Influence of major histocompatibility complex haplotype on the mitogenic response of T cells to staphylococcal enterotoxin B.

The abilities of antigen-presenting cells (APC) from nine independent major histocompatibility complex haplotypes and a number of intra-H-2 recombinant congenic strains of mice to present staphylococcal enterotoxin B (SEB) and induce proliferation in murine T-cell receptor V beta 8+ T-cell clones were compared. SEB presented by APC of all haplotypes tested induced significant responses in each of the T-cell clones. The magnitude of response was similar for most haplotypes, but there were limited quantitative differences between certain haplotypes. SEB presented by APC from H-2b mice as well as the intra-H-2 recombinant strains B10.GD and B10.A(4R), which do not express cell surface I-E (designated I-E-), induced the poorest T-cell responses. However, APC from AfE-, AsE-, and AqE- mice were as potent in SEB presentation as APC expressing both I-A and I-E. Antibodies against I-E were more effective than anti-I-A antibodies at inhibiting responses to SEB presented by APC expressing both I-A and I-E, whereas responses induced by APC expressing I-A but not I-E were blocked by antibodies against I-A. Thus, our results show that I-A can present SEB efficiently but that expression of both I-A and I-E on the same APC results in presentation of SEB predominantly by I-E. In addition, experiments using four distinct I-E- strains of mice indicate that I-A alleles differ in their ability to present SEB.     

Pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 superantigen

Superantigens (SAg) are proteins of bacterial or viral origin able to activate T cells by forming a trimolecular complex with both MHC class II molecules and the T cell receptor (TCR), leading to clonal deletion of reactive T cells in the thymus. SAg interact with the TCR through the beta chain variable region (Vbeta), but the TCR alpha chain has been shown to have an influence on the T cell reactivity. We have investigated here the role of the TCR alpha chain in the modulation of T cell reactivity to Mtv-7 SAg by comparing the peripheral usage of Valpha2 in Vbeta6(+) (SAg-reactive) and Vbeta8.2(+) (SAg non-reactive) T cells, in either BALB/D2 (Mtv-7(+)) or BALB/c (Mtv-7(-)) mice. The results show, first, that pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 SAg. Second, there is a strikingly different distribution of the Valpha2 family members in CD4 and CD8 populations of Vbeta6 but not of Vbeta8.2 T cells, irrespective of the presence of Mtv-7 SAg. Third, the alpha chain may play a role in the overall stability of the TCR/SAg/MHC complex. Taken together, these results suggest that the Valpha domain contributes to the selective process by its role in the TCR reactivity to SAg/MHC class II complexes, most likely by influencing the orientation of the Vbeta domain in the TCR alphabeta heterodimer.     

T cell receptor usage by HLA-DQw8-specific T cell clones.

To investigate whether T cells recognizing the same HLA molecule may demonstrate homology in parts of their TCR, five different HLA-DQw8-specific T lymphocyte clones (TLC) were studied. The TCR alpha and -beta genes of four alloreactive, HLA-DQw8-specific and one antigen-specific TLC were sequenced. All TLC used different V alpha and V beta genes. However, four of the TLC shared a certain CDR1 beta motif and all five used either J beta 2.3 or -2.5. In addition, two used the same J alpha. The results indicate a possible preferential usage of certain TCR structures by T cells specific for DQw8.     

Genetic analysis of the effects of Mtv-2 on the T cell repertoire in the WXG-2 mouse strain.

In this report, the T cell repertoire was studied in a natural genetic model system using a novel mouse strain (WXG-2) carrying a single pathogenic mouse mammary tumor virus (MMTV) provirus (Mtv-2) on an otherwise MMTV-free genetic background. The Mtv-2 provirus has complete biological activity, produces infectious milk-transmitted virus, and contributes to mammary carcinogenesis by an insertion mutation mechanism. In mice carrying the Mtv-2 provirus, T cells expressing V beta 14 were specifically deleted in mice with a functional MHC class II I-E gene but not in I-E- controls. The deletion of V beta 14+ T cells was more rapid in mice with the Mtv-2 provirus than in Mtv-2-free control mice infected with exogenous MMTV. In addition, the Mtv-2 deletion phenotype was age dependent. A slow depletion of V beta 14+ T cells was observed, and greater than 95% of the V beta 14+ T cells were eliminated by 6 months of age. These experiments indicate that (i) the Mtv-2 provirus encodes or regulates expression of a V beta 14-specific superantigen, (ii) interactions between Mtv-2 and other MMTV proviruses are not necessary for the V beta 14 deletion phenotype, (iii) the presence of a retroviral superantigen in all cells is not sufficient for T cell depletion during neonatal development in the thymus, and (iv) the Mtv-2 provirus and its associated exogenous provirus have the same V beta specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient presentation of endogenous superantigen by H-2Aq

