis of a positive clone identified a 4.4-kb fragment containing the entire coding region of the gene plus 2.4 kb upstream. Sequencing revealed the gene to encode a 621-amino acid protein, bearing 56% identity with the Escherichia coli dnaK gene product and 47% and 46% identity with the human and Caenorhabditis elegans hsp70, respectively. Comparison with the C-terminal 203 amino acids of the Mycobacterium tuberculosis 71-kDa Ag yielded 70% identity. Recombinant M. leprae p70 was produced in E. coli as a fusion protein (rp70f) with a portion of the schistosomal glutathione-S-transferase, using the expression vector, pGEX-2T. Cleavage with thrombin resulted in the release of a 70.0-kDa protein (rp70c) from the glutathione-S-transferase. Examination of the proteins by immunoblotting demonstrated that anti-M. leprae mAb, L7, and sera from lepromatous leprosy patients bound to both the cleaved and fusion proteins. We compared the T cell reactivity of the M. leprae recombinant proteins with that of mAb affinity-purified bacille Calmette-Guerin (BCG) 70-kDa Ag using proliferation assays. PBMC of BCG vaccinees responded to both M. leprae cleaved and fusion p70, though more subjects responded to the rp70c (18 of 20) than to rp70f (13 of 20). Responses were generally higher to rp70c than to rp70f, however all responses to the M. leprae recombinant proteins were lower than to mAb affinity-purified BCG p70. Thus, the M. leprae 70-kDa heat shock protein elicits T and B cell responses in subjects exposed to mycobacteria, despite its homology with the human hsp70.     

Cell mediated immune response elicited in mice after immunization with the P64k meningococcal protein: epitope mapping

The P64k protein of Neisseria meningitidis has been reported as an immunological carrier for weak immunogens. This investigation was aimed at characterizing the T-cell response produced in primed mice and at identifying T helper cell epitopes within this molecule. BALB/c mice subcutaneously immunized with the recombinant antigen provided inguinal lymph node cells (LNC) that proliferated in the presence of P64k in a dose-dependent manner. Proliferating cells secreted IL-4 while the concentration of IL-12 remained unaltered in the culture supernatant. By testing a panel of 59 overlapping synthetic peptides spanning the entire sequence of the antigen a T-cell determinant was localized. Prime-boost and lymphoproliferation experiments, conducted with highly purified synthetic peptides, confirmed that the segment including amino acids 470-485 comprises a T-cell epitope within the P64k molecule.     

Mycobacterium leprae-specific T cells from a tuberculoid leprosy patient suppress HLA-DR3-restricted T cell responses to an immunodominant epitope on 65-kDa hsp of mycobacteria.

The polar tuberculoid type (TT) of leprosy, characterized by high T cell reactivity to Mycobacterium leprae, is associated with HLA-DR3. Surprisingly, DR3-restricted low T cell responsiveness to M. leprae was found in HLA-DR3-positive TT leprosy patients. This low responsiveness was specifically induced by M. leprae but not by M. tuberculosis and was seen only in patients and not in healthy controls. We studied this patient-specific, M. leprae-induced, DR3-restricted low T cell responsiveness in depth in one representative HLA-DR3-positive TT leprosy patient by using T cell clones. From this patient two types of T cell clones were obtained: one type was cross-reactive with M. tuberculosis and recognized an immunodominant epitope (amino acids 3 to 13) on the 65-kDa heat shock protein (hsp) the other type was M. leprae specific and reacted to a protein other than the 65-kDa one. To examine whether these M. leprae-specific T cell clones were responsible for the DR3-restricted low responsiveness to M. leprae, we tested them for the ability to suppress the proliferation of the DR3-restricted, 65-kDa, hsp-reactive clones. The DR3-restricted, M. leprae-specific T cells completely suppressed the proliferative responses of DR3-restricted, cross-reactive T cell clones to the 65-kDa hsp from the same patient as well as from other individuals. Also, DR3-restricted responses to an irrelevant Ag were suppressed by the M. leprae-specific T cell clones. However, no suppression of non-DR3-restricted T cell responses was seen. Although the mechanism must still be elucidated, this M. leprae-induced, DR3-restricted immunosuppression may at least partly explain the observed DR3-associated low T cell responsiveness in TT leprosy patients.     

