Autoreactive human T cell lines recognizing ribosomal protein L7.
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Sera of patients suffering from systemic lupus erythematosus (SLE) frequently contain oligoclonal IgG autoantibodies with high affinity for the ribosomal protein L7 (rpL7). The humoral autoimmune response to rpL7 apparently is driven by antigen and T cell dependent. In order to analyze the T cell response to rpL7 we cultured peripheral blood lymphocytes of healthy individuals and SLE patients in the presence of recombinant rpL7. After 10 days, the cytokine response to re-stimulation with rpL7 was examined using a spot-ELISA. Measuring IFN-gamma secretion, the T cells of two patients and four healthy donors showed a significant increase in the number of spots as compared to control cells. Secretion of IL-4 or IL-10 was not detected. From the antigen-stimulated primary cultures we established by limiting dilution cloning six rpL7-reactive, IFN-gamma-secreting T cell lines which show a CD3+CD4+CD8- phenotype. One line additionally was shown to be positive for HLA-DR and CD45R0, but negative for CD27 and CD31. The cell lines carry alphabeta TCR chains which differ from each other in sequence and specificity. rpL7 fragments rich in basic amino acids could be identified as epitopes recognized by the TCR of three cell lines. Recognition of rpL7 is HLA-DR6 restricted or respectively HLA-DP restricted in the two cell lines analyzed. (+info)
Differential effects of manipulating signaling in early T cell development in intestinal intraepithelial lymphocytes and thymocytes.
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A pre-TCR-CD3 signal is required for the efficient maturation of CD4- CD8- thymocytes to the CD4+ CD8+ stage. This study addressed whether a similar signal is required for maturation of intestinal intraepithelial lymphocytes (IEL) that may develop extrathymically. We have shown previously that IEL from mice deficient for CD3- associated zeta chains include an immature population of CD3- CD8alphaalpha+ cells expressing cytoplasmic TCR beta chains but lacking detectable surface TCRalphabeta, CD16 and B220. Here we stimulated the appearance of such IEL in epsilon+/- zeta-/- mice by expression of an activated Lck transgene or in vivo treatment with anti-CD3epsilon. Anti-CD3epsilon treatment of RAG-deficient animals also yielded CD16- B220- IEL. In contrast, expression of a TCRbeta transgene in rag-1(-/-) mice did not stimulate the appearance of CD3- CD8alphaalpha+ CD16- B220- cells. Taken together these data indicate that although anti-CD3epsilon treatment and LckF505 assist in catalyzing a CD16+ B220+ --> CD16- B220- transition, these manipulations are not equivalent to a pre-TCR signal in IEL lymphocytes. (+info)
Mutual activation of Ets-1 and AML1 DNA binding by direct interaction of their autoinhibitory domains.
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The transcription factors Ets-1 and AML1 (the alphaBl subunit of PEBP2/CBF) play critical roles in hematopoiesis and leukemogenesis, and cooperate in the transactivation of the T cell receptor (TCR) beta chain enhancer. The DNA binding capacity of both factors is blocked intramolecularly but can be activated by the removal of negative regulatory domains. These include the exon VII domain for Ets-1 and the negative regulatory domain for DNA binding (NRDB) for alphaB1. Here we report that the direct interaction between the two factors leads to a reciprocal stimulation of their DNA binding activity and activation of their transactivation function. Detailed mapping revealed two independent contact points involving the exon VII and NRDB regions as well as the two DNA binding domains. Using deletion variants and dominant interfering mutants, we demonstrate that the interaction between exon VII and NRDB is necessary and sufficient for cooperative DNA binding. The exon VII and NRDB motifs are highly conserved in evolution yet deleted in natural variants, suggesting that the mechanism described is of biological relevance. The mutual activation of DNA binding of Ets and AML1 through the intermolecular interaction of autoinhibitory domains may represent a novel principle for the regulation of transcription factor function. (+info)
Regulation of T cell receptor (TCR) beta gene expression by CD3 complex signaling in immature thymocytes: implications for TCRbeta allelic exclusion.
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During alphabeta thymocyte development, clonotype-independent CD3 complexes are expressed at the cell surface before the pre-T cell receptor (TCR). Signaling through clonotype-independent CD3 complexes is required for expression of rearranged TCRbeta genes. On expression of a TCRbeta polypeptide chain, the pre-TCR is assembled, and TCRbeta locus allelic exclusion is established. We investigated the putative contribution of clonotype-independent CD3 complex signaling to TCRbeta locus allelic exclusion in mice single-deficient or double-deficient for CD3zeta/eta and/or p56(lck). These mice display defects in the expression of endogenous TCRbeta genes in immature thymocytes, proportional to the severity of CD3 complex malfunction. Exclusion of endogenous TCRbeta VDJ (variable, diversity, joining) rearrangements by a functional TCRbeta transgene was severely compromised in the single-deficient and double-deficient mutant mice. In contrast to wild-type mice, most of the CD25(+) double-negative (DN) thymocytes of the mutant mice failed to express the TCRbeta transgene, suggesting defective expression of the TCRbeta transgene similar to endogenous TCRbeta genes. In the mutant mice, a proportion of CD25(+) DN thymocytes that failed to express the transgene expressed endogenous TCRbeta polypeptide chains. Many double-positive cells of the mutant mice coexpressed endogenous and transgenic TCRbeta chains or more than one endogenous TCRbeta chain. The data suggest that signaling through clonotype-independent CD3 complexes may contribute to allelic exclusion of the TCRbeta locus by inducing the expression of rearranged TCRbeta genes in CD25(+) DN thymocytes. (+info)
Unique superantigen activity of staphylococcal exfoliative toxins.