Endogenous superantigens encoded by mouse mammary tumor viruses associate with MHC class II and interact with T cells bearing particular Vβ gene segments. H-2E is more efficient at presentation than H-2A, indeed A^q has not been shown to be capable of presenting endogenous superantigens. Atypically, the superantigen vSAG-3 encoded by Mtv-3 is presented efficiently in non-obese diabetic (H-2^g7) mice by H-2A; we have examined the independent contributions of vSAG-3 and A^g7 to this process. A^g7 was not found to have a more general ability to efficiently present endogenous superantigens other than Mtv-3. Examination of Mtv-3-mediated thymic deletion of Vβ 3⁺ thymocytes in the presence of H-2^q additionally demonstrated the efficient presentation of vSAG-3 by A^q. Interaction of vSAG-3 with A^q and A^g7 is likely to reflect the unique sequence of Mtv-3 within the second polymorphic region previously implicated in MHC class II binding. The demonstration that mouse endogenous superantigens can be presented by a wider range of MHC haplotypes than previously thought is further evidence for their immunological impact on the mouse population.     

Binding of toxic shock syndrome toxin-1 to murine major histocompatibility complex class II molecules

The staphylococcal exotoxin toxic shock syndrome toxin-1 (TSST-1) has potent stimulatory effects on murine and human lymphocytes. This is the consequence of TSST-1 binding to major histocompatibility complex (MHC) class II molecules and the engagement in a V beta-restricted fashion of the T cell receptor by the TSST-1-MHC class II complex. Using radioligand and functional assays we have recently shown that TSST-1 binds to all HLA-DR (n = 14), HLA-DQ (n = 2) and HLA-DP (n = 2) phenotypes tested. In this study, we have examined the ability of murine MHC class II molecules to bind TSST-1. Specific high-affinity binding of TSST-1 was detectable to unfractionated BALB-c (H-2d) and C57BL/6 (H-2b), but not to C3H (H-2k) spleen cells. The Kd of this binding estimated from Scatchard analysis was in the same nanomolar range as the Kd of binding of TSST-1 to HLA-DR. Binding of 125I-labeled TSST-1 to BALB/c-derived B cell lymphoma lines and to L cell transfectants correlated with the expression of I-A molecules, but not with the expression of I-E molecules. Furthermore, I-A+, I-E- cells but not I-A-, I-E+ cells were able to support TSST-1-induced T cell proliferation. The binding affinity of TSST-1 for I-Ak appears to be much lower than for I-Ad. L cell transfectants expressing hybrid DR alpha: I-E beta k molecules, but not those expressing I-E alpha k: DR1 beta molecules, could bind TSST-1 and efficiently support TSST-1-induced T cell proliferation. This suggests that minor differences in the highly homologous I-E alpha and DR alpha chains are critical in determining the affinity of the MHC class II molecule for TSST-1. These results demonstrate that the binding of TSST-1 to MHC class II molecules in the mouse, in contrast to humans, is strongly influenced by phenotype. Analysis of the molecular basis of these differences may help to localize staphylococcal exotoxin binding sites on MHC class II molecules.     

Role of MHC, Mls and TCR in immune tolerance

MHC class II molecules and self antigens, such as Mls, influence T-cell selection by clonal deletion of potentially self-reactive T cells. In order to examine the role of various class II molecules in the T-cell receptor-self antigen interaction, class II transgenic and recombinant mice were analysed for TCR expression. Our studies indicate that the A alpha and E alpha chains can present Mls gene products for the clonal deletion of V beta 6-bearing T cells, and that the A alpha q chain is defective in this process. We have also shown that E alpha A beta heterodimer in transgenic and recombinant mice is expressed and functions to delete I-E reactive V beta llT cells, demonstrating again the role of the E alpha molecule.     

