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.     

Th1 clones that suppress IgG2ab specifically recognize an allopeptide determinant comprising residues 435–451 of γ2ab

We have previously reported that γ2a^b/I-A^d-specific Th1 clones from BALB/c mice (γ2a^a, H-2^d) mediated a long-lasting, selective suppression of serum IgG2a^b levels when transferred to newborn (BALB/c X B10.D2)F₁ (γ2^a/b, H-2^d) mice (Bartnes, K. and Hannestad, K. Eur. J. Immunol. 1991. 21: 2365). We here analyze the peptide specificity of hybridomas derived from two suppressive T cell clones. The shortest synthetic peptide with optimal antigenicity comprises γ2a^b residues 435–451 (Kabat numbering). The determinant core encompasses the γ2a^b 440–446 (KLRVQKS) sequence which contains an I-A^d allele-specific motif. Challenge with single amino acid-substituted γ2a^b 435–447 analogs revealed that residues K⁴⁴⁰, R⁴⁴² and K⁴⁴⁵ which are shared by the autologous and allogeneic γ2a, as well as residues Q⁴⁴⁴ and S⁴⁴⁶ which represent allogeneic differences, are critical for recognition. We obtained evidence that K⁴⁴⁰, R⁴⁴² and Q⁴⁴⁴ are epitope residues, while K⁴⁴⁵ and S⁴⁴⁶ contribute to anchoring of the peptide to I-A^d. Amino acids located outside of the core also influence antigenicity, the most striking effect being a 340–870-fold augmentation of potency when γ2a^b 437–451 is extended by F⁴³⁶. IgG2a^b required processing in order to stimulate the hybridomas. The data support the contention that the Th1 clones specific for Fc of γ2a^b mediated IgG2a^b suppression by cognate interaction with sIgG2a^b+ B cells that presented a Cγ2a^b peptide(s) derived from their endogenous Ig on major histocompatibility complex class II. The T cells cross-reacted weakly with peptide 435–451 of the autologous γ2a^a allotype. This opens the possibility that self-peptides from Ig C regions can target B cells for regulatory interactions with autologous Th cells.     

Vβ11+ T-lymphocyte expansion by toxic shock syndrome toxin-1 differs in mice bearing H-2q versus H-2b haplotypes

We have recently demonstrated that toxic shock syndrome toxin-1 (TSST-1) expanded Vβ11⁺ T lymphocytes contribute to Staphylococcus aureus arthritis and sepsis-induced mortality. Interestingly, Vβ11⁺ T-cell mediated joint pathology varies in different mouse strains. In this study, we characterized the in vitro pattern of Vβ11⁺ T-cell expansion by TSST-1 in mice with various genetic backgrounds. Mice expressing major histocompatibility complex (MHC) class II I-E molecules did not expand Vβ11⁺ T cells upon stimulation with TSST-1. Using B10 congeneic I-E negative mouse strains, we found that the TSST-1-expanded Vβ11⁺ T cells in B10Q (H-2^q) and B10M (H-2^f) mice but not in B10B (H-2^b) mice. Antigen-presenting cells (APC) from B10Q mice, L cells and lymphoma cell line transfected with a q gene did not restore the deficient Vβ11⁺ T-cell expansion by TSST-1 in purified T cells from B10B mice. In contrast, I-A^b APC were able to stimulate Vβ11⁺ T cells from H-2^q mice. Furthermore, Vβ11⁺ T cells in H-2^b mice did expand when exposed to staphylococcal enterotoxin A (SEA). These findings suggest that the T-cell repertoire, skewed by clonal deletion and inactivation of self-reactive T cells, accounts for the different magnitude of Vβ11⁺ T-cell expansion among the different mouse strains.     

