Characterization of a new minor lymphocyte stimulatory system. I. Cluster of self antigens recognized by "I-E-reactive" V beta s, V beta 5, V beta 11, and V beta 12 T cell receptors for antigen.

In the mouse, two sets of V beta gene products have been shown to be associated with T cell recognition of endogenous self Ag. One of these is the set of V beta associated with T cell reactivities to stimulatory Mls gene products, Mlsa (V beta 6, V beta 8.1, V beta 9) or Mlsc (V beta 3); another is the set of V beta, such as V beta 5, V beta 11, V beta 12, or V beta 17a, which were originally found to be related to I-E recognition. Although the Mls system has been well characterized, little is known about the nature of the ligands for the second set of V beta. In this work, we describe the evidence that the natural ligand or ligands of V beta 5, V beta 11, and V beta 12 may be novel Mls determinants that are recognized by naive T cells at a high precursor frequency and function as the ligand for clonal deletion of self-reactive T cells by negative selection. However, surprisingly, unlike the conventional Mls system, in which all V beta associated with Mlsa recognition or Mlsc recognition are uniformly deleted in those animals expressing the relevant Mls type, expression of these three V beta segregates independently among strains. Based on these observations, the nature of T cell recognition for this new Mls gene product(s) is discussed.     

H-2K molecules positively select V beta 17a+ CD4(-)8+ T cells in bone marrow and thymic chimeras

Population size of V beta 17a brightly positive cells among CD4(-)8+ thymocytes was analyzed in thymic chimeras as well as bone marrow (BM) chimeras in which SWR/J mice were used as BM donors and various strains of mice including H-2Kb mutant (bm) mice as recipients. It was shown that the proportion of V beta 17a+ CD4(-)8+ thymocytes was determined by H-2K molecules expressed on thymic epithelial cells. The highest proportion was observed in Ks and Kb thymuses, the intermediate proportion in Ks/q and Kk, and the lowest in Kq thymuses. Fine analysis of the H-2Kbm molecules involved in the positive selection revealed that the region important to the selection was located on the beta-pleated floor of antigen recognition site. According to the three-dimensional class I structure, this site appears not to be directly accessible to the T cell antigen receptor. Thus, the present finding suggests that the substitutions of amino acids at this site alter the shape and charge of the peptide binding site and eventually influence the positive selection of the V beta 17a+ T cell repertoire during differentiation.     

Preferential use of a T cell receptor V beta gene by acetylcholine receptor reactive T cells from myasthenia gravis-susceptible mice.

Experimental autoimmune myasthenia gravis (EAMG) is an important model for testing current concepts in autoimmunity and novel immunotherapies for autoimmune diseases. The EAMG autoantigen, acethylcholine receptor (AChR), is structurally and immunologically complex, a potential obstacle to the application of therapeutic strategies aimed at oligoclonal T cell populations. Inasmuch as we had previously shown that the clonal heterogeneity of T cell epitope recognition in EAMG was unexpectedly limited, we examined TCR V beta expression. AChR primed lymph node T cells and established AChR reactive T cell clones from EAMG-susceptible C57BL/6 (B6; H-2b, Mls-1b) mice showed preferential utilization of the TCR V beta 6 segment of the TCR. After in vivo priming and in vitro restimulation for 7 days with AChR or a synthetic peptide bearing an immunodominant epitope, V beta 6 expressing lymph node cells (LNC) were expanded several-fold, accounting for up to 75% of recovered viable CD4+ cells. The LNC of B6.C-H-2bm12 (bm12; H-2bm12, Mls-1b) mice, which proliferated in response to AChR but not to the B6 immunodominant peptide, failed to expand V beta 6+ cells. Inasmuch as nonimmune bm12 and B6 animals had similar numbers of V beta 6+ LNC (4-5%), this suggested that structural requirements for TCR recognition of Ag/MHC complexes dictated V beta usage. Results concerning peptide reactivity and V beta 6 expression among T cells from (B6 x bm12)F1 animals also suggested that structure-function relationships, rather than negative selection or tolerance, accounted for the strain differences between B6 and bm12. To examine the potential effects of thymic negative selection of V beta 6+ cells on the T cell response to AChR, CB6F1 (H-2bxd, Mls-1b; V beta 6-expressing) and B6D2F1 (H-2bxd, Mls-1axb; V beta 6-deleting) strains were analyzed for AChR and peptide reactivity and V beta 6 expression. Both F1 strains responded well to AChR but the response of B6D2F1 mice to peptide was significantly reduced compared to CB6F1. Short and long term cultures of peptide-reactive B6D2F1 LNC showed no expansion of residual V beta 6+ cells, although similar cultures of CB6F1 LNC were composed of more than 60% V beta 6+ cells. The results from the F1 strains further indicated that the T cell repertoire for peptide was highly constrained and that non-V beta 6 expressing cells could only partially overcome Mls-mediated negative selection of V beta 6+ TCR capable of recognizing peptide.(ABSTRACT TRUNCATED AT 400 WORDS)     

