Distinct orientation of the alloreactive monoclonal CD8 T cell activation program by three different peptide/MHC complexes

We have characterized three different programs of activation for alloreactive CD8 T cells expressing the BM3.3 TCR, their elicitation depending on the characteristics of the stimulating peptide/MHC complex. The high-affinity interaction between the TCR and the K(b)-associated endogenous peptide pBM1 (INFDFNTI) induced a complete differentiation program into effector cells correlated with sustained ERK activation. The K(bm8) variant elicited a partial activation program with delayed T cell proliferation, poor CTL activity and undetectable ERK phosphorylation; this resulted from a low-avidity interaction of TCR BM3.3 with a newly identified endogenous peptide, pBM8 (SQYYYNSL). Interestingly, mismatched pBM1/K(bm8) complexes induced a split response in BM3.3 T cells, with total reconstitution of T cell proliferation but defective generation of CTL activity that was correlated with strong but shortened ERK phosphorylation. Crystal structures highlight the molecular basis for the higher stability of pBM8/K(bm8) compared to pBM1/K(bm8) complexes that exist in two conformers. This study illustrates the importance of the stability of both peptide/MHC and peptide/MHC-TCR interactions for induction of sustained signaling required to induce optimal CTL effector functions. Subtle allelic structural variations, amplified by peptide selection, may thus orient distinct outcomes of alloreactive TCR-based therapies.     

Serological analysis of B6.C-H-2bm12 (I-A mutant)

The B6.C-H-2bm12 has been examined serologically with a new set of reagents and several complementation studies were performed to determine the extent of the mutation. The results show that: (a) the mutation has also affected the site(s) bound by xenogeneic anti-Ia antibodies; (b) the IJb region was not affected; (c) complementation studies with stains bearing a, b, d, k and s haplotypes did not complement bm12 for the expression of the lost I-Ab specificities, suggesting a structural (rather than regulatory) gene alteration in bm12; (d) H-2 haplotypes b and bm12 could complement d to establish the Ia.22 specificity, indicating that Ia-1 and Ae are separate genes in the I-A subregion. In addition, an antibody to the gained specificity on bm12 is described.     

A structural basis for LCMV immune evasion: subversion of H-2D(b) and H-2K(b) presentation of gp33 revealed by comparative crystal structure.Analyses

LCMV infection of H-2(b) mice generates a CD8(+) CTL response mainly directed toward three immunodominant epitopes. One of these, gp33, is presented by both H-2D(b) and H-2K(b) MHC class I molecules. The virus can escape immune recognition in the context of both these MHC class I molecules through single mutations of the peptide. In order to understand the underlying structural mechanism, we determined the crystal structures of both complexes. The structures reveal that the peptide is presented in two diametrically opposed manners by H-2D(b) and H-2K(b), with residues used as anchor positions in one MHC class I molecule interacting with the TCR in the other. Importantly, the peptide's N-terminal residue p1K protrudes from the binding cleft in H-2K(b). We present structural evidence that explains the functional consequences of single mutations found in escape variants.     

Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2^bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2K^b, H-2D^b, and H-2K^bm1 class I molecules, although the sequence lacks the usual structural K^b and D^b peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2^b) targets as well as the L cell transfectants, L + K^b, L + D^b, and L + K^bm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro⁴⁴ and Thr⁴⁷, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2K^b, H-2D^b, and mutant H-2K^bm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.     

The immunization of A2/K(b) transgenic mice with the KMP11-HSP70 fusion protein induces CTL response against human cells expressing the T. cruzi KMP11 antigen: identification of A2-restricted epitopes