Differential expression of CLIP: MHC class II and conventional endogenous peptide: MHC class II complexes by thymic epithelial cells and peripheral antigen-presenting cells

Major histocompatibility complex (MHC) class II molecules expressed by thymic epithelial cells are involved in positive selection of CD4 T cells, whereas the high-avidity interaction of T cell receptors with the endogenous peptide : MHC class II complexes expressed on bone marrow (BM)-derived antigen-presenting cells (APC) and, to a lesser extent, on thymic epithelial cells mediate negative selection. To understand better the generation of the CD4 T cell repertoire both in the thymus and in the periphery we analyzed relative levels of expression of specific endogenous peptide: MHC class II complexes in thymic epithelial cells (TEC) and peripheral APC. Expression of Eα52–68: I-A^b and class II-associated invariant chain peptide (CLIP): I-A^b complexes in thymic epithelial cells and in the bone-marrow derived splenic APC, i.e. B cells, was studied using YAe and 30-2 monoclonal antibodies which are specific for the corresponding complexes. To distinguish between expression of both complexes in radioresistant thymic epithelial elements and radiation sensitive BM-derived APC, radiation BM chimeras were constructed. Using immunohistochemical and immunochemical approaches we demonstrated that the level of expression of Eα52–68:I-A^b complexes in thymic epithelial cells is approximately 5–10 % of that seen in splenic cells whereas total class II levels were comparable. In contrast, CLIP: I-A^b complexes are expressed at substantially higher levels in TEC vs. splenic APC. This result demonstrates quantitative differences in expression of distinct peptide: MHC class II complexes in thymic epithelial cells and peripheral splenic APC.     

Altered Th1/Th2 balance associated with the immunosuppressive/protective effect of the H-2Ab allele on the response to allo-4-hydroxyphenylpyruvate dioxygenase

The H-2A^b allele exerts a dominant down-regulatory effect on the anti-allo-HPPD (4-hydroxyphenylpyruvate dioxygenase) antibody response, through a hitherto unknown mechanism. In the present study, the allo-variable peptide bound to responder H-2A^k molecules with higher affinity than to H-2A^b ones, arguing against the operation of an affinity hierarchy. Quantitative polymerase chain reaction revealed differences in cytokine mRNA expression between suppressed and high-responder mice. Lymph node cells of responder but not suppressed mice contained high levels of interleukin (IL)-4 mRNA as early as 11 h post-immunization and continued to do so for at least 8 days; this early burst was paralleled by a small burst in transforming growth factor (TGF)-β mRNA level. Differences in IL-12 mRNA were not detected, although an early IL-12 effect could not be excluded. Interferon (IFN)-γ appeared to contribute to the suppression at later time points. Early treatment of responder mice with anti-IL-4 monoclonal antibody (11B11) down-regulated the antibody response. The proliferative T cell response from hyperimmunized mice was reduced but still detectable in the presence of an H-2A^b allele. Thus, in the presence of this allele, the Th1 response is enhanced and that of Th2 cells suppressed, apparently as a result of the bias of H-2A^b-restricted T cells in favor of the Th1 subset.     

N-terminal elongation of a peptide determinant beyond the first primary anchor improves binding to H-2 I-Ad and HLA-DR1 by backbone-dependent and aromatic side chain-dependent interactions,respectively

The IgG2a^b heavy chain allopeptide determinant γ2a^b 436 – 451 (Kabat numbering) presented by the major histocompatibility complex (MHC) class II molecule I-A^d is recognized by T cells which cross-react with a corneal self antigen and with the UL6 protein of the herpes simplex virus which induce autoimmune keratitis, and is the target of Th1 clones that suppress IgG2a^b production in vivo. In the γ2a^b peptide/I-A^d complex, tyrosine⁴³⁸ is the first primary anchor (P1) and residues 440 – 445 encompass the T cell receptor contact residues. Amino-terminal elongation of γ2a^b 437– 451 by a single residue (P-2) augmented the I-A^d binding capacity 10-fold and the antigenicity 55 –195-fold. This was a function of the peptide main chain, since non-conservative substitutions were accepted. The γ2a^b peptide also bound HLA-DR1, and amino-terminal extension by a single aromatic amino acid at P-3 augmented binding 15-fold. The interaction between HLA-DR1 and P-3 specifically required an aromatic peptide side chain, and computer simulations indicated that the aromatic ring at P-3 engaged conserved HLA-DR1 phenylalanine residues at the edge of the peptide binding groove. Thus, these data demonstrate that residues amino terminal to P1 may substantially increase peptide affinity for MHC class II by main chain-dependent as well as side chain-dependent interactions, and imply that the HLA-DR1 motif should be extended to include an aromatic amino acid at P-3.     