A T cell receptor V beta segment that imparts reactivity to a class II major histocompatibility complex product

We have identified in mice an allele of a new T cell receptor V beta gene, V beta 17a, whose product is bound by the monoclonal antibody KJ23a. Over 90% of T cell hybridomas prepared from V beta 17a+ T cells of SWR mice respond to allogeneic forms of the IE class II MHC protein, indicating that V beta 17a has an appreciable affinity for IE regardless of the other components of the T cell receptor. These results suggest a bias in the germ-line T cell receptor repertoire toward recognition of MHC proteins and indicate that the V beta portion of the receptor may form the most important contact points with MHC ligands.     

Failure of (SJL/J x PL/J)F1 hybrid mice to develop immunologic tolerance to V beta 17a+ T cells results in F1 anti-SJL mixed lymphocyte reaction.

It has been reported previously that spleen cells from (SJL x PL) F1 hybrid mice are not tolerant to SJL parental cells as assessed by a one-way MLR. The possibility that the F1 anti-SJL reaction was due to the effect of lymphokines produced by the irradiated SJL T cells in response to I-Eu expressed on the F1 hybrid cells was eliminated since inclusion of anti-I-E mAb was without effect. Cell separations showed the responder cells to be plastic and nylon wool nonadherent Ia- T cells. Separation of the SJL spleen cells showed that the stimulator cells were nonadherent, passed through a nylon wool column, and were Ia-. the F1-anti-SJL MLR was blocked 70 to 90% by inclusion of mAb KJ23a in the culture medium that indicated that the stimulatory cell population was V beta 17a+ T cells. This was confirmed by the use of V beta 17a+ and V beta 17a-T cell clones as stimulators. To determine whether failure to develop tolerance to this T cell subset in F1 hybrid mice might be responsible for the F1-anti-parent MLR, (SJL x PL)F1 mice were treated at birth and 48 h thereafter with anti-I-E mAb for 7 wk. Spleen cells from antibody-treated F1 mice were nonreactive with irradiated SJL parental cells in contrast to spleen cells from control mice which indicated that V beta 17a+ T cells were eliminated by negative selection before the development of tolerance.     

V beta gene polymorphism and a major polyclonal T cell receptor idiotype

Genetic polymorphism in the beta variable gene pool (V beta) is responsible for the strain-specific distribution of the KJ16 T cell receptor (TcR) marker on 20% of peripheral T cells. KJ16+ strains carry two homologous V beta genes that are absent in KJ16- strains. All functional KJ16+ T cell hybrids tested express a member of this V beta subset. mRNA hybridizing to this variable-region probe can be easily detected in total splenic T cells of a prototype KJ16+ strain. Thus, 20% of the TcR from the cytotoxic and helper T cell population, with various MHC restrictions and antigen reactivities, can be generated from two V beta genes. However, their deletion appears to have no effect on the functionality of the T cell repertoire.     

Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy

Experimental allergic encephalomyelitis (EAE) is a paralytic autoimmune disease induced in susceptible animals by active immunization with myelin basic protein (MBP) or by passive transfer of MBP-specific T helper (TH) lymphocytes. We have analyzed the T cell receptor genes of 33 clonally distinct TH cells specific for a nonapeptide of MBP inducing EAE in B10.PL (H-2u) mice. All 33 TH cells used two alpha variable gene segments (V alpha 2.3, 61%; V alpha 4.2, 39%), the same alpha joining gene segment (J alpha 39), and two V beta and J beta gene segments (V beta 8.2-J beta 2.6, 79%; V beta 13-J beta 2.2, 21%). The anti-V beta 8 monoclonal antibody F23.1 was found to block completely recognition of the nonapeptide by V beta 8 TH cells in vitro and to reduce significantly the susceptibility of B10.PL mice to peptide-induced EAE.     

The T cell receptor V beta 6 domain imparts reactivity to the Mls-1a antigen

A monoclonal antibody secreting hybridoma was established by fusing spleen cells from a rat immunized with a murine T cell clone, OI11, which has I-Ab restricted specificity for the male H-Y antigen and unrestricted specificity for the minor lymphocyte stimulating antigen, Mls-1a, to the mouse myeloma P3X63AG8.653 and screening for the capacity of the hybridoma supernatants to stimulate the OI11 T cell clone. An antibody (RR4-7) was found to be specific not only for the immunizing T cell clone but virtually for all T cells using the V beta 6 TCR gene product as part of their surface antigen receptor. When the expression of the V beta 6 gene in various strains of mice was analyzed, it was found that strains expressing the Mls-1a antigen contained few T cells expressing V beta 6-encoded TCRs. The majority of T cell hybridomas which expressed V beta 6-encoded TCRs were found to be reactive to the Mls-1a antigen. These data confirm the finding of H. R. MacDonald et al. (Nature (London) 332, 40, 1988) that most TCRs encoded by the V beta 6 gene have a biased specificity for the Mls-1a antigen.     

Mouse T lymphocyte response to acetylcholine receptor determined by T cell receptor for antigen V beta gene products recognizing Mls-1a.

Mice of strain B6, but not AKR/J, respond to immunization with Torpedo acetylcholine receptor (AChR) by manifesting in vitro an Ag-specific T lymphocyte proliferative response. Our analysis of (AKR x B6)F1 mice reveals that the T cell unresponsiveness of AKR/J is inherited as a dominant trait, possibly associated with expression of the Mls-1a allele. Mice derived from backcrossing (AKR x B6)F1 x B6 were selected for H-2b homozygosity and were classified as Mls-1a or Mls-1b according to the relative numbers of peripheral blood T cells that expressed the TCR V beta 6 gene product. After challenge by injection with AChR in CFA, lymph node cells from mice classified as having less than 2% of V beta 6+ peripheral T cells had low responsiveness to AChR, whereas mice with greater than 7% V beta 6+ peripheral T cells had high T cell responsiveness to AChR. These results are consistent with the notion that regulation of the T cell repertoire by Mls loci may be a determinant of susceptibility to autoimmunity.     

Thymic deletion of V beta 11+, V beta 5+ T cells in H-2E negative, HLA-DQ beta+ single transgenic mice

DQw6b transgenic mice have been generated by microinjecting a linearized cosmid clone containing 34-kb DQb genomic DNA, isolated from HLA-homozygous B cell line AKIBA (DR2, Dw12, DQw6), into embryos of (CBA x B10.M)F2 or (SWR x SJL)F2. Among 85 mice screened, eight mice were transgene-positive. The transgene in seven of eight founders was germline-transmitted. FACS analysis and immunohistochemical studies with DQ beta-specific mAb demonstrated that DQ beta molecules in association with mouse A alpha f molecules are expressed on peripheral mononuclear cells, spleen cells, and in thymic medulla. More interestingly, V beta 11-, V beta 5.1-, and V beta 5.2-bearing T cells, but not V beta 8.2-bearing T cells, were clonally deleted in the H-2E-negative but DQ beta+ progeny of two selected founders (260-23 and 258-10). The deletion was found to take place intrathymically during the transition stage from immature to mature thymocyte development. We postulate that although human DQ genes are more homologous to mouse H-2A genes, A alpha f/DQ beta hybrid molecules may possess the same self-peptide- (or superantigen)-presenting epitope as E alpha/E beta molecules critical for deletion of V beta 11-, V beta 5.1-, and V beta 5.2-bearing T cells in thymus. Our results also confirm the previous findings that accessory molecules on thymocytes such as CD4 may be involved in thymic selection, and further suggest that an interaction of mousE CD4 and mouse A alpha chain is required for the clonal deletion.     