Self-peptides that bind with low affinity to the diabetes-associated I-A(g7) molecule readily induce T cell tolerance in non-obese diabetic mice

Although non-obese diabetic (NOD) mice spontaneously develop T cell autoimmunity, it is not clear whether this phenomenon results from a defect in tolerance to self-Ag. Furthermore, as autoimmunity has been postulated to result from T cell responses directed toward self-peptides that bind with low affinity to NOD I-A(g7) MHC class II molecules, it is important to determine whether the expression of such peptides induces tolerance. We have constructed NOD transgenic (Tg) mice expressing the Leishmania antigen receptor for C kinase (LACK) Ag in either the thymus or pancreatic beta cells. We identified LACK peptides that were the targets of T cells in LACK-immunized NOD mice while binding to I-A(g7) with low affinity. While CD4(+) T cells from NOD mice secreted IFN-gamma, IL-4, IL-5 and IL-10 in response to LACK, those from LACK-expressing Tg mice secreted reduced levels of cytokines. Experiments using peptide/MHC multimers showed that LACK-expressing Tg mice exhibited self-reactive CD4(+) T cells with impaired proliferation capabilities. Hence, even self-peptides that bind to I-A(g7) with low affinity can induce tolerance in NOD mice. This result is important in light of the commonly held hypothesis that T cells reacting to peptides that bind to MHC with low affinity escape tolerance induction and cause autoimmunity.     

H-2 I-E molecules isolated from MIs1a stimulatory cells do not activate Mls1a-responsive T cells but do present exogenous staphylococcal enterotoxins

The T cell response to allogeneic murine Mls determinants is not H-2 restricted but is dependent on H-2 class II molecules on the Mls-expressing stimulator cells. We have tested planar membranes containing H-2 class II I-E molecules alone or with I-A molecules for their ability to activate a panel of Mls1^a-specific T hybrids. Despite the ability of the planar membranes to activate an alloreactive T hybrid and to present staphylococcal enterotoxins or an antigenic peptide to appropriately responsive T hybrids, they failed to stimulate the Mls1^a-specific T hybrids. These findings, in the light of the various controls demonstrating sufficiency of the I-E molecules in the planar membranes, indicate that Mls1^a determinants are not covalently bound to I-E molecules; the two molecular species are thus either not physically associated or are linked by a relatively weak interaction. In addition, our experiments show that isolated I-E molecules but not I-A molecules present staphylococcal enterotoxins A and B to two independently derived T hybrids expressing T cell receptor Vβ1, Vβ8.2 and Vβ6 elements.     

Dominant TCR V alpha usage by virus and tumor-reactive T cells with wide affinity ranges for their specific antigens

We have studied the TCR features and functional responses of three sets of human cytolytic T cell (CTL) clones, recognizing antigenic peptides presented by HLA-A2 and derived from the Epstein-Barr virus proteins BMLF1 and BRLF1 and from the melanoma protein Melan-A/MART-1. Within each set, a majority of clones used a recurrent V alpha region, even though they expressed highly diverse TCR beta chains and V(D)J junctional sequences. Functional assays and peptide/MHC multimer binding studies indicated that this restricted V alpha usage was not associated with the affinity/avidity of the CTL clones. The V alpha dominance, which may be a frequent feature of antigen-specific T cells, likely reflects a restricted geometry of TCR/peptide/MHC complexes, primarily determined by V alpha CDR.     

Molecular profile of the T cell receptors of regulatory and effector CD4+ T cells recognizing overlapping determinants on glutamic acid decarboxylase (524-543)