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.     

Mercury-induced autoimmunity in the absence of IL-4

In susceptible H-2^s mice, mercuric chloride (HgCl₂) induces an autoimmune syndrome characterized by production of anti-nucleolar antibodies (ANoA) and increased serum levels of IgG1 and IgE antibodies. The increase in serum IgG1 and IgE, which are under IL-4 control, suggests a role for the Th2 subset in the induction of this syndrome. We have previously shown that administration of IL-12, a potent Th1-promoting cytokine, resulted in a dramatic reduction of the HgCl₂-induced anti-nucleolar antibody titres and inhibited serum IgG1 increase. These results suggest that Th1 T cells can down-regulate ANoA, and support a role for the Th2 subset in ANoA production, possibly via IL-4. To examine the role of IL-4 in this syndrome, C57Bl/6 mice (H-2^b) with a targeted deletion of the IL-4 gene were mated with A.SW mice (H-2^s) to yield H-2^s mice lacking IL-4. We then analysed ANoA and serum immunoglobulin levels in these mice after HgCl₂ treatment. While mercury-treated IL-4^−/− H-2^s mice had virtually no detectable serum IgG1 or IgE, and very low levels of IgG1 ANoA, these mice had levels of IgG2a and IgG2b class ANoA comparable to mercury-treated IL-4⁺ H-2^s mice, indicating that IL-4 is not required for the ANoA response in mercury-induced autoimmunity.     

Restricted expression of transgenic HLA-DRA gene in thymic epithelial cells and its role in acquisition of T cell tolerance to self-superantigens and processed DRα-derived peptide

We have established a set of transgenic mouse lines in which the HLA-DRA gene was expressed in different cell types. In one line (DRα-24), DRαEβ^b molecules were expressed on thymic medullary and cortical epithelial cells and all lineages of bone marrow-derived antigen-presenting cells (APC) except for thymic macrophages. By contrast, expression of the molecules in another line (DRα-30) was found on thymic medullary and cortical epithelial cells but not on bone marrow-derived APC in the thymus and periphery. To evaluate the role of thymic epithelial cells in acquisition of T cell tolerance, comparative analysis of DRα-24 and DRα-30 was performed. In DRα-30, T cells expressing TcR Vβ5 and Vβ11 were eliminated to comparable levels to those in DRα-24, suggesting that expression of the DRαEβ^b molecules on thymic epithelial cells are sufficient for clonal deletion of the self-superantigen-reactive T cells. In addition, CD4⁺ T cells from DRa-30 as well as those from DRα-24 were tolerant to DRα-derived peptide/I-A^b complex expressed on spleen cells from DRα-24 even in the presence of exogenous interleukin-2. These observations suggest that expression of the DRα chain in thymic epithelial cells could induce T cell tolerance directed toward naturally processed DRα-derived peptide bound to I-A^b molecules, probably via clonal deletion of the self-reactive T cells.     