Evidence that Mls-2 antigens which delete V beta 3+ T cells are controlled by multiple genes

V beta 3+ T cells are eliminated in Mls-2a mice carrying some, but not all, H-2 types. Analysis of AKXD and BXD recombinant inbred strains showed that Mls-2a (formerly Mlsc) was not the product of a single gene and suggested that at least two non-H-2 genes control V beta 3 levels. Studies of the progeny of a B10.BR x (C3H/HeJ x B10.BR)F1 backcross confirmed the existence of two V beta 3+ T cell deleting genes: one unlinked and one linked to Ly-7, which we propose be called Mls-2 and Mls-3, respectively. Mls-2a induces partial deletion of V beta 3+ T cells with a bias toward deleting CD4+ cells. It stimulates V beta 3+ hybrids and may be linked to Mtv-13 on chromosome 4. A third non-H-2 gene is implicated in enhancing the presentation of Mls-2a. Mls-3a causes elimination of all V beta 3+ T cells in H-2k and H-2d mice but poorly stimulates V beta 3+ hybrids.     

Multiple, autoreactive TCR V beta genes utilized in response to a small pathogenic peptide of an autoantigen in EAU.

The restricted usage of particular T cell receptor beta chain genes in autoimmune disease was studied in LEW rats using T cell hybridomas specific for an immunodominant sequence of bovine retinal S-Ag, which induces experimental autoimmune uveoretinitis. T cell hybridomas from a pathogenic T cell line, R858, specific for residues 273-289 of bovine retinal S-Ag were analyzed in order to determine the contribution of their TCR V beta to self specificity as determined by recognition of the pathogenic epitope represented in the autologous rat S-Ag sequence. Six different, functional TCR rearrangements were expressed by the panel of hybridomas, including two distinct V beta 8.2 rearrangements and functional V beta 10, V beta 14, V beta 19 rearrangements, and an unidentified V beta gene. All hybridomas were Ag specific and reacted both to nonself-peptide derivatives as well as to self-peptide homologues. No unique pattern of peptide reactivity distinguished V beta 8.2+ hybridomas from V beta 8.2- hybridomas; all of the hybridomas were most reactive to the nonself sequences and reacted to self peptide with one to three orders of magnitude less sensitivity. However, all V beta 8.2+ hybridomas were much better responders overall and were activated by lower concentrations of all peptides than were V beta 8.2- hybridomas. Although V beta 8.2 gene usage is strongly associated with autoimmune pathology, these data show that in LEW rats several different TCR V beta genes are utilized in response to a short pathogenic sequence of this autoantigen and show that V beta 8.2 receptors are not uniquely self-reactive. However, the enhanced reactivity to Ag of V beta 8.2+ hybridomas relative to V beta 8.2- hybridomas specific for the same peptide may help explain the close association of V beta 8.2 TCR gene usage with pathogenicity found in autoimmune disease models.     

T cell tolerance to self antigens in New Zealand hybrid mice with lupus-like disease