Glutamic acid decarboxylase (GAD65) is one of the autoantigens that initiates pathogenic T cell responses against insulin-secreting pancreatic beta cells in Type 1 diabetes (T1D). Previously it was shown that spontaneously arising pathogenic T cell responses in the NOD mouse model are confined to GAD530-543 (p530). However, regulatory T cell subpopulations, which can prevent diabetes, can also be generated, for example, by immunization with GAD524-538 (p524) or GAD524-543. Interestingly, two functionally distinct subpopulations of T cells which recognize overlapping determinants of GAD524-543, p524 and p530, utilize distinct TCR Vbeta families, Vbeta4 for pathogenic, and Vbeta12 for regulatory T cells. We characterized T cell receptors (TCRs) from each subpopulation of T cells and visualized p524-specific TCR/p524/I-A(g7) and p530-specific TCR/p530/I-A(g7) complexes via molecular modeling to help us understand, at a molecular level, the in vivo expansion of p524- or p530-specific T cells in the NOD model of T1D. The absolute restriction in Vbeta usage but not Valpha usage and conserved CDR3beta lengths for both T cell subpopulations demonstrates that the beta chains are main contributors in shaping both p524/I-A(g7) and p530/I-A(g7) restricted TCRs. However, only Vbeta4+ T cells but not Vbeta12+ T cells contain a common motif (DWG) in CDR3beta and may involve all of CDR1beta, CDR2beta, and CDR3beta in the recognition of the C-terminus of p530. These observations imply that the spontaneously arising p530-restricted TCRs may be selected under stringent structural frameworks to bind p530/I-A(g7) with high affinity. Thus, the pathogenic p530-specific T cells may arise from a small pool of autoreactive T cells upon breaking tolerance.     

Evidence for shared recognition of a peptide ligand by a diverse panel of non-obese diabetic mice-derived,islet-specific,diabetogenic T cell clones

MHC class II-restricted autoreactive T cells play a major role in the development of autoimmune diabetes mellitus in both human and mouse. Two of our groups previously established panels of islet-reactive CD4+ T cell clones from prediabetic non-obese diabetic (NOD) mice. These clones express distinct sets of TCR V alpha , V beta , J alpha and J beta , and also differ in the structure of the junctional region of TCR. All of the T cell clones have been shown to cause insulitis and several induce diabetes when transferred to various recipients. The antigen specificities of these T cell clones have not been determined, but they do not react with defined islet cell antigens such as glutamic acid decarboxylase. To identify the peptide ligands recognized by these clones, we examined the reactivity of the T cell clones to peptide mixtures in which anchor residues for H2-A g7 were fixed. Most of the clones showed similar reactivity to the peptide mixtures. To further determine the peptide ligands of the T cell clones, we synthesized several peptides based on the favored amino acid motifs and examined clone reactivity to the synthetic peptides. Some of the peptides, e.g. HLAI-RM and HIPI-RM, could stimulate most of the T cell clones tested, even though the clones expressed different TCR. The results suggest that our islet-reactive T cell clones recognize in islet beta cells a natural ligand that is similar to these peptides.     

Mls-1 and Mls-2 superantigens do not control susceptibility to collagen-induced arthritis in HI and HII mice.

The HI mouse line is sensitive to collagen-induced arthritis (CIA), whereas HII is refractory, although both express the H-2q permissive haplotype. The two lines also share the same T-cell receptor (TcR) gene haplotypes for alpha and beta chains. The distribution of mouse mammary tumour viruses (MMTV), which encode endogenous superantigens (SAg) such as minor leucocyte-stimulating antigens (Mls) known to modulate the available TcR-V beta repertoire, was investigated in the two lines. Mls-1 is present in HI-susceptible mice, while Mls-2 and Mls-2-like SAg are absent in both lines. This suggests that Mls antigens play no significant role in the resistance to CIA. Moreover, HI and HII exhibit close V beta gene usage as assessed by fluorescence staining with 11 V beta-specific monoclonal antibodies (mAb). These results indicate that mechanisms other than clonal deletion based on V beta expression and induced by SAg are involved in the resistance of H-2q-positive mice to experimental arthritis. Yet, a slightly reduced level of V beta 5+ T cells is observed in HII animals which might correlate with the presence of Mtv-6 and Mtv-9 proviruses.     

