Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. Labeling of the alpha-chain correlates with J alpha segment usage

Using a direct binding assay based on photoaffinity labeling, we studied the interaction of T cell receptor (TCR) with a Kd-bound photoreactive peptide derivative on living cells. The Kd-restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was reacted NH2-terminally with biotin and at the TCR contact residue Lys259 with photoreactive iodo, 4-azido salicylic acid (IASA) to make biotin-YIPSAEK(IASA)I. Cytotoxic T lymphocyte (CTL) clones derived from mice immunized with this derivative recognized this conjugate, but not a related one lacking the IASA group nor the parental PbCS peptide. The clones were Kd restricted. Recognition experiments with variant conjugates, lacking substituents from IASA, revealed a diverse fine specificity pattern and indicated that this group interacted directly with the TCR. The TCR of four clones could be photoaffinity labeled by biotin-YIPSAEK(125IASA)I. This labeling was dependent on the conjugates binding to the Kd molecule and was selective for the TCR alpha (2 clones) or beta chain (1 clone), or was common for both chains (1 clone). TCR sequence analysis showed a preferential usage of J alpha TA28 containing alpha chains that were paired with V beta 1 expressing beta chains. The TCR that were photoaffinity labeled at the alpha chain expressed these J alpha and V beta segments. The tryptophan encoded by the J alpha TA28 segment is rarely found in other J alpha segments. Moreover, we show that the IASA group interacts preferentially with tryptophan in aqueous solution. We thus propose that for these CTL clones, labeling of the alpha chain occurs via the J alpha-encoded tryptophan residue.     

The glutamate dehydrogenase, E74 and putative actin gene loci in the Drosophila montium subgroup. Chromosomal homologies among the montium species and D. melanogaster

Myasthenia gravis (MG) is a T-cell-regulated autoimmune disease in which a pathological autoantibody response is mounted against the nicotinic acetylcholine receptor of the neuromuscular junction. Our laboratory previously identified a T cell epitope, p195-212, derived from the human acetylcholine receptor alpha subunit, which triggered PBL to proliferate from about 70% of MG patients tested. p195-212 was also found to be an immunodominant T cell epitope in SJL mice and a cryptic epitope in C3H.SW mice. Inoculation of naive SJL mice with cells from a p195-212-specific syngeneic T cell line caused MG-related autoimmune manifestations in those mice. In these studies we analyzed TCR alpha and beta chain sequences used by T cell lines and clones from both high- and low-responder mouse strains in response to p195-212. T cell lines generated from either strain expressed single TCR V beta gene segments (V beta 17 in SJL mice and V beta 8 in C3H.SW mice). By deleting V beta 17-expressing T cells in p195-212-immunized SJL mice we established a T cell line that expressed the V beta 6 gene product, suggesting that SJL mice are not limited to using a single V beta gene segment in response to p195-212. In addition, we found that N- and/or C-terminal-truncated peptides of p195-212, presented under the same culture conditions to different clones bearing the same TCR alpha beta chain, could elicit very different proliferative responses from the clones. Thus, even within a constrained system, factors other than TCR sequence contribute to the differential stimulation of T cell responses.     

Inactivation of T cell receptor peptide-specific CD4 regulatory T cells induces chronic experimental autoimmune encephalomyelitis (EAE)

T cell receptor (TCR)-recognizing regulatory cells, induced after vaccination with self-reactive T cells or TCR peptides, have been shown to prevent autoimmunity. We have asked whether this regulation is involved in the maintenance of peripheral tolerance to myelin basic protein (MBP) in an autoimmune disease model, experimental autoimmune encephalomyelitis (EAE). Antigen-induced EAE in (SJL x B10.PL)F1 mice is transient in that most animals recover permanently from the disease. Most of the initial encephalitogenic T cells recognize MBP Ac1-9 and predominantly use the TCR V beta 8.2 gene segment. In mice recovering from MBP-induced EAE, regulatory CD4+ T cells (Treg) specific for a single immunodominant TCR peptide B5 (76-101) from framework region 3 of the V beta 8.2 chain, become primed. We have earlier shown that cloned B5-reactive Treg can specifically downregulate responses to Ac1-9 and also protect mice from EAE. These CD4 Treg clones predominantly use the TCR V beta 14 or V beta 3 gene segments. Here we have directly tested whether deletion/blocking of the Treg from the peripheral repertoire affects the spontaneous recovery from EAE. Treatment of F1 mice with appropriate V beta-specific monoclonal antibodies resulted in an increase in the severity and duration of the disease; even relapses were seen in one-third to one-half of the Treg-deleted mice. Interestingly, chronic disease in treated mice appears to be due to the presence of Ac1-9-specific T cells. Thus, once self-tolerance to MBP is broken by immunization with the antigen in strong adjuvant, TCR peptide-specific CD4 Treg cells participate in reestablishing peripheral tolerance. Thus, a failure to generate Treg may be implicated in chronic autoimmune conditions.     