Gene complementation in the T lymphocyte proliferative response to poly(Glu56Lys35Phe9)nFunctional evidence for a restriction element coded for by both the I-A and I-E subregions

The T lymphocyte proliferative response to poly(Glu⁵⁶Lys³⁵Phe⁹)_n (GLΦ) is under the control of two immune response genes, Ir-GLΦ-β and Ir-GLΦ-α, mapping in the I-A and I-E/C subregions of the major histocompatibility complex, respectively. Previous studies have demonstrated that in order to generate a response to GLΦ, both gene products must be expressed in the antigen-presenting cell (APC) but that neither responder allele has to be present in the responding T lymphocyte, provided that the T cell has matured in a responder environment. These results suggested that both gene products function as restricting elements in GLΦ presentation by APC. In this report, we provide further evidence for this model from experiments designed to test histocompatibility restrictions in antigen presentation at the I-E/C subregion. Genetic identity at the I-A subregion between T cells and APC was required for GLΦ presentation. To assess the requirements at I-E/C, B10. A(5R) T cells (I-A^b, I-E^k) primed to GLΦ were stimulated in vitro with GLΦ-pulsed spleen cells from F₁ hybrids between C57BL/10 (B10: I-A^b, I-E^b) which made the cells compatible at I-A, and a variety of B10 congenics bearing other H-2 haplotypes. Although none of the parental spleen cells could present GLΦ to B10.A(5R) T cells, spleen cells from F₁ hybrids between B10 and strains possessing H-2I of k, d, p and r presented GLΦ, whereas hybrids with strains possessing H-2I of f, q and s failed to present. This pattern of complementation for GLΦ presentation could not be explained on the basis of the responder status of the I-E/C donating parental haplotypes nor by invoking inhibitory stimuli from mixed lymphocyte reactions induced by the F_l APC. Rather, the pattern correlated with the presence of the serologic marker Ia.7 coded for by the I-E subregion of the complementing parental haplotype and the possession of an I-E-encoded a chain which has been shown by peptide mapping to be very similar in strains bearing the k, d, p and r haplotypes. These results suggest that the restriction element involved in the presentation of GLΦ to B10.A(5R) T cells is composed of a β chain encoded in I-A^b and an a chain encoded in I-E for which the allelic products of the k, d, p and r haplotypes are functionally equivalent. This correlation between structure and function represents the strongest evidence so far that Ia antigen-bearing molecules are the Ir gene products.     

Schistosoma mansoni Soluble Egg Antigens Enhance T Cell Responses to a Newly Identified HIV-1 Gag H-2b Epitope

Schistosome infection induces significant T helper type 2 (Th2) and anti-inflammatory immune responses and has been shown to negatively impact vaccine efficacy. Our goal was to determine if the administration of schistosome soluble egg antigens (SEA) would negatively influence the induction of cytotoxic T lymphocyte (CTL) and Th1-type T cell responses to an HIV candidate vaccine in the Th1-biased C57BL/6 mouse strain. Initial experiments failed, as we were unable to detect any response to the defined class I epitope for HIV-1 IIIB Gag. Therefore, we initiated an epitope mapping study to identify C57BL/6 (H-2^b) T cell epitopes in HIV-1 IIIB Gag in order to perform the experiments. This analysis defined two previously unreported minimal class I H-2^b and class II I-A^b epitopes for HIV-1 IIIB Gag. The newly defined HIV-1 IIIB Gag epitopes were used to evaluate the influence of SEA on the generation of CTL and Th1-type HIV-1 IIIB Gag responses. Surprisingly, in contrast to our hypothesis, we observed that the coadministration of SEA with a Listeria monocytogenes vector expressing HIV-1 IIIB Gag (Lm-Gag) led to a significantly increased frequency of gamma interferon (IFN-γ)-producing CD8⁺ and CD4⁺ T cells in C57BL/6 mice compared to mice immunized with Lm-Gag only. These observations suggest that SEA contains, in addition to Th2-type and immune-suppressive molecules, substances that can act with the Lm-Gag vaccine to increase CTL and Th1-type vaccine-specific immune responses.     