Adoptive transfer of islet antigen-autoreactive T cell clones to transgenic NOD.Ea(d)mice induces diabetes indicating a lack of I-E mediated protection against activated effector T cells

Transgenic insertion of the MHC class II Ea(d)gene in NOD mice restores I-E expression and prevents T-cell-mediated autoimmune diabetes (IDDM). The specific molecular and cellular mechanisms responsible for the diabetes resistance of transgenic NOD.Ea(d)mice remain unclear. We adoptively transferred islet antigen-specific T cell clones into NOD and transgenic NOD.Ea(d)mice to evaluate the level of protection provided by I-E expression against activated effector T cells. We have found that neither neonatal or 3-5-week-old I-E-expressing NOD.Ea(d)mice can completely inhibit the diabetogenic activities of activated islet antigen-specific T cell clones. These data indicate that Ealpha protein expression in NOD antigen presenting cells (APC) does not reduce islet autoantigen presentation in the context of I-A(g7)below the threshold required for stimulation of effector/memory diabetogenic T cells. Our results suggest that the mechanism of Ealpha protein-mediated diabetes resistance in NOD mice may be "antigen ignorance," in which the quantity of islet autoantigens presented in the context of I-A(g7)by APC is reduced below the threshold required to activate nai;ve islet antigen-specific T cells.     

Effector T cells have a lower ligand affinity threshold for activation than naive T cells

It has been previously established that effector and memory T cells are more sensitive to antigen stimulation than naive T cells. In this study, we compared the effect of ligand affinity on the activation of naive and effector T cells derived from pigeon cytochrome c (PCC)-specific TCR transgenic mice by stimulating these cells with a variety of ligands with widely differing antigenicity. The data obtained indicated the following. (i) The differences in antigen dose requirements for activation of naive and effector cells widened as the affinity of the antigen decreased. Most dramatically, peptides that were TCR antagonists for naive T cells were recognized as agonists by effector T cells. (ii) While both naive and effector T cells were activated by the bacterial superantigen staphylococcal enterotoxin A, specific for the transgenic TCR V(beta)3 chain, effector, but not naive, T cells were stimulated to proliferate by toxic shock syndrome toxin-1, a superantigen not previously described to be stimulatory for V(beta)3 T cells. (iii) Effector T cells, but not naive cells, proliferated in response to endogenous self-peptides presented by antigen-presenting cells in a syngeneic mixed lymphocyte reaction. Taken together these data indicate that effector T cells have a lower affinity threshold for activation than naive T cells. Further studies demonstrated that the heightened reactivity of effector T cells to low-affinity ligands declined progressively with repeated stimulations by antigen such that after repeated stimulation effector T cells were no longer stimulated by low-affinity ligands but recognized them as TCR antagonists similar to naive T cells.     

Permissive recognition of a mycobacterial T-cell epitope: localization of overlapping epitope core sequences recognized in association with multiple major histocompatibility complex class II I-A molecules.

Most T-cell epitopes are recognized in the context of a single or limited number of major histocompatibility complex (MHC) class II molecules. We have shown previously, however, that the immunodominant p61-80 epitope from the Mycobacterium tuberculosis 19,000 MW protein is recognized in a genetically permissive manner. In this study, permissive recognition of p61-80 was analysed in three murine MHC haplotypes (H-2b,d and k) with respect to: (i) T-cell-epitope core structure; (ii) I-A/I-E class II MHC restriction; and (iii) the identification of critical amino acid residues within the core region. Overlapping epitope core sequences composed of 6 to 8 amino acids were identified for each of the three H-2 haplotypes by T-cell epitope scanning (PEPSCAN) using peptide-specific T-cell lines. The epitope core sequences recognized by peptide and 19,000 MW protein-specific T cells were similar. In all three haplotypes, responses to p61-80 were restricted by class II MHC I-A molecules. To identify residues within the epitope core critically required for recognition, single substitution (alanine or leucine) analogue peptides were tested for their capacity to stimulate p61-80-specific T-cell hybridomas. A heterogeneous pattern of reactivity was observed, even among individual hybridomas derived from the same H-2 haplotype. Although every core residue could be defined as critical for at least one hybridoma, only one critical substitution (74Val-->Ala) was common to all hybridomas. The identification and structural analysis of genetically permissive epitopes of mycobacteria may be a useful strategy for the rational design of peptide-based vaccines for tuberculosis.     