Cytotoxic T lymphocyte response against Jurkat-A2/K(b) cells expressing the T. cruzi KMP11 protein has been evaluated after immunization of C57BL/6-A2/K(b) transgenic mice with the KMP11 and KMP11-HSP70 recombinant proteins. The results show that mice immunized with KMP11 covalently fused to the T. cruzi HSP70 protein, but not mice immunized with KMP11 alone, elicit a CTL response against the Jurkat-A2/K(b) cells expressing the KMP11 protein. The data also show that spleen cells from mice immunized with the fusion protein and stimulated with the K1 peptide induce lysis of both the Jurkat-A2/K(b) cells transfected with the KMP11 coding gene and the Jurkat-A2-K(b) cells pulsed with the K1 peptide. Splenocytes stimulated with the K3 peptide induce lysis of the Jurkat-A2/K(b) cells loaded with the K3 peptide but they do not recognize the target cells expressing the KMP11 protein. Similar results were obtained using lymph node from mice immunized with the peptides. Thus, we believe there are two cytotoxic T cell epitopes restricted to the A2 molecule (K1(KMP11) (4-12) and K3(KMP11) (41-50)) in the KMP11 protein, and suggest that the K1 peptide could be considered an immunodominant antigen whilst the K3 peptide may be regarded as a cryptic epitope. The fact that the CTL lines induced in B6-A2/K(b) mice recognize human cells expressing KMP11 protein, indicates that the KMP11 antigen fused to HSP70 could be a good candidate for the induction of immunoprotective cytotoxic responses against T. cruzi natural infection.     

Threshold tolerance in H-2Kb-specific TCR transgenic mice expressing mutant H-2Kb: conversion of helper-independent to helper-dependent CTL.

To evaluate the role of the structure of the class I molecule and associated peptide(s) in intrathymic selection and tolerance, mice expressing as a transgene (tg) a TCR specific for the H-2Kb alloantigen were crossed with mice expressing the mutant class I molecule H-2Kbm1 or H-2Kbm8. In H-2k/k TCR tg mice (in a situation of exclusive positive selection), peripheral tg TCR expressing (Ti+) CD8+ T cells showed high, suboptimal, and an absence of reactivity for H-2Kb, H-2Kbm1, and H-2Kbm8, respectively. In the peripheral lymphoid organs of TCR tg H-2k/k, H-2k/bm8, H-2k/bm1, and H-2k/b mice respectively, the tg TCR was expressed on T cells with decreasing intensity of surface CD8. Thymic subpopulations of TCR tg mice presented a pattern of negative selection with decreasing intensity from H-2k/b to H-2k/bm1 and H-2k/bm8. This suggests that a weak interaction between the TCR and H-2Kbm8 exists which partially results in negative, but not in positive, intrathymic selection. Results further indicate that expression of H-2Kbm8 does not induce tolerance to H-2Kb. In H-2k/bm1 mice, the peripheral Ti+ CD8lo cells express two distinct types of 'threshold' tolerance in vitro: (i) they generate cytotoxic T lymphocytes (CTL), in the presence of exogenous IL-2, which fail to respond to H-2Kbm1 but remain reactive to H-2Kb; and (ii) they do not make significant titers of IL-2 and do not significantly proliferate in response to H-2Kb, unlike the Ti+ CD8+ T cells from H-2k/k TCR tg mice which respond efficiently. These results show that tolerance is induced up to a level of non-reactivity within a given MHC environment: for the same TCR, CTL reactivity to H-2Kbm1 is totally lost, whereas CTL reactivity to H-2Kb is only slightly reduced. Additionally, proliferation and IL-2 production by Ti+ CD8+ cells in response to H-2Kb were strongly affected in H-2k/bm1 mice. Thus, in H-2k/k mice the Ti+ CD8+ cells behave as helper-independent, whereas in H-2k/bm1 mice CD8+ cells expressing the same TCR behave as helper-dependent CTL.     

Immunogenicity of the Mutated H-2Kbm1 Antigen(s). Test of Thyroid Graft Rejection between B6.C-H-2bm1 and C57BL/6 Mice Following Reciprocal Immunization with Normal Versus Malignant Cells

