Co-dominant restriction by a mixed-haplotype I-A molecule (alpha k beta b) for the lysozyme peptide 52-61 in H-2k x H-2b F1 mice

Helper (CD4+) T lymphocytes recognize protein Ag as peptides associated to MHC class II molecules. The polymorphism of class II alpha- and beta-chains has a major influence on the nature of the peptides presented to CD4+ T lymphocytes. For instance, T cell responses in H-2k and H-2b mice are directed at different epitopes of the hen egg lysozyme (HEL) molecule. The current studies were undertaken with the aim of defining the role of mixed haplotype I-A (alpha k beta b and alpha b beta k) molecules in T cell responses to HEL in (H-2k x H-2b)F1 mice, as well as the nature of the immunogenic peptides of HEL recognized in the context of I-A alpha k beta b and I-A alpha b beta k. A series of HEL-reactive T cell lines and hybridomas derived from MHC class II heterozygous (C57BL/6 x C3H F1) mice were established. Their responsiveness to HEL and synthetic HEL peptides was analyzed with the use of L cells transfected with either I-A alpha k beta b or I-A alpha b beta k as APC. Out of 28 clonal T cell hybridomas tested, 13 (46%) only responded to HEL presented by I-A alpha k beta b, 11 (40%) by I-A alpha b beta k (and to a minor extent I-A alpha k beta k), only 4 (14%) were primarily restricted by I-Ak, and none by I-Ab. All the I-A alpha k beta b-restricted T cell hybridomas responded to the HEL peptide 46-61 and to its shorter fragment 52-61, even at concentrations as low as 0.3 nM. As this determinant has been previously defined as immunodominant for I-Ak but not for I-Ab mice, these results suggest a role for the I-A alpha k chain in the selection and immunodominance of HEL 52-61 in H-2k mice. The fine specificity of I-A alpha k beta b-restricted T cell hybridomas for a series of different HEL peptides around the sequence 52 to 61 suggests that peptide 52-61 binds to I-A alpha k beta b with higher affinity than to I-A alpha k beta k. The peptides recognized in the context of I-A alpha b beta k and I-A alpha k beta k were not identified.     

IgM induction in murine B hybridomas with Ia-restricted T cell clones: a monoclonal model for T and B cell interactions in antibody response

The mechanism of MHC-restricted T and B cell interactions in antibody response was studied with IgM-inducible B hybridomas and antigen-specific helper T cell clones. B hybridomas were prepared by fusion between splenic B cells from (CBA/N (H-2k) X BALB/c (H-2d)) F1 (NBF1) male mice and a B lymphoma cell line, M12.4.5. A B hybridoma clone, 1M70, which expressed I-Ad but not I-Ak determinants was chosen in the present study. IgM secretion was induced in 1M70 when it was cocultured with a "resting" KLH-specific and H-2d restricted helper T cell clone in the presence of KLH. A "resting" KLH-specific and H-2k restricted T cell clone did not induce IgM secretion in 1M70 even in the presence of KLH. However, when these KLH-specific T cell clones were activated by KLH and appropriate antigen presenting cells, both H-2d and H-2k restricted T cell clones induced IgM secretion in 1M70 even in the absence of KLH. A monoclonal anti-I-Ad antibody inhibited IgM secretion induced by a "resting" H-2d restricted T cell clone, but not by an "activated" T cell clone. These results indicated that T cell clones recognized antigens in the context of Ia molecules on B hybridomas in a MHC-restricted manner and were activated to produce B cell stimulatory factors which in turn acted on B hybridomas in a non-MHC-restricted manner and induced differentiation of B hybridomas into IgM secreting cells.     

Immune response gene control of determinant selection. II. Genetic control of the murine T lymphocyte proliferative response to insulin.

The genetic control of the murine T cell proliferative response to insulin was examined. It was found for two responder strains of mice that each recognizes a different determinant on the insulin molecule. H-2b mice recognize a determinant in the A chain loop of insulin whereas H-2d mice recognize a determinant that resides in the B chain, possibly in the last eight amino acids. Using H-2 recombinant strains of mice, the location of Ir gene control of the response to both determinants was mapped to the K region and/or I-A subregion of H-2. The possibility of non-MHC regulation of MHC-controlled immune responses is suggested by studies of recombinant inbred strains of mice.     