T-cell receptor V region beta-chain gene expression in the autoimmune thyroiditis of non-obese diabetic mice

The non-obese diabetic (NOD) mouse develops both a spontaneous T-cell-mediated autoimmune insulitis and, in addition, a well characterized thyroiditis. We have examined the repertoire of murine T-cell receptor (TCR) variable (V) beta-chain genes used by intrathyroidal T cells with specific oligonucleotides that amplified 17 murine V beta gene families in cDNA samples prepared from intact NOD thyroid tissues. Normal NOD thyroid tissue contained only low levels of TCR V gene mRNA. In contrast, NOD mice with histologic thyroiditis showed the marked expression of up to 3 TCR V beta genes consistent with a restricted T-cell invasion. Sequencing of amplified TCR V beta cDNA showed that within each NOD thyroid sample at least one of the overexpressed V beta gene families was clonally expanded. However, the clonally expanded T-cell V gene family was not consistent in all animals. Even within the same TCR V beta gene families, various D and J segments had been rearranged with open reading frames and together with insertions and deletions gave no significant homology at the nucleotide or amino acid level. In summary, these data showed that the intrathyroidal T-cell infiltrate in NOD mice was markedly biased towards the use of a single, but variable, TCR V gene family within each animal. It also appeared that the choice of the TCR V beta chain determined the intrathyroidal infiltrative process rather than the choice of D and/or J regions. However, there was no consistent use of a single TCR V beta chain. As thyroiditis does not occur uniformly in apparently genetically homogeneous animals, reared under similar environmental conditions, it may not be surprising that different TCR V genes are involved in different animals.     

Role of TCR V alpha 24 J alpha Q+ T cells in autoimmune diseases

We investigated the role of T cell receptor (TCR) V alpha 24+ T cells in the pathogenesis of systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). The invariant V alpha 24 J alpha Q was expanded and comprised 50-90% of the total V alpha 24 in healthy individuals. In contrast, patients with SSc and SLE showed the selective reduction of the invariant V alpha 24 J alpha Q with oligoclonal expansion of V alpha 24 TCR other than V alpha 24 J alpha Q. Because human V alpha 24 J alpha Q TCR is analogous to murine invariant V alpha 14 J alpha 281 TCR which is the major genotype of NK T cells, these results suggest that the selective reduction of T cells with invariant V alpha 24 J alpha Q TCR might play an important role of the generation of autoreactive T cells including T cells bearing other V alpha 24 TCR in autoimmune disease patients.     

A TCR alpha chain transgene induces maturation of CD4- CD8- alpha beta+ T cells from gamma delta T cell precursors

The proportion of CD4- CD8- double-negative (DN) alpha beta T cells is increased both in the thymus and in peripheral lymphoid organs of TCR alpha chain-transgenic mice. In this report we have characterized this T cell population to elucidate its relationship to alpha beta and gamma delta T cells. We show that the transgenic DN cells are phenotypically similar to gamma delta T cells but distinct from DN NK T cells. The precursors of DN cells have neither rearranged endogenous TCR alpha genes nor been negatively selected by the MIsa antigen, suggesting that they originate from a differentiation stage before the onset of TCR alpha chain rearrangements and CD4/CD8 gene expression. Neither in-frame V delta D delta J delta nor V gamma J gamma rearrangements are over-represented in this population. However, since peripheral gamma delta T cells with functional TCR beta gene rearrangements have been depleted in the transgenics, we propose that the transgenic DN population, at least partially, originates from the precursors of those cells. The present data lend support to the view that maturation signals to gamma delta lineage-committed precursors can be delivered via TCR alpha beta heterodimers.     