Determination of amino acids on agretopes of pigeon cytochrome c-related peptides specifically bound to I-A allelic products

In our prior study it was demonstrated that residues 46 and 54 on a synthetic peptide, AEGFSYTVANKNKGIT (50V), work as an agretope (site contacts with major histocompatibility complex molecules) and residues 50 and 52 function as an epitope (site contacts with T cell receptor), when tri-molecular complexes are formed among 50V, I-A^b and the T cell receptor. 50V was composed of residues 43 to 58 of pigeon cytochrome c (p43--58) except that the aspartic acid (D) at residue 50 was substituted by valine (V). Substitution of agretopic residues on 50V changed this I-A^b-binding peptide to an I-A^k-binding peptide, suggesting that positions 46 and 54 work as an agretope in I-A^k-restricted T cell responses. In the present study we examined whether residues 46 and 54 of 50V worked as agretopes in T cell responses restricted to other I-A haplotypes. The 50V-related peptides with phenylalanine (F) at position 46 and alanine (A) at position 54 bound tightly to I-A^b, I-A^d, I-A^q and I-A^s molecules and stimulated T cells most potently in mice bearing these I-A haplotypes. In contrast, 50V-related peptides carrying D at position 46 and A at position 54 bound most potently to I-A^k molecules, and the peptides with arginine (R) at position 46 and A at position 54 bound most efficiently to I-A^v molecules. The present findings, thus, demonstrate that the agretopic positions on the p43--58 related peptides are preserved in T cell responses restricted to each I-A haplotype studied, and that the specific amino acids on the agretopic positions exist a priori for each I-A allele-specific structure.     

Allogeneic recognition of class I molecules: Anti-H-2Ld repertoire of H-2b mice includes T cells recognizing mutant class II H-2b (Abm12) molecules

Two major histocompatibility complex (MHC) class I-reactive T cell clones derived from H-2^b mice, generated against the allogeneic L^d molecule, were found to recognize the H-2^b class II mutant A^bm12 molecule as well. In addition, these clones also recognize the class II A^s molecule, and display a class II-dependent reactivity to staphylococcal enterotoxin B. Neither the class I nor the class II alloreactivities of the clones were found to be dependent on other MHC molecules. Both clones express CD4+CD8^? phenotypes. The CD4 molecule appears to be involved in their class II reactivity, while little or no role for CD4 could be detected in the class I reactivity. This is the first report of a class I/class II cross-reactivity being mediated by CD4⁺ T cells. The structural basis for this cross-reactivity is discussed.     

Engagement of the B lymphocyte antigen receptor induces presentation of intrinsic immunoglobulin peptides on major histocompatibility complex class II molecules

By means of the clonotypic variable region, the immunoglobulin (Ig) is a tumorspecific antigen on B cell neoplasms. We report that engagement of the B cell antigen receptor (BcR) promotes presentation of peptides derived from the B cell's intrinsic Ig to major histocompatibility complex (MHC) class II-restricted T cells. Thus, anti-Ig endowed normal, ex vivo B lymphocytes from H-2^d, Ig constant heavy chain allotype b (IgC_H^b) mice with the capacity to stimulate an I-A^d-restricted T cell clone which recognizes the γ2a^b 435–451 allopeptide. The corresponding self γ2a^a peptide is cryptic and 6000-fold less antigenic than the γ2a^b allopeptide. Even so, the syngeneic B cell lymphoma A20 which expresses surface (s) IgG2a^a, was also recognized by the T cells after BcR ligation. Thus, anti-Ig triggered the disclosure of a cryptic tumor antigen determinant. We propose that autoantigens, by engaging the BcR of self-reactive B cells, induce presentation of intrinsic Ig peptides to which the T helper cell (Th) repertoire is not tolerant. In this way, B cells with anti-self potential may be activated without Th recognition of nominal autoantigen.     