Immune response to hepatitis B virus core antigen (HBcAg): localization of T cell recognition sites within HBcAg/HBeAg

Hepatitis B virus nucleocapsid particles (HBcAg) can function as a T cell-independent antigen when injected into athymic mice. However, immunization of euthymic mice with HBcAg results in dramatically increased anti-HBc titers. Therefore we have examined the murine T cell response to HBcAg in terms of immunogenicity, the influence of H-2-linked genes, and the fine specificity of T cell recognition using synthetic peptide analogs. The HBcAg was shown to be an extremely efficient immunogen in terms of T cell activation as measured by the in vivo dose required to induce T cell sensitization (1.0 microgram), and the minimal in vitro concentration required to elicit interleukin 2 (IL 2) production (0.03 ng/ml). The degree of T cell immunogenicity of HBcAg and its ability to directly activate B cells most likely explain the enhanced humoral response to HBcAg in euthymic mice and HBV-infected patients. The influence of H-2-linked genes on the humoral response to HBcAg was discernable, and high responder (H-2k,s,d), intermediate responder (H-2b,f), and low responder (H-2p) haplotypes were identified. The H-2-linked regulation of the T cell response correlated with in vivo anti-HBc production. Examination of the fine specificity of T cell recognition revealed HBcAg-specific T cells from a variety of strains recognize multiple but distinct sites within the HBcAg/HBeAg sequence. T cell recognition sites were defined by small (16 to 21 residue) synthetic peptides. Each strain recognized a predominant T cell determinant, and the fine specificity of this recognition process was dependent on the H-2 haplotype of the responding strain. For example H-2s,b strains recognized p120-140, H-2f,q strains recognized p100-120, and H-2d mice recognized p85-100 predominantly. Because these sequences are common to both HBcAg and a nonparticulate form of the antigen termed HBeAg, these results indicate that HBcAg and HBeAg are highly cross-reactive at the T cell level although they are serologically distinct. These findings may have clinical relevance, because T cell sensitization to HBeAg and the subsequent seroconversion to anti-HBe status correlates with viral clearance during hepatitis B infection.     

T cells from nonresponder mice. MHC restricted and unrestricted recognition of insulin

Two types of insulin-reactive T cell hybridomas expressing TCR-alpha beta were derived from nonresponder H-2b mice immunized with pork insulin. One type had characteristics of conventional class II-restricted Th cells. These CD4+ CD8- I-Ab-restricted T cells recognized a self determinant, present within the insulin B-chain. This determinant was distinct from the immunodominant A-chain loop determinant that is recognized by the majority of T cells induced after immunization with normally immunogenic beef insulin. Our results suggest that this determinant is readily generated during immunologic processing of insulins, including nonimmunogenic pork insulin and self insulin. A second type of T cell lacking CD4 and CD8 recognized a distinct B-chain determinant of insulin in a class II-dependent, but MHC unrestricted, fashion. These cells may represent a novel subpopulation which has bypassed conventional selection during development in the thymus.     

Major histocompatibility complex class II-regulated immunity to murine leukemia virus protects against early T- but not late B-cell lymphomas

We studied the relative importance of class I and class II major histocompatibility complex (MHC) immunoregulation in the control of T- and B-cell lymphomas induced by murine leukemia virus. Previously, we have described a mink cell focus-inducing (MCF) murine leukemia virus, MCF 1233, which induces not only lymphoblastic T-cell lymphomas but also follicle center cell or lymphoblastic B-cell lymphomas. We now report that the outcome of neonatal infection with MCF 1233 in H-2-congenic C57BL/10 and C57BL/6 mice is decisively influenced by the H-2 I-A locus. A total of 64% of H-2 I-Ak, d mice [B10.BR, B10.D2, B10.A(2R), B10.A(4R), and B10.MBR] developed T-cell lymphomas after MCF 1233 infection (mean latency, 37 weeks). In contrast, H-2 I-Ab [B10, B10.A(5R), B6], H-2 I-Ab/k [(B10.A x B10)F1 and (B10 x B10.A)F1], and H-2 I-Abm12 (bm12) mice were resistant against T-cell lymphomagenesis, but 65% of these H-2 I-Ab, b/k, bm12 animals developed B-cell lymphomas (mean latency, 71 weeks). Animals of T-cell lymphoma-susceptible strains that escaped from T-cell lymphomagenesis developed B-cell lymphomas with similar frequency as animals of T-cell lymphoma-resistant strains, but with a shorter latency. H-2 class II-determined regulation of antiviral immunity was reflected in the presence of high titers of antiviral envelope antibodies in T-cell lymphoma-resistant B-cell lymphoma-susceptible H-2 I-Ab, b/k, bm12 mice, whereas in T-cell lymphoma-susceptible H-2 I-Ak,d mice no antiviral antibodies were found. At week 4 after neonatal MCF 1233 infection, a high percentage of thymocytes were virally infected in both T-cell lymphoma-susceptible and -resistant mice. However, T-cell lymphoma-resistant animals cleared the thymic infection between weeks 4 and 10 of age, coinciding with a sharp rise in serum levels of antiviral antibodies. We conclude that the pleiotropic effects of MCF 1233 infection in H-2-congenic mice result from MHC class II I-A-determined T-cell response differences.     