The immunogenic properties of one (or few) selected antigen(s) encoded by the mouse major histocompatibility complex was studied using the C57BL/6(B6) mouse strain and its descendant B6.C-H-2 6m1(bm1) mutant. These strains differ in a point mutation in the H-2K region. We compared the immunogenic and antigenic expression of the mutated antigen on different bm1 tissues by testing the vulnerability of these tissues to graft rejection response in B6 recipients. Previous results demonstrated that B6 and bm1 mice do not reject reciprocal thyroid transplants, despite the acute rejection of reciprocal skin grafts. Thyroid grafts were rejected, however, after presensitizing the recipients with skin graft syngeneic with the thyroid, but not after sensitization with spleen cells. In the present work we induced tumors in bm1 mice by treating them with a chemical carcinogen (3-methylcholanthrene). We found that two out of four tumors demonstrated strict strain specificity and were rejected by all mouse strains (including the B6 recipients) except by their strain of origin. All tumors were found to be sensitive to in vitro lysis by B6 anti-bm1 effector cells. HZ1-A and HZ1-B tumor cells were rejected by B6 recipient mice but could not immunize B6 mice against a subsequent bm1 thyroid graft. When testing the immunogenicity of B6 originated EL4 leukemia cells (which are fatal to B6 mice), we found that the tumor cells were rejected by bm1 recipients, but, unlike B6 skin grafts, were incapable of inducing the rejection of a subsequent B6 thyroid transplant. The results demonstrated that an H-2K molecule may exhibit different immunological properties when expressed on cells of different tissues. The different expression of the mutated antigen on different cell types, its ability to trigger T cells but not B cells responses and the potential involvement of the tissue specific differentiation molecules in the graft rejection response are discussed.     

Enhancement of MHC class I-stimulated alloresponses by TNF/TNF receptor (TNFR)1 interactions and of MHC class II-stimulated alloresponses by TNF/TNFR2 interactions

In vivo TNF inhibition has been observed to ameliorate the disease process attributed to T cell-dependent immune responses such as those generated during graft-vs.-host disease. The present studies were designed to evaluate whether TNF/TNF receptor (TNFR)1 and TNF/TNFR2 interactions were involved in the generation of allospecific T cell responses. Splenic lymphocyte populations were obtained from TNFR1- or TNFR2-deficient B6 mice and from control B6 mice. These responder cells were cultured with irradiated MHC class II-disparate B6.C-H-2bm12 (bm12) or MHC class I-disparate B6.C-H-2bm1 (bm1) or irradiated syngeneic stimulator cells for 3 days before assay of [3H]thymidine incorporation. IL-2 levels of the mixed lymphocyte culture (MLC) supernatants were assessed by enzyme-linked immunosorbent assay. With MHC class II-disparate bm12 stimulator cells, a significant reduction in T cell proliferation was observed utilizing TNFR2-deficient CD4+ responder T cells, but not when using TNFR1 -deficient CD4+ responder T cells. A significant decrease in proliferation of TNFR1-deficient CD8+ responder cells, but not of TNFR2-deficient CD8 responder T cells was observed after stimulation with MHC class I-disparate bm1 stimulator cells. IL-2 levels were lower in MLC utilizing MHC class I stimulators and TNFR1-deficient responders or MHC class II stimulators and TNFR2-deficient responders. These results indicate that TNF/TNFR2 interactions promote MHC class II-stimulated alloresponses, while TNF/TNFR1 interactions promote MHC class I-stimulated alloresponses.     

Breaking tolerance in hepatitis B surface antigen (HBsAg) transgenic mice by vaccination with cross-reactive, natural HBsAg variants

Processing exogenous hepatitis B surface antigen (HBsAg) of the hepatitis B virus (HBV) generates the K(b)-binding S(208-215) epitope 1; processing endogenous HBsAg generates the K(b)-binding S(190-197) epitope 2. Cross-reactive CD8(+) T cell responses were primed to epitope 1 but not epitope 2 when mice were immunized with natural HBsAg(ayw), or HBsAg(adw2) variants differing within both epitopes by one or two residues. Expression of HBsAg(ayw) from a transgene in the liver renders (HBs-tg) mice tolerant to epitope 1 of HBsAg(ayw). CD8(+) T cells specific for epitope 1 could be primed in HBs-tg mice by HBsAg(adw2); these specific CD8(+) T cells cross-reacted with epitope 1 processed from the transgene-encoded HBsAg(ayw). The liver of vaccinated HBsAg(ayw) transgenic mice showed severe histopathology and contained functional (IFNgamma-producing), cross-reactive CD8(+) T cells, and vaccinated HBs-tg mice showed reduced antigenemia. Hence, vaccination with natural HBsAg variants from different HBV sero/genotypes can prime cross-reactive, specific CD8(+) T cell immunity that breaks tolerance to HBsAg.     