Studies on the mechanism of the enhancement of delayed-type hypersensitivity by pertussigen

The potentiation of delayed-type hypersensitivity (DTH) reactions by pertussigen, a protein toxin from Bordetella pertussis, has been studied in adoptive transfer assays. Lymph node or spleen cells from mice treated with or without pertussigen at the time of immunization with protein antigens were transferred to naive, syngeneic recipients that were challenged with antigen. Cells from donors treated with pertussigen had the capacity to transfer vigorous, antigen-specific DTH reactions. Cells from immunized donors not given pertussigen transferred little or no DTH. These results indicate that pertussigen is able to augment DTH reactions by potentiating the antigen reactivity of cell populations in lymphoid organs. The phenotype of the effector cells induced by pertussigen was Thy-1 positive, L3T4 positive, and Ly-2 negative. Cells from mice given pertussigen and an irrelevant antigen had no influence on specific DTH responses, suggesting that pertussigen enhances the activity of the antigen-specific cell type mediating DTH. The effect of pertussigen and of immunization on the lymphocyte subpopulations present in the lymph nodes was studied by analysis of suspensions of lymph node cells by flow cytometry. In immunized and in nonimmune mice, pertussigen increased the ratio of Ly-2-negative:Ly-2-positive T cells, and reduced the overall proportion of B cells. In immunized mice, pertussigen induced a much higher proportion of large dividing cells from 5 days after sensitization onwards. The relevance of these changes in lymphocyte behavior to the development of enhanced and prolonged DTH in mice given pertussigen is discussed.     

Determination of T cell epitopes on the S1 subunit of pertussis toxin.

To understand the immunologic characteristics of pertussis toxin molecule and to explore the possibility of developing a synthetic vaccine, T cell epitopes on the enzymatic S1 subunit of pertussis toxin were studied by measuring the proliferative response of immune murine lymph node cells and T cell lines to Ag and to synthetic peptides. The maximum in vitro T cell proliferative response was obtained by stimulating immune lymphoid cells with 20 nM of the enzymatic S1 subunit. When the T cell proliferative response of murine lymphoid cells with different MHC backgrounds was tested, only mice bearing the H-2d haplotype were high responder to the S1 subunit. To determine T cell epitopes on the S1 subunit, the proliferative response of BALB/c immune lymphoid cells to several synthetic S1 peptides was measured. Only the peptide containing amino acid residues, 65-79, was recognized by BALB/c lymphoid cells and was confirmed to contain a T cell epitope by generating S1 specific BALB/c T cell line. By using this T cell line, the response of BALB/c mice to the S1 subunit as well as to peptide 65-79 was shown to be restricted to the I-Ad sublocus of class II Ag. Finally, we showed that lymph node cells of mice immunized with peptide 65-79 respond to the native S1 subunit.     

Identification of a minimal T cell epitope recognized by antinucleosome Th cells in the C-terminal region of histone H4

Autoreactive T cells responding to systemic autoantigens have been characterized in patients and mice with autoimmune diseases and in healthy individuals. Using peptides covering the whole sequence of histone H4, we characterized several epitopes recognized by lymph node Th cells from nonsystemic lupus erythematosus-prone mice immunized with the same peptides, the H4 protein, or nucleosomes. Multiple T epitopes were identified after immunizing H-2d BALB/c mice with H4 peptides. They spanned residues 28-42, 30-47, 66-83, 72-89, and 85-102. Within the region 85-102, a minimal CD4+ T epitope containing residues 88-99 was characterized. Although Abs to peptide 88-99 recognized H4, this peptide does not contain a dominant B cell epitope recognized by anti-H4 Abs raised in BALB/c mice or Abs from NZB/NZW H-2d/z lupus mice. Th cells primed in vivo with H4 responded to H4, but not to peptide 88-99. However, this peptide was able to stimulate the proliferation and IL-2 secretion of Th cells generated after immunization with nucleosomes. H488-99 thus represents a cryptic epitope with regard to H4 and a supradominant epitope presented by nucleosome, a supramolecular complex that plays a key role in lupus. This study shows that in the normal repertoire of naive BALB/c mice, autoreactive Th cells specific for histones are not deleted. The reactivity of these Th cells seems to be relatively restricted and resembles that of Th clones generated from SNF1 ((SWR x NZB)F1; I-Ad/q) lupus mice described earlier.     

The antibody repertoire to proteins of Mycobacterium leprae. Genetic influences at the antigen and epitope level.

The effects of both H-2 and non-H-2 genes on the antibody response to the proteins of Mycobacterium leprae were investigated. Using a B10 series of congenic mice we found that the repertoire of antibody responses was under H-2 gene control, and that non-H2 genes were also involved. By Western blotting, differences in the number and m.w. of proteins recognised by mice of different genetic background were apparent. Such differences were also reflected in the total antibody response to a soluble extract of M. leprae (M. leprae sonicate), as measured by ELISA. Concentrating on one particular Ag, the 65-kDa heat shock protein, we found that all strains of mice developed antibodies following immunization with the purified recombinant protein, although there was a continuous distribution in the titer of antibodies obtained, with differences between individual strains indicating both H-2 and non-H-2 effects. Using a library of overlapping peptides based on the amino acid sequence of this protein, we have mapped the B cell epitopes in the different strains of mice. H-2 genes had no effect on the structure and number of epitopes recognized, although this was influenced by non-H-2 genes. There was a high level of concordance between actual epitopes recognized and those predicted by calculations of antigenic index, and B cell epitopes were located in similar positions to previously determined T cell epitopes.