We determined if self-reactive T cells are able to escape thymic tolerance in autoimmune New Zealand mice. T cells utilizing V beta 17a and V beta 11 encoded receptors have been shown to be clonally eliminated in nonautoimmune mice expressing I-E because of their potential self-reactivity. Similarly, V beta 8.1+ and V beta 6+ T cells are tolerized in the thymus of nonautoimmune mice that express Mls-1a. These T cell subsets were quantitated in the lymph nodes and spleens of (NZB x NZW)F1 and (NZB x SWR)F1 mice. In young mice from both autoimmune strains, deletion was similar to that observed in control animals matched for I-Ed and Mls-1a expression. Furthermore, older female autoimmune mice with elevated levels of IgG antinuclear antibodies and severe lupus-like renal disease did not demonstrate evidence of a global tolerance defect. We also found that the levels of residual V beta 17a+ cells in MHC-matched control F1 strains were further reduced by up to 80% in autoimmune (NZB x SWR)F1 mice. The greater in vivo elimination corresponded to an enhanced ability of NZB spleen cells, compared with other H-2d spleen cells, to stimulate V beta 17a+ hybridomas in vitro. The increased stimulation in culture could not be attributed to quantitative differences in I-E Ag expression. The results suggest that autoreactive T cells have been eliminated in these autoimmune mice by normal mechanisms of self-tolerance. Furthermore, the data demonstrate the existence of an NZB minor locus not present in other H-2d strains that influences T cell repertoire and enhances stimulation of T cells potentially reactive to self class II MHC Ag.     

Characterization of a new, potent, immunopathogenic epitope in S-antigen that elicits T cells expressing V beta 8 and V alpha 2-like genes.

Experimental autoimmune uveoretinitis (EAU) is a predominantly T cell-mediated autoimmune disease induced in susceptible animals by active immunization with human or bovine retinal S-Ag or by passive transfer of activated S-Ag or peptide-specific CD4+ T cells. During the course of studies aimed at the identification of T cell and B cell recognition sites in bovine and human S-Ag, a new potent uveitogenic region, located near the carboxy terminus of the molecule, was identified and characterized. Analysis of several synthetic peptides from this region showed that a 14 amino acid residue peptide, BSAg339-352, was highly uveitogenic when injected with adjuvants into Lewis rats. A uveitogenic T cell line, R737, was raised by in vitro selection of lymphocytes from animals immunized with peptide BSAg333-352. Northern blot analysis of mRNA from the R737 T cell line was positive for the rat homologs of murine V beta 8 and V alpha 2 T cell receptor gene probes. Whereas peptide BSAg339-352 defined the pathogenic site, nonpathogenic, proliferative sites were found in close physical association. This region is immediately adjacent to previously characterized pathogenic and proliferative sites contained in residues BSAg352-364. These results, as well as our previous observations, show S-Ag to be a complex molecule with several highly conserved amino acid sequences that can elicit pathogenic T cells with restricted T cell receptor V gene usage capable of active and passive elicitation of experimental autoimmune uveoretinitis.     

I-E-independent deletion of V beta 17a+ T cells by Mtv-3 from the nonobese diabetic mouse.

Analysis of TCR beta-chain V region (V beta) frequency among NOD lymphocytes reveals a profound depletion of V beta 3+ T cells, and a recent study has linked this phenomenon to the Mtv-3 insertion on chromosome 11. When the V beta 17a gene segment is introduced into mice with an nonobese diabetic mouse background, T cells bearing the TCR encoded by this gene segment are also dramatically reduced in frequency. Deletion of V beta 17a+ T cells segregates with deletion of T cells bearing V beta 3 and occurs in the absence of I-E, which had been shown in previous studies to be a major deleting element for V beta 17a+ thymocytes.     

Overexpression of select T cell receptor V beta gene families within CD4+ and CD8+ T cell subsets of myasthenia gravis patients: a role for superantigen(s)?