Polyclonal activation of xid B cells by auto-Ia-reactive T cell clones

The mechanism of T cell-dependent activation of xid B cells into Ig-producing cells was studied by employing H-2-restricted, antigen-specific T cell clones. Helper factors (B cell stimulatory factors, BSF) released from KLH-specific T cell lines could induce polyclonal Ig production in B cells from (CBA/N X BALB/c)F1 (NBF1) female mice but not from CBA/N or NBF1 male mice. Direct addition of helper T cell lines induced Ig production in xid B cells from CBA/N or NBF1 male mice. A T cell clone, MK6, which was derived from NBF1 male mice and specific against Iad determinant, could activate NBF1 male but not CBA/N B cells. Another clone, CK4, derived from CBA/N mice and having specificity against KLH plus I-Ak determinant could activate both CBA/N and NBF1 male B cells into IgM- and IgG-producing cells in the absence of KLH, and monoclonal anti-I-Ak antibody specifically blocked such activation. These results suggest that xid B cells are able to be activated by the signal provided by the recognition of Ia molecules on B cells by auto-Ia-reactive T cells. Xid B cells from CBA/N mice that had been co-cultured with a T cell line specific against I-Ak determinant for 24 hr became reactive to BSF and capable of differentiating into Ig-producing cells in the presence of BSF. The results showed that even xid B cells could be responsive to BSF if they were in a certain activation stage.     

Ir gene control of immune responses to insulins. II. Phenotypic differences in T cell activity among nonresponder strains of mice

Immune responses by mice to heterologous insulins are controlled by H-2 linked Ir genes. In studies to determine the mechanisms responsible for nonresponsiveness, we found that although pork insulin failed to stimulate antibody or proliferative responses in H-2b mice, it did prime T cells that can express helper activity in adoptive recipient mice. This helper activity was insulin-specific in both elicitation and expression. In studies presented in this paper, we have extended this analysis to the response patterns of helper T cells stimulated by sheep, horse, and rat insulins in mice bearing different H-2 haplotypes. The results demonstrate that nonresponder forms of insulin, including rat insulin, prime T cells in H-2b and H-2d, but not H-2k, mice. These results suggest that regulation of nonresponsiveness to insulin appears to be through different pathways in mice bearing different H-2 haplotypes.     

Modulation of F1 cytotoxic potentials by graft-vs-host reaction. Cooperative non-H-2- and H-2D region-gene control of F1 natural resistance to graft-vs-host reaction-associated immunosuppression