Importance of a conserved TCR J alpha-encoded tyrosine for T cell recognition of an HLA B27/peptide complex

Human HLA B27-restricted cytotoxic T lymphocytes (CTL) specific for the influenza A epitope NP383-391 use similar TCR alpha and beta chains, with two closely related J alpha segments used by six of nine CTL clones from three unrelated donors (Bowness et al., Eur J. Immunol. 1993. 23: 1417-1421). The role of TCR complementarity-determining region (CDR)3alpha residues 93 and 100-102 was examined by site-directed mutagenesis, following expression of the TCR alpha and beta extracellular domains from one clone as a TCR zeta fusion heterodimer in rat basophil leukemia (RBL) cells. For the first time we have measured direct binding of tetrameric HLA B*2705/NP383-391 complexes to transfected TCR. Independently peptide-pulsed antigen-presenting cells (APC) were used to induce TCR-mediated degranulation of RBL transfectants. Our results show a key role for the conserved TCRalpha CDR3 J alpha-encoded residue Y102 in recognition of HLA B27/NP383-391. Thus the Y102D mutation abolished both tetramer binding and degranulation in the presence of peptide-pulsed APC. Even the Y102F mutation, differing only by a single hydroxyl group from the native TCR, abolished detectable degranulation. Further mutations F93A and S100R also abolished recognition. Interestingly, the N101A mutation recognized HLA B27/NP in functional assays despite having significantly reduced tetramer binding, a finding consistent with "kinetic editing" models of T cell activation. Modeling of the GRb TCR CDR3alpha loop suggests that residue Y102 contacts the HLA B*2705 alpha1 helix. It is thus possible that selection of germ-line TCRAJ-encoded residues at position 102 may be MHC driven.     

Preferential but not exclusive T cell receptor V beta chain utilization of myelin basic protein and peptide-specific T cell clones in mice

The repertoire of T cell receptor (TCR) V beta chain utilization was investigated in PL/J, CXJ-1, SJL/J and B10.S-->SJL/J chimeric mice in response to either myelin basic protein (MBP) or the strain-specific encephalitogenic peptide. Our analysis showed that there was an overlapping predominance in the TCR V beta gene utilization in the MBP-specific responses, which were independent of the major histocompatibility complex (MHC) class II haplotype present, and the immunodominant peptide region recognized in these different strains. In those mice having the TCR V beta b haplotype (PL/J, CXJ-1, and the B10.S-->SJL chimera) either the TCR V beta 4, 8, and 13 or the TCR V beta 4, 6, and 13 predominated. In contrast, in mice with TCR V beta a haplotype (SJL/J) V beta 4, 6, and 17a were found. However, the quantitative distribution of these preferentially utilized TCR V beta chains in each strain was defined by the MHC class II haplotype and the immunodominant peptide recognized. The expression of the V beta 8 gene product in the peripheral TCR repertoire did not always correlate with predominant V beta 8 utilization in the MBP-specific response.     

Recurrent T cell receptor rearrangements in the cytotoxic T lymphocyte response in vivo against the p815 murine tumor

P815 is a murine mastocytoma of DBA/2 origin which, although immunogenic, rapidly develops as a tumor in immunocompetent syngeneic hosts. In this report, we have studied, by a molecular approach, the in vivo alpha/beta T cell response to P815. Both situations of tumor growth after engraftment of naive animals or tumor rejection by preimmunized animals have been analyzed. The spectrum of T cell receptor beta chain rearrangements in the tumor-infiltrating lymphocytes was found to be highly variable among individual tumor-bearing mice. However, two rearrangements, one using V(beta)1 and J(beta)1.2 segments and one using the V(beta)1 and J(beta)2.5 segments, with conserved junctional regions, reproducibly emerge in most individuals. These two rearrangements thus correspond to "public" (recurrent) T cell clones, as opposed to "private" ones, which emerge in a seemingly stochastic fashion in immunized animals. Importantly, these public cells are observed in situations of either growth or rejection of the tumor. Quantification provides a clear increase in public T cells in secondary responses, but no obvious correlation provides between their level and primary tumor rejection. The V(beta)1- J(beta)1.2 rearrangement is borne by CTL directed against an antigen derived from P1A, a nonmutated mouse self protein which is expressed in P815 but not