The detection of public Ia antigens on the surface of B6-C.H-2bm12 blast cells

The B6-C.H-2bm12 (bm12) strain has previously been described as a spontaneously occurring mutant wherein the parental (C57BL/6 Iab specificities (Ia.3, 8, 9, 15 and 20) are absent or reduced in amount. To further characterize the bm12 mutant the antigenic phenotype of LPS-activated bm12 blast cells was determined with monoclonal and conventional anti-Ia antibodies. By direct testing and absorption the public Ia.8, 9 and 15 specificities were readily detected on both parental C57BL/6 and mutant bm12 blast cells, and the amounts of these antigens on activated bm12 cells were similar to or less than those found on C57BL/6 blast cells. The findings demonstrate that the Ia-I mutation has not resulted in an absolute loss of these specificities, but has altered their expression, or their accessibility to antibody on resting cells. By contrast, the private Ia. 20 specificity and another specificity (designated Iab) could not be detected on bm12 blast cells.     

A yeast display system for engineering functional peptide-MHC complexes

In a cellular immune response, antigenic peptides derived by intracellular processing of foreign pathogens are bound to the class I major histocompatability complex (MHC I) and presented to CD8(+) cytotoxic T cells. Although the crystal structures of several different MHC products have been solved, many MHC molecules, including some associated with diseases, have not been amenable to biochemical and structural studies. The variability in this success is based largely on the fact that peptide-MHC complexes vary extensively in their stability. These properties also are intimately tied to the biological activity of the complexes. The ability to apply the techniques of directed evolution to this system in order to engineer stable complexes has been complicated by the trimeric structure of peptide-MHC complexes, requiring association of three polypeptides: the heavy chain, beta2-microglubulin (beta2m), and a short peptide. We show here that single-chain forms of peptide-MHC complexes can be expressed as Aga-2 fusions on the surface of yeast. Three different complexes, SIYRYYGL-K(b)-beta2m (SIYR-K(b)), EQYKFYSV-K(b)-beta2m (dEV8-K(b)), and SIINFEKL-K(b)-beta2m (OVA-K(b)), were expressed on yeast and detected by flow cytometry with a conformation-specific anti-K(b) antibody (B.8.24.3). In addition, yeast displaying K(b) loaded with exogenous SIYR and OVA peptides were recognized by a high-affinity T cell receptor that is specific for SIYR-K(b) and by an antibody (25.D1-16) that is specific for OVA-K(b), respectively. Finally, yeast that display the SIYRYYGL-K(b) also directly stimulated CD69 up-regulation on naive 2C T cells. Hence, yeast display represents a technology that can be used for directed evolution of any of the components of the trimeric pep-MHC complex.     

The assembly of functional beta(2)-microglobulin-free MHC class I molecules that interact with peptides and CD8(+) T lymphocytes

Functional MHC class I molecules are expressed on the cell surface in the absence of beta(2)-microglobulin (beta(2)m) light chain that can interact with CD8(+) T lymphocytes. Whether their assembly requires peptide binding and whether their recognition by CD8(+) T lymphocytes involves the presentation of peptide epitopes remains unknown. We show that beta(2)m-free H-2D(b) assembles with short peptides that are approximately 9 amino acid residues in length, akin to ligands associated with completely assembled beta(2)m(+) H-2D(b). Remarkably, a subset of the peptides associated with the beta(2)m-free H-2D(b) has an altered anchor motif. However, they also include peptides that contain a beta(2)m(+)H-2D(b) binding anchor motif. Further, the H-2K(b)- and H-2D(b)-restricted peptide epitopes derived from SV-40 T antigen also assemble with H-2(b) class I in beta(2)m-deficient cells and are recognized by epitope-specific CD8(+) T lymphocytes. Taken together our data reveal that functional MHC class I molecules assemble in the absence of beta(2)m with peptides and form CD8(+) T lymphocyte epitopes.     