Our previous study revealed that in F1 mice raised by crossing C3H/He or AKR/J mice with various H-2-congenic B10-series strains, parental H-2k spleen cells (SC) could not induce the graft-vs-host reaction (GvHR)-associated immunosuppression (GAIS). We also elucidated that a limited number of non-H-2 genes of parental C3H/He or AKR/J mice that had been incorporated into the F1 hybrids determined the F1 resistance to the GAIS, and the present study was done to explore the mechanism implicated in this type of F1 resistance to GAIS. SC from B10.AL mice carrying an rH-2 (K:k I:k S:k D:d) haplotype but not SC from H-2K B10.BR (k k k k) mice induced GAIS of in vitro CTL responses to third-party alloantigens in H-2k/d (C3H/He x B10.D2)F1 recipients mice. Further, SC from H-2k/a (C3H/He x B10.A)F1 mice carrying heterozygous C3H/B10 non-H-2 background but not SC from the same H-2k/a (B10.BR x B10.A)F1 mice but carrying homozygous B10/B10 background induced GAIS in H-2k/d (C3H/He x B10.D2)F1 recipients. Although C3H/He-, B10.BR-, and C3H.OH (d d d k)-SC were incapable of inducing GAIS in (C3H/He x B10.D2)F1 (k/d k/d k/d k/d) recipients, they were all good inducers of GAIS in (C3H.OH x B10.BR)F1 (d/k d/k d/k k/k) recipients. Exactly the same pattern of co-operative non-H-2 AKR and H-2D region-gene control of GAIS was observed on GvHR induced in H-2k/d (AKR/J x B10.D2)F1 recipients. These results suggest that the non-H-2 genes of C3H/He or AKR/J strain inhibit the functional expression of certain antigenic determinant(s) when it is encoded by heterozygous but not homozygous gene(s) linked tightly to H-2D region of k haplotype. Thus, the F1 resistance to GAIS is mediated by immune response of F1 recipients who miss the antigenic determinant(s) against that expressed on cell surface of GvHR-inducing T lymphocytes.     

Cytotoxic T lymphocyte response to minor H-43a alloantigen in H-43b mice. Privileged H-2Kb restriction to the response is not due to immunodominance or epistatic effect but due to Ir gene function of H-2Kb itself

Previous study demonstrated that anti-H-43a cytotoxic T lymphocyte (CTL) response of H-43b CWB (H-2b) stain carrying non-major histocompatability complex (MHC) genes of C3H and F1 strains raised by crossing CWB with various H-43b strains was restricted exclusively by self H-2Kb (Kb). In the present study, newly produced C3W strain (H-2k, H-43b), which is H-43-congenic to C3H/HeN (H-2k, H-43a), was used as H-43b mice, and possibility of immunodominance of Kb was examined. No anti-H-43a CTL response could be induced in C3W strain and F1 strains raised by crossing C3W with other H-43b strains not carrying Kb. Thus, the possibility of immunodominance of Kb over the other MHC class I alleles could not be supported. We also examined possibility of epistatic effect of I region genes and non-MHC genes on the Kb restriction. (C3W x C57BL/6)F1(I-Ak/b) and (C3W x B6.CH-2bm12)F1(I-Ak/bm12)mice showed equally anti-H-43a CTL response restricted exclusively by self Kb, and (C3W x B10.MBR)F1(Ik/k) mice also showed anti-H-43a CTL response restricted solely by self Kb. Cold target competition experiments demonstrated that H-43b C57BL/10 or A.BY mice, which do not have non-MHC genes of C3H mounted anti-H-43a CTL response restricted solely by self Kb. Thus, no relation of I region genes or non-MHC genes to the Kb restriction was shown. All the results indicate that H-43b mouse strains, including F1, can not achieve anti-H-43a CTL response unless they carry Kb allele. Notably, (C3W x C57BL/6)F1 mice mounted self Kb-restricted anti-H-43a CTL response, whereas (C3W x B6.CH-2bm1)F1 mice carrying mutated Kb could not mount anti-H-43a CTL response at all. These findings indicate strongly that Kb itself is classical Ir gene of anti-H-43a CTL response and directs self Kb restriction of the response.     

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.     

Expression of hemopoietic histocompatibility antigens on H-2-loss variants of F1 hybrid lymphoma cells: evidence consistent with trans gene regulation