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Certain strains of Staphylococcus aureus express one or both of two related, but immunologically distinct, exfoliative toxins (ETA and ETB). These toxins induce the symptoms associated with staphylococcal scalded skin syndrome. Both ETs have been shown to stimulate T cell proliferation. Recently, it was reported that ETA is a superantigen that stimulates T cells bearing human Vbeta2 or several murine Vbetas. However, other investigators have proposed that the superantigenicity reported for ETA resulted from contaminants in commercial preparations. This present study addresses those conflicting reports by assessing the biological and immunologic activities of highly purified rETs. ETA and ETB required APCs to induce selective polyclonal expansion of several human Vbetas (huVbetas), although, neither toxin expanded huVbeta2. ETB induced expansion of murine T cells bearing Vbetas 7 and 8, those that have the highest homology to the huVbetas expanded by ETA and ETB. Although flow cytometry of ETB-stimulated T cells matched PCR results, stimulation by ETA reduced percentages of T cells positive for several huVbetas that had been shown to have increased levels of mRNA transcripts. ETA and ETB induced contrasting reactions in vivo. In rabbits, ETB was moderately pyrogenic and enhanced susceptibility to lethal shock, while ETA lacked both activities. Predictions based on comparisons with other superantigens suggest molecular regions potentially involved in receptor binding in the ETA crystal structure and a modeled ETB three-dimensional structure. These results show that ETs are superantigens with unique properties that could account for the discrepancies reported. (+info)
Two mechanisms for the non-MHC-linked resistance to spontaneous autoimmunity.
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Genetic susceptibility and resistance to most autoimmune disorders are associated with highly polymorphic genes of the MHC and with non-MHC-linked polygenic modifiers. It is known that non-MHC-linked polymorphisms can override or enhance the susceptibility to an autoimmune disease provided by pathogenic MHC genes, but the mechanisms remain elusive. In this study, we have followed the fate of two highly diabetogenic beta cell-specific T cell receptors (Kd and I-Ag7 restricted, respectively) in NOR/Lt mice, which are resistant to autoimmune diabetes despite expressing two copies of the diabetogenic MHC haplotype H-2g7. We show that at least two mechanisms of non-MHC-linked control of pathogenic T cells operate in these mice. One segregates as a recessive trait and is associated with a reduction in the peripheral frequency of diabetogenic CD8+ (but not CD4+) T cells. The other segregates as a dominant trait and is mediated by IL-4- and TGF-beta1-independent immune suppressive functions provided by lymphocytes that target diabetogenic CD4+ and CD8+ T cells, without causing their deletion, anergy, immune deviation, or ignorance. These results provide explanations as to how non-MHC-linked polymorphisms can override the susceptibility to an autoimmune disease provided by pathogenic MHC haplotypes, and demonstrate that protective non-MHC-linked genes may selectively target specific lymphoid cell types in cellularly complex autoimmune responses. (+info)
Dramatic influence of V beta gene polymorphism on an antigen-specific CD8+ T cell response in vivo.
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According to recent crystallographic studies, the TCR-alpha beta contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1 beta (CDR1 beta) and the hypervariable (D)J-encoded CDR3 beta and CDR3 alpha domains. To evaluate directly the relative importance of CDR1 beta polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic V beta a and V beta b mouse strains that differ by a CDR1 polymorphism in the V beta 10 gene segment. The V beta 10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in V beta a compared with V beta b mice, as measured either by the expansion of V beta 10+ cells or by the binding of MHC-peptide tetramers. These data indicate that V beta polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1 beta-peptide interactions. (+info)
Control of V(D)J recombinational accessibility of the D beta 1 gene segment at the TCR beta locus by a germline promoter.
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The germline promoter region upstream of the D beta 1 gene segment in the murine TCR beta locus was deleted to assess its role in controlling V(D)J recombination. Associated with diminished D beta 1 region germline transcription, rearrangement of the D beta 1 but not the D beta 2 gene segment was reduced 10- to 20-fold. A corresponding reduction in RAG-mediated cleavage at the D beta 1 and J beta 1 signal sequences was apparent only when purified CD4- CD8- thymocytes were analyzed because, as we demonstrate, cleavage at these gene segments also occurred in CD4+ CD8+ thymocytes. These findings suggest that germline promoters regulate localized accessibility of gene segments for recombination and that in CD4+ CD8+ thymocytes TCR beta allelic exclusion does not result from inaccessibility of D beta gene segments. (+info)