BACKGROUND: The principal symptoms of myasthenia gravis (MG), muscle weakness and fatigue due to impaired neuromuscular transmission, are caused by autoantibodies to the muscle nicotinic acetylcholine receptor (AChR). The mechanisms underlying the autoimmune response, however, appear to be initiated by activation of specific HLA class II-restricted CD4+ T lymphocytes. Thus, central to elucidating the causation of MG is determining how T cells are recruited to contribute to misguided immunological assaults on the major autoantigenic target, AChR. MATERIALS AND METHODS: By combining a polymerase chain reaction (PCR)-based strategy and Southern blot technique, we have analyzed the frequency of expression of 22 individual T cell receptor (TCR) V beta gene subfamilies in CD4+ and CD8+ peripheral blood T cell subsets derived from eight MG patients and seven healthy controls. The quantification of relative usage of individual TCR J beta gene segments was performed by hybridization of PCR-amplified products (specifically V beta 1-C beta) with a complete panel of 32P-5''-end-labeled J beta-specific oligonucleotide probes, followed by scanning analysis of autoradiographs. RESULTS: Comparisons of data obtained from V beta analyses of T cells from MG patients with those from healthy individuals established that MG patients significantly overexpressed V beta 1, V beta 13.2, V beta 17, and V beta 20 gene family members within both CD4+ and CD8+ T cell subpopulations. Moreover, analysis of the relative utilization of individual TCR J beta gene segments in V beta 1+/CD4+ and V beta 1+/CD8+ T lymphocytes revealed distribution patterns in patients indistinguishable from those recorded in the corresponding cell subsets derived from controls. CONCLUSIONS: T lymphocytes from MG patients displayed a biased overexpression of four TCR V beta gene segments: V beta 1, V beta 13.2, V beta 17, and V beta 20. The relative frequencies of association of individual V beta 1 (D beta) J beta combinations revealed that J beta gene usage in the V beta 1-over-represented T cell subsets had normal distribution patterns. It can thus be deduced that J beta gene segment products appear not to have a selective effect on the process leading to overexpression of V beta 1 exons in MG patients. Hence, our observations suggest a possible role for superantigen(s) in the T cell activation in MG patients.     

Interactions of thrombospondins with alpha4beta1 integrin and CD47 differentially modulate T cell behavior

Thrombospondin (TSP)-1 has been reported to modulate T cell behavior both positively and negatively. We found that these opposing responses arise from interactions of TSP1 with two different T cell receptors. The integrin alpha4beta1 recognizes an LDVP sequence in the NH2-terminal domain of TSP1 and was required for stimulation of T cell adhesion, chemotaxis, and matrix metalloproteinase gene expression by TSP1. Recognition of TSP1 by T cells depended on the activation state of alpha4beta1 integrin, and TSP1 inhibited interaction of activated alpha4beta1 integrin on T cells with its counter receptor vascular cell adhesion molecule-1. The alpha4beta1 integrin recognition site is conserved in TSP2. A recombinant piece of TSP2 containing this sequence replicated the alpha4beta1 integrin-dependent activities of TSP1. The beta1 integrin recognition sites in TSP1, however, were neither necessary nor sufficient for inhibition of T cell proliferation and T cell antigen receptor signaling by TSP1. A second TSP1 receptor, CD47, was not required for some stimulatory responses to TSP1 but played a significant role in its T cell antigen receptor antagonist and antiproliferative activities. Modulating the relative expression or function of these two TSP receptors could therefore alter the direction or magnitude of T cell responses to TSPs.     

Thymic selection defines multiple T cell receptor V beta 'repertoire phenotypes' at the CD4/CD8 subset level

We describe here the use of a sensitive and accurate multiprobe V beta RNase protection assay in characterizing the expression levels of 17 V beta genes in separated CD4+ and CD8+ subsets of selected mouse strains. The IE-reactive V beta genes (V beta s 11, 12, 5.1 and 16) showed various patterns of skewed subset expression in different strains, suggesting additional influences of IA, class I, and non-MHC genes in the selection process. Clonal deletion of V beta 11- and V beta 12-bearing T cells, among others, was skewed strongly towards the CD4+ subset in many IE+ mouse strains, supporting the notion that negative selection can cause incomplete, subset biased, V beta clonal deletions. Broad analysis in separated CD4+ and CD8+ subsets gave improved resolution of V beta repertoire selection, and revealed significant strain and/or subset specific skewing for additional V beta genes; with consistent bias towards higher expression of V beta 7 and V beta 13 in the CD8+ subset, and V beta 15 in the CD4+ subset of most mouse strains. The influence of diverse non-MHC ligands in V beta repertoire selection was further illustrated by the identification of unique V beta repertoires for six different MHC-identical (H2k) strains. Such polymorphisms in TCR repertoire expression may help to define better disease susceptibility phenotypes.