H-2 heterozygous marrow stem cells, lymphoid progenitor cells, and leukemia/lymphoma cells do not express hemopoietic or hybrid histocompatibility (Hh) antigens, which are important transplantation antigens recognized during the rejection of normal or neoplastic hemopoietic cells. The Hh-1b determinant of the H-2b haplotype maps to the D region of H-2. We have tested the hypothesis that gene(s) at or near H-2D of the H-2d haplotype down-regulate the expression of Hh-1b in the trans configuration. We used Abelson leukemia virus-transformed pre-B lymphoma cells (ACCb) of BALB/c X BALB.B (H-2d X H-2b) origin, as well as variant lines of ACCb, which were selected for resistance to monoclonal anti-H-2 antibodies plus complement. B6D2F1 (H-2b X H-2d), C3B6F1 (H-2k X H-2b), or B6 (H-2b) mice were infused with inocula of 5 X 10(6) B6 bone marrow cells (BMC). Proliferation of donor-derived marrow cells was judged in terms of DNA synthesis by measuring the splenic incorporation of 5-iodo(125I)-2'-deoxyuridine (IUdR) 5 days after cell transfer. B6 BMC grew much better in B6 than in F1 hybrid host mice, an expression of "hybrid resistance". As observed previously, the injection of EL-4 (H-2b, Hh-1b) tumor cells prior to infusion of B6 (H-2b, Hh-1b) BMC enhanced the growth of B6 BMC in F1 hybrid mice. Therefore, this in vivo "cold target cell competition" type of assay can be used to detect the expression of Hh-1b antigens. Unlike EL-4 (H-2b) cells, hybrid resistance was not affected by prior infusion of (H-2b X H-2d) heterozygous ACCb cells. In contrast, three ACCb variant cell lines, H-2d-, Ld-Dd-, and Dd-, enhanced the growth of B6 BMC in F1 hosts. The ACCb H-2b- cell line did not affect hybrid resistance to B6 BMC. The loss of gene expression on the H-2d chromosome at or very near the H-2Dd locus is correlated with the appearance Hh-1b, as determined by the in vivo cold target competition assay. These results support the hypothesis that heterozygous cells possess trans-acting, dominant, down-regulatory genes mapping near H-2D that control the Hh-1 phenotype of lymphoid tumor cells.     

MHC class II A alpha and E alpha molecules determine the clonal deletion of V beta 6+ T cells. Studies with recombinant and transgenic mice

Interactions between MHC class II genes and minor lymphocyte stimulating (Mls) associated products are responsible for clonally deleting self-reactive T cells in mice. Here we demonstrate the role of the intact I-A and I-E molecules as well as the individual A alpha and E alpha chains in the deletion of cells bearing the V beta 6 TCR. DBA/1 (H-2q, Mls-1a) mice were crossed with various inbred congenic, recombinant, and transgenic strains and the F1's were screened for V beta 6 expression. All I-E+ strains were fully permissive in deleting V beta 6+ T cells. I-E- strains expressing I-A b,f,s,k,p permitted only partial deletion, while I-Aq strains showed no deletion. Recombinant I-Aq and I-Af strains which expressed E kappa alpha chain in the absence of E beta chain showed a decrease in V beta 6+ T cells as compared to their H-2q and H-2f counterparts. Furthermore, transgenic mice expressing E kappa alpha Aq beta gene in an H-2q haplotype (E kappa alpha Aq beta?) gave similar results to that of the recombinants in deleting V beta 6 T-cells. The role of the 1-A molecule was also shown by the partial deletion of V beta 6+ T cells in H-2q mice expressing transgenic I-Ak molecules. These results demonstrate that the E alpha chain is important in the deletion of V beta 6 T-cells in Mls-1a mice. The role of A alpha chain is also implied by the permissiveness of E kappa alpha Aq beta but not Aq alpha Aq beta molecules in the deletion of V beta 6+ T cells.     

Synthesis and immunochemical study of the antigenic determinant of the H-2Db molecule of the murine major histocompatibility complex

Primary structure of murine class I histocompatibility antigens has been analysed to select possible antigenic determinant. Hexapeptide Leu-Gln-Gln-Leu-Ser-Gly, homologous to the region 95-100 of the H-2Db antigen heavy chain, was synthesised by stepwise elongation of peptide chain beginning from the COOH-terminal Gly. Rabbit anti-hexapeptide antibodies were obtained and shown to interact specifically with purified H-2Db antigen as well as with the native antigen on cell surface. These antibodies bind to lymphocytes of H-2b haplotype (C57BL/6 mice) but not H-2d (BALB/c) or H-2k (CBA). These data suggest that the region 95-100 is responsible for serologic differences between the alleles of H-2 antigens, i.e. it may be a xenotypic as well as an allotypic antigenic determinant. The latter was confirmed by study of interaction of the hexapeptide with allogeneic monoclonal antibodies specific to H-2Db antigen.     

Insertion of the B2 sequence into intron 13 is the only defect of the H-2k C4 gene which causes low C4 production.