IL-2 and IL-7 differentially induce CD4-CD8- alpha beta TCR+NK1.1+ large granular lymphocytes and IL-4-producing cells from CD4-CD8- alpha beta TCR+NK1.1- cells: implications for the regulation of Th1- and Th2- type responses

Effector functions of CD4-CD8- double negative (DN) alpha beta TCR+ cells were examined. Among mouse DN alpha beta TCR+ thymocytes, NK1.1+ cells expressing a canonical V alpha 14/J alpha 281 TCR but not NK1.1- cells produce IL-4 upon TCR cross-linking and IFN-gamma upon cross- linking of NK1.1 as well as TCR. Production of IL-4 but not IFN-gamma from DN alpha beta TCR+NK1.1+ cells was markedly suppressed by IL-2. Whereas V alpha 14/J alpha 281 TCR+ cells express NK1.1+, these cells are not the precursor of DN alpha beta TCR+NK1.1+CD16+B220+ large granular lymphocytes (LGL). IL-2 induces rapid proliferation and generation of NK1.1+ LGL from DN alpha beta TCR+NK1.1- but not from DN alpha beta TCR+NK1.1+ cells. LGL cells exhibit NK activity and produce IFN-gamma but not IL-4 upon cross-linking of surface TCR or NK1.1 molecules. In contrast to IL-2, IL-7 does not induce LGL cells or NK activity from DN alpha beta TCR+NK1.1- cells but induces the ability to produce high levels of IL-4 upon TCR cross-linking. Our results show that DN alpha beta TCR+ T cells have several distinct subpopulations, and that IL-2 and IL-7 differentially regulate the functions of DN alpha beta TCR+ T cells by inducing different types of effector cells.      

Role of the first external domain of I-A beta chain in immune responses and diabetes in non-obese diabetic (NOD) mice.

Diabetes in the non-obese diabetic (NOD) mouse is a multigenic autoimmune disease and is possibly controlled by three recessive loci, including one that is linked to the major histocompatibility complex (MHC). The first external domain of the Class II MHC I-A beta chain in these mice is unique and has been suggested as being responsible for autoimmunity. The I-A alpha chain in these mice is I-A alpha d, and they lack the expression of I-E molecules. We have investigated immune responses to various Ir gene control antigens in NOD mice to determine the influence of the NOD Ia and particularly the I-A beta chain. We find that sheep insulin is highly immunogenic while other insulins are weakly immunogenic in these mice. Hen egg lysozyme, pigeon cytochrome C and the synthetic polypeptide Poly 18, Poly EYK(EYA)5 antigen produce good antibody responses. Apart from H-2d, NOD are the only mice where Poly 18 antigen is immunogenic. In these mice Poly 18 induced good T-cell proliferative response, which was inhibited by anti-Ia antibody, and the mice were able to respond to tyrosine-containing polypeptide Poly EYA but not to the phenylalanine-containing antigen Poly EFA. We also found that synthetic peptide 48-60 of the NOD I-A beta chain is highly immunogenic in syngeneic NOD mice both for T cells and B cells. Using an I-A beta chain-specific monoclonal antibody, we are able to prevent induction of diabetes when the antibody was administrated in prediabetic, young mice. Our results suggest that the immune response to various antigens and autoimmune diabetes in NOD mice is directly influenced by the I-A beta chain.     

Failure of T cell receptor V beta negative selection in murine intestinal intra-epithelial lymphocytes.

The intestinal intra-epithelial lymphocytes (IEL) are divided into several subsets on the basis of expression of T cell receptor (TCR) alpha beta and gamma delta, intensity of Thy-1 expression and expression of Lyt-3 chain. To investigate the differentiation pathway of the IEL, we examined the repertoire of V beta segments of T cells in the IEL in BALB/c (H-2d, MIs-1b2a) or AKR/J (H-2k, MIs-1a2b) mice. Among freshly isolated IEL, an appreciable number of T cells bearing V beta 3 or V beta 11, which recognize MIs-2a- or MHC IE-encoded molecules respectively, were detected in BALB/c mice. Similarly, in AKR/J mice, IEL contained appreciable levels of V beta 6-bearing T cells. V beta 3- or V beta 11-bearing T cells in the IEL in BALB/c mice increased to a significant level when incubated with staphylococcal enterotoxin A which specifically stimulates V beta 3- and V beta 11-bearing T cells. Most of IEL without clonal deletion expressed Lyt-2 but not Lyt-3 antigens. Such T cells were hardly detected in other organs, including liver. Our results indicate that TCR alpha beta-bearing intestinal IEL that have not undergone negative selection may have differentiated outside the thymus, presumably at a local site of the intestine and can respond normally to the signal via their TCR.