Characterization of Bombyx mori nucleopolyhedrovirus with a deletion of bm118

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF118 (bm118) is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF142, one of the core genes existing in all baculovirus genomes sequenced to date, suggesting that Bm118 plays a critical role in viral infection. In this study, the primary role of Bm118 was investigated by using homologous recombination in Escherichia coli to generate a bm118 knockout bacmid containing the BmNPV genome. In addition, the bm118 rescue bacmid was constructed by transposing a bm118 gene cassette into the polh locus of the bm118 knockout bacmid. Transfection assays demonstrated that the bm118 knockout bacmid was incapable of producing budded virion (BV). Nevertheless, this defect could be partially recovered by a rescue bacmid. Electron microscopy analysis revealed that the bm118 knockout produced aberrant capsids characterized by translucent, elongated nucleocapsids present as bundles within the nuclei. This construct also produced polyhedra lacking virions. These results reveal that Bm118 is essential for BV production and nucelocapsid maturation.     

Ductal lesions of exocrine glands and insulitis induced by L3T4+ T cells following graft-versus-host reaction due to major histocompatibility complex class II disparity

Recently, we have demonstrated characteristic hepatic lesions resembling primary biliary cirrhosis (PBC) in semiallogeneic F1 hybrid mice with major histocompatibility complex (MHC) class II-disparate graft-versus-host reaction (GVHR). In the present study, we tried to reveal other ductal lesions in extrahepatic organs, including salivary glands and pancreas. Murine strains used are C57BL/6 (B6), B6 mutant bm1, and bm12. bm1 carries a mutant gene at the H-2K locus of MHC and bm12 carries a mutant gene at the I-A locus of MHC of the B6 strain. The (B6 x bm1)F1, (B6 x bm12)F1, and (bm1 x bm12)F1 mice were injected intravenously with 1 x 10(7) B6 L3T4+ or Lyt-2+ T cells and were sacrificed on the 14th day postinjection for histological examinations. Mononuclear cell infiltration was detected around the ducts of salivary glands only in (B6 x bm12)F1 mice injected with B6 L3T4+ T cells. A moderate to marked level of cell infiltration was demonstrated in pancreas of (B6 x bm12)F1 recipients similar to nonobese diabetic (NOD) mice. By immunohistochemical examinations, infiltrating cells were shown to consist not only of L3T4+ but also of Lyt-2+ T cells, even after the inoculation of L3T4+ cells. These results are discussed in reference to mechanisms of organ-specific autoimmune diseases, especially insulitis in NOD mice which show insulin-dependent diabetes mellitus.     

Differential genetic requirements for in vivo and in vitro induction of T-killer and T-suppressor cells in the mutant H-2Kb system and the cross-reactivity of the T-killer clones

Differences in the generation of anti-H-2Kb wild-type specific cytotoxic T lymphocytes (CTL) and specific suppressor T cells (SSTC) were investigated in H-2Kbm mutant mouse strains. To this end, optimal conditions for in vivo induction of highly active CTL in this mutant system were found and the T-cell origin of CTL and SSTC was confirmed using the anti-Thy-1.2 monoclonal antibody G4. Unlike the CTL, which were generated in vivo by any of the mutant strains tested (bm1, bm3, and bm4), the SSTC were only produced by bm3, whose H-2Kbm3 antigen, in contrast to the other H-2Kbm molecules, differs from wild type by serologically defined determinants. The high activity of anti-wild type bm4 CTL induced in vivo contrasted with a low activity of such CTL induced in mixed lymphocyte culture (MLC). This appeared to be the property of bm4 only, but not of bm1 or bm3, and it was reproduced in the reciprocal system B10 anti-bm4. CTL generation could be restored in the MLC by the addition of concanavalin A supernate or a mixture of bm4 and bm12 stimulator cells. Three of the six in vivo induced and in vitro propagated bm3 anti-B6 CTL clones demonstrated selective cross-reactivity to only one of the third-party H-2K molecules used, either Kk, Kd, or Kbm4. The present results indicate that (a) the SSTC and antibody recognize similar H-2Kb epitopes; (b) the H-2Kb epitopes recognized by the CTL and SSTC are not identical; (c) the genetic control of CTL generation in vivo is distinct from that in MLC, and (d) the affinities of antigen-specific receptors on T-cell clones of the same specificity may be different, leading to their individual cross-reactivity patterns.     