The serum level of the fourth component of complement (C4) in mice bearing H-2k haplotype is only 1/10 of that of non-H-2k mice. H-2k bearing mice, but not non-H-2k bearing mice, have an insertion of the B2 sequence into intron 13 of the C4 gene, and aberrant C4 mRNA in liver apparently generated by abnormal RNA splicing caused by the insertion of the B2 sequence. To test the possible causal relationship between the B2 insertion and low C4 production in H-2k mice directly, we constructed the H-2k C4 gene without the B2 insertion and the H-2w7 (non-H-2k) C4 gene with the B2 insertion by exchanging a part of intron 13 between these two genes. Transfection of the intact H-2w7 C4 gene or the chimeric H-2k gene without the B2 insertion into HepG2 cells resulted in the production of only normal C4 mRNA at the normal level. On the other hand, the intact H-2k C4 gene or the chimeric H-2w7 C4 gene with the B2 insertion directed production of both aberrant and a decreased amount of normal C4 mRNA. These results demonstrated that the insertion of B2 sequence into intron 13 of the C4 gene is the only determinant of low C4 production by H-2k mice through aberrant RNA processing.     

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.     

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

Apparent lack of H-2 restriction of allograft rejection.

BALB/c ByJ (H-2d) mice immunized with tail skin grafts of either B10.D2 (H-2d) or B10 (H-2b) demonstrated similar second set rejection of B10.D2 tail skin. This apparent lack of H-2 restriction was not due to the induction of a new population of cytotoxic T lymphocytes (Tc) since 450R given 24 hr before the challenge graft did not abrogate the second set reactivity. Host macrophage processing or anti-Qa-2 reactivity was also not the explanation for the lack of H-2 restriction since immunization of BALB/c Li mice with either B10.D2 or B10 tail skin grafts resulted in second set rejection of B10 tail skin. Shared public H-2 specificities of H-2d and H-2b may result in cross-reactive Tc, thus causing the apparent lack of H-2 restriction. However, no H-2 restriction of allograft rejection is observed when only one public H-2 specificity is shared between the recipient and the allogeneic challenge graft (H-2f and H-2k combination). These results suggest that H-2 restriction of T cell cytotoxicity has little relevance in allograft rejection because 1) one public H-2 specificity is sufficient to cause cross-reactivity or 2) Tc are not the major effectors of allograft rejection.     

Localization of T cell epitope regions of chicken ovomucoid recognized by mice

We localized the T cell epitope regions of chicken ovomucoid (OVM), a potent egg allergen, with the overlapping pin-peptides covering the entire sequence of OVM and three strains of mice with different haplotypes. In C3H/He (H-2k) mice, the T cells recognized relatively broad regions on OVM; the dominant regions were 49-93 and 97-114 residues, and the subdominant regions were 7-21, 37-48, 94-96, 115-123 and 145-177 residues. In contrast, a more limited number of T cell epitope regions were localized in BALB/c (H-2d) and C57BL/6 (H-2b) mice. The T cells from BALB/c mice recognized 100-114 and 157-171 residues, and the T cells from C57BL/6 mice recognized only 157-180 residues. These results were confirmed by using peptides separately synthesized and purified on the putative epitope regions. The roles of the carbohydrate moieties and cysteine residues involved in the disulfide bridges of OVM were also examined, and we found that they were not important in recognition by the T cell/antigen presenting cell.     

H-2 linked Ir gene control of antibody responses to porcine insulin. I. Development of insulin-specific antibodies in some but not all nonresponder strains injected with proinsulin

Cell-mediated and humoral immune responses to heterologous insulins in mice are controlled by H-2 linked, dominant, immune response (Ir) genes. For example, mice bearing the H-2d haplotype develop T cell proliferative responses and produce antibody after injection with porcine insulin, whereas mice bearing other H-2 haplotypes do not. Data presented in this communication demonstrate that homozygous and heterozygous H-2d mice produce insulin-binding antibodies when immunized with porcine insulin or proinsulin. Some (H-2b,k,s) insulin-nonresponder mice produce insulin-binding antibodies after injection of proinsulin, whereas other insulin-nonresponder strains (H-2q) do not. All strains, except homozygous H-2q mice, produce antibodies specific for proinsulin, suggesting that the response to porcine proinsulin is also controlled by H-2-linked Ir genes. More importantly, F1 hybrids between insulin-nonresponder C57BL/10 (H-2b) and DBA/1 (H-2q) produce no insulin-binding antibodies when injected with proinsulin, despite the fact that proinsulin-binding antibodies are produced by these mice.