The P9 peptide sidechain specificity of I-A

The murine MHC class II variant I-A^d confers susceptibility to herpes simplex virus (HSV)-induced keratitis and relative protection against type 1 diabetes mellitus. The association to these autoimmune diseases appears to be largely determined by the peptide sidechain specificity of the P9 pocket, which we therefore have analyzed in detail. Assessment of T-cell responses and I-A^d binding capacity of position 446-substituted analogs of an IgG2a allotype b (IgG2a^b) heavy chain peptide demonstrates that engagement of the P9 pocket is crucial for effective peptide presentation. Sidechain size rather than charge decides the capacity to engage the P9 pocket. Thus, small, uncharged sidechains are accepted, whereas acidic and aromatic amino acids as well as lysine and arginine are disfavored. The specificity of the P9 pocket of I-A^d (serine^β57) is distinct from that of the diabetes-associated I-A^g7 (aspartic acid^β57), supporting the contention that the polymorphism at residue β57 influences diabetes susceptibility via P9-specific effects on the repertoires of self peptides presented to T cells. Furthermore, the data rationalize the susceptibility to HSV-induced keratitis conferred by the a and the protection conferred by the b allotypes of the IgG2a heavy chain. Keratitogenic T cells, which cross-react with the viral UL6 protein and a corneal antigen, are silenced in IgG2a^b mice because of antigenic mimicry with γ2a^b 435–451. Our finding that the lysine P9 residue of the corresponding γ2a^a allopeptide precludes high-affinity binding to I-A^d indicates that the susceptibility of IgG2a^a mice reflects inefficient thymic presentation of autologous IgG2a and thus failure to purge the T-cell repertoire of the pathogenic clones.     

Mechanisms by which the I-ABM12 Mutation Influences Susceptibility to Experimental Myasthenia Gravis: a Study in Homozygous and Heterozygous Mice

The I-A^bm12 mutation in C57B1/6 (B6) mice yields the B6. C-H-2^bm12 (bm12) strain, which is resistant to Experimental Myasthenia Gravis (EMG) induced by immunization with Torpedo acetylcholine receptor (TAChR), while the parental B6 strain is highly susceptible to EMG. CD4⁺ cells from bm12 mice immunized with TAChR do not recognize three sequence regions of the TAChR Q subunit which dominate the CD4⁺ cell sensitization in B6 mice. We immunized with TAChR bm12, B6 and (bm12B6)Fl mice. B6 and F1 mice developed EMG with comparable frequency. Their CD4⁺ cells recognized the same TAChR α subunit peptide sequences (Tα150–169, Tα181–200 and Tα360–378). CD4⁺ cells from TAChR-sensitized Fl mice were challenged with TAChR and α subunit epitope peptides, using F1, B6 or bml 2 APC. B6 and F1 APC presented all these Ag efficiently, while bm 12 APC presented TAChR and peptide Tα150–169 poorly and erratically. Anti-TAChR and anti-α subunit epitope CD4⁺ lines propagated from Fl and B6 mice had similar TcR Vβ usage. All lines but those specific for the sequence Tα150–169 had unrestricted Vβ usage. Anti-Tα150–169 lines from both B6 and Fl mice had a strong preferential usage of Vβ6. Anti-Tα150–169 lines from Fl mice had also a slightly higher Vβ14 usage. B6, bm12 and Fl mice developed similar anti-TAChR Ab titres, and had Ab bound to muscle AChR in comparable amounts. Therefore EMG resistance of bm12 mice must be due to a subtle shift in the anti-AChR Ab repertoire, and absence of special Ab able to cause destruction and/or dysfunction of muscle AChR. This is probably related to the absence of CD4⁺ cells sensitized to epitopes within the sequence Tα 150–160, consequent to the inability of the I-A^bm12 molecule to present this sequence.     

Prevention of murine lupus by an I-E α chain transgene: Protective role of I-E α chain-derived peptides with a high affinity to I-Ab molecules