Characterization of a dominant anti-Ia idiotype using the IA mutant mouse strain B6.C-H-2bm12

Initial studies of antibody recognition of Ia molecules using the IA mutant mouse strain bm12 suggested that two anti-Ia monoclonal antibodies (mAbs), 25-9-17 and 34-5-3, share several features: (1) indistinguishable serologic specificity including a lack of reactivity with Iabm12, (2) binding of the same spatial epitope (cluster), and (3) definition of a cross-reactive idiotype (CRI) as defined by xenogeneic antisera. In the present study we characterize a rabbit anti-idiotype (anti-Id) to 25-9-17 by affinity chromatography, and demonstrate that it detects at least two distinct idiotopes, one shared by 25-9-17 and 34-5-3 designated CRI (25-9-17) and one unique for 25-9-17 molecules. Experiments were also undertaken to determine whether CRI (25-9-17) represents a measurable component of allogeneic humoral responses to Iab antigens. By both absorption analyses of a polyspecific antiserum and production of antigenically-restricted antisera using bm12 mice, CRI (25-9-17) was found to represent a significant proportion of the antibodies to Iab. By several criteria it was shown that the CRI (25-9-17)+ molecules were among the antibodies defining the serologic lesion of bm12 mice. In preparation for future studies to alter in vivo T-cell responses involving recognition of Ia (e.g. graft vs host disease and allogeneic transplant rejection), various immunization protocols and mouse strains were tested for induction of Id (25-9-17) following in vivo administration of various anti-idiotypic reagents. Rabbit anti-Id (25-9-17) successfully induced CRI (25-9-17) positive molecules in all strains tested regardless of IA or Ig genotype. Moreover, some of these treated mice produced antibodies to an Ia determinant missing on bm12 cells, suggesting that they recognize the same serologic determinant as mAb 25-9-17.     

Recognition of H-2K(b) by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function

Ly49Q is a member of the polymorphic Ly49 family of NK cell receptors that displays both a high degree of conservation and a unique expression pattern restricted to myeloid lineage cells, including plasmacytoid dendritic cells (pDC). The function and ligand specificity of Ly49Q are unknown. Here, we use reporter cell analysis to demonstrate that a high-affinity ligand for Ly49Q is present on H-2(b), but not H-2(d), H-2(k), H-2(q), or H-2(a)-derived tumor cells and normal cells ex vivo. The ligand is peptide-dependent and MHC Ia-like, as revealed by its functional absence on cells deficient in TAP-1, beta(2)m, or H-2K(b)D(b) expression. Furthermore, Ly49Q is specific for H-2K(b), as the receptor binds peptide-loaded H-2K(b) but not H-2D(b) complexes, and Ly49Q recognition can be blocked using anti-K(b) but not anti-D(b) mAb. Greater soluble H-2K(b) binding to ligand-deficient pDC also suggests cis interactions of Ly49Q and H-2K(b). These results demonstrate that Ly49Q efficiently binds H-2K(b) ligand, and suggest that pDC function, like that of NK cells, is regulated by classical MHC Ia molecules. MHC recognition capability by pDC has important implications for the role of this cell type during innate immune responses.     

Immune recognition of insulin by H-2b mice: the mutation in the I-A gene of the B6.C-H-2bm12 mouse alters the self-I-A-restricted T cell repertoire