The expression of a transgene encoding the I-E α chain prevents a lupus-like autoimmune syndrome in BXSB mice. However, it had not been elucidated whether the Eα^d transgene-mediated protective effect results from I-E expression or from the generation of I-E α chain-derived peptides (Eα peptide) displaying high affinity for the I-A^b molecule. To address this question, two different BXSB lines expressing the transgene at low or high levels were crossed with lupus-prone MRL mice; this resulted in three types of (MRL × BXSB)F₁ mice, differing in the expression levels of I-E molecules and of Eα peptides presented by I-A^b molecules. Comparative analysis of these three (MRL × BXSB)F₁ mice as well as several BXSB transgenic lines showed that the Eα^d transgene-mediated protection paralleled the expression levels of Eα peptide presented by I-A^b molecules, but not of I-E molecules on B cells. In addition, use of transgenic and nontransgenic double bone marrow chimeras showed a selective activation of nontransgenic B cells during I-A^b-restricted T cell-dependent immune responses, while both transgenic and nontransgenic B cells were comparably activated during T cell-independent responses. These results favor a model of autoimmunity prevention based on competition for antigen presentation, in which excessive generation of Eα peptides prevents, because of their high affinity to the I-A molecules, activation of potential autoreactive T and B cells.     

GENETIC CONTROL OF THE IMMUNE RESPONSE TO HEN'S EGG-WHITE LYSOZYME IN MICE: I. ANTIBODY AND T-LYMPHOCYTE PROLIFERATIVE RESPONSES TO THE NATIVE PROTEIN*

We have initiated studies to determine whether the antibody and T-lymphocyte proliferative responses to lysozyme and its antigenic sites is genetically controlled in mice. Mice of the H-2^f, H-2^k and H-2^p were high responders, while haplotypes H-2^b, H-2^d, H-2^r and H-2^s were low responders. Studies with recombinants indicated that the immune response is controlled by two H-2I region loci, one being in the I-A subregion and the other may be in the I-C subregions.     

Induction or prevention of immunopathological disease by cloned cytotoxic T cell lines specific for lymphocytic choriomeningitis virus

Cloned lymphocytic choriomeningitis virus (LCMV)-specific cytotoxic T lymphocyte (CTL) lines were prepared from spleens of 129/J (H-2^b) mice immunized 7-9 months earlier with LCMV (UBC strain), or of C57BL/10J (H-2^b) mice immunized 4 to 5 weeks earlier with LCMV (Armstrong strain). One uncloned and 3 cloned cytotoxic T cell lines were assessed for their respective abilities to produce, or protect against, fatal disease upon transfer to appropriate recipients or to induce specific footpadswelling reaction. The effects of all lines were essentially identical. In recipient mice acutely infected with LCMV and immunosuppressed either by irradiation (750-990 rds) or treatment with cyclophosphamide, cloned T cells administered intracerebrally (i.c.) caused a convulsive disease and death within 1-4 days. No disease was produced when the same CTL were transferred to uninfected recipients or when they had been frozen and thawed prior to transfer to infected recipients. When admixed with 500 plaque-forming units of LCMV and transferred i.c. to immunocompetent H-2^b mice, the T cell clones prevented overt disease. Allogeneic (H-2^k) recipients of this same admixture all developed typical LCM disease as did H-2^b recipients of the admixture after T cells had been frozen and thawed. Inoculation of cloned CTL into preinfected footpads induced a specific footpad-swelling reaction, which reached maximum levels after about 36 h. Irradiated and infected recipients of cloned LCMV-specific T cells showed the footpad-swelling reaction only when they had been reconstituted with bone marrow cells. In contrast, cloned T cells induced LCM disease in i.c. infected and irradiated mice independent of bone marrow reconstitution. These findings indicate that both fatal LCMV-induced neurologic disease and protection against it are mediated directly by virus-specific CTL.     

A novel T‐cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice

We used a newly generated T‐cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10‐mer) or SSVWNATTA (9‐mer) in complex with class I MHC antigen H‐2D^b, were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8⁺ T cells can detect 10‐ and 9‐mer length variants similarly. This result varies from the idea that a given T‐cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10‐mer was presented on the surface of uninfected and infected CD8α⁺CD11c⁺ dendritic cells, which suggests the use of direct and cross‐presentation pathways. In contrast, CD11b⁺CD11c^? cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell‐type specific mechanisms of antigen presentation in vivo.