The response to heterologous insulin in H-2^b mice is restricted to the A chain loop determinant(s) of beef insulin. The recognition of this specificity requires the expression of the immune response (Ir) gene epitope Ia. W39 which is absent from the I-A^b mutant B6.C-H-2^bm12 (bm12) mice. This restriction could reflect the inability of H-2^b antigen-presenting cells (APC) to present other insulin determinants or may reflect “self-major histocompatibility complex”-dependent influences on the generation of the T cell repertoire. To assess these possibilities we analyzed the genetic control and fine specificity of the insulin-specific T cell repertoire of H-2^b mice by fusing the AKR thymoma BW5147 with T cells of C57BL/6 mice which had been immunized in vivo and challenged in vitro with beef insulin. The cloned hybridomas that we have produced respond to APC either alone or in conjunction with insulin by the production of interleukin 2. The insulin-specific hybridomas vary in their fine specificity such that some clones recognize a determinant(s) shared by beef, sheep and pork insulin and the isolated B chain, while other clones recognize a determinant(s) shared by beef and sheep insulin only, likely to involve amino acids 8 and/or 10 of the A chain loop. The presentation of insulin to these hybridomas is restricted by I-A^b, but not by Ia.W39. This analysis revealed that the insulin-specific immune potential in H-2^b mice is of greater scope than previously defined and led us to consider, whether insulin nonresponder bm12 mice also possess a latent insulin-specific immune potential. Our study of the insulin-specific immune recognition by bm12 mice shows that these nonresponders do possess insulin-specific T cell clones. Despite the fact that the I-A^b and I-A^bm12 gene products differ only by 3 amino acids, insulin-specific C57BL/6 and bm12 hybridomas are restricted to recognize exogenous antigen only in the context of C57BL/6 and bm12 APC, respectively. Furthermore, upon direct analysis of autoreactive subclones, a similar although not complete, restriction was observed. The implications of these findings for understanding the mechanism of Ir gene control are discussed.     

Promiscuous T cell recognition of an H-2 IA-presented mycobacterial epitope

Genetically permissive T cell epitopes are an important prerequisite for the development of peptide-based vaccines or immunodiagnostic reagents. We have investigated the structural requirements of permissive T cell recognition of peptide p350—369 from the 38-kDa antigen of Mycobacterium tuberculosis. This peptide was found to be immunogenic in mice of the H-2^{b, bm12, d. s and k}, but not of the H-2^f genotype. T cell responses were restricted by I-A class II molecules. The same epitope core was recognized in the H-2^{b, d and k} genotypes. T cell hybrids from BALB/c and C57BL/10 mice were used to determine: (i) the critical residues using substituted peptide derivatives and (ii) the degree of T cell promiscuity. Two out of five BALB (H-2^d)-derived hybridomas tested displayed promiscuous peptide recognition in the context of H-2^b and H-2^bm12 antigen-presenting cells. The recognition of critical residues was found to be uniform for all five hybridomas when tested with syngeneic antigen-presenting cells; additional critical residues were identified when the peptide was recognized in the context of allogeneic antigen-presenting cells. Only one of the four tested C57BL/10 (H-2^b) hybridomas showed promiscuity in the context of H-2^bm12. Each of these C57BL/10-derived clones had a distinct response profile toward the critical residues. We propose that the demonstrated T cell promiscuity involves peptide interaction with polymorphic H-2 I-A residues.     

Thymic immune response gene function in radiation chimeras reconstituted with purified hemopoietic stem cells

Thymectomized (C57BL/6[B6] X bm1)F1 mice and thymectomized (B6 X bm12)F1 mice were engrafted with neonatal parental thymus of either B6 type [H-2b mouse, Sendai virus cytotoxic T cell (Tc) responder] or bm1 type (H-2Kb mutant, Sendai virus Tc nonresponder) and B6 type (H-Y Tc responder) or bm12 type (H-2 I-Ab mutant, H-Y Tc nonresponder), respectively. All mice were irradiated and reconstituted with highly purified syngeneic pluripotent hemopoietic stem cells. All types of thymus engraftment resulted in a restored T cell immunocompetence. The Tc reaction to Sendai virus in (B6 X bm1)F1 mice engrafted with both responder type B6 and nonresponder, type bm1 neonatal thymus allowed maturation of Sendai-specific, H-2Kb-restricted Tc. For the Tc reaction to H-Y, only responder type B6 thymus restored the Tc response, whereas this was not achieved with nonresponder type bm12 thymuses. We conclude from this study that in this radiation stem cell chimera system the radioresistant component of the thymus dictates major histocompatibility complex (MHC) specificity and immune response phenotype of T cells restricted to class II MHC molecules but not of T cells restricted to class I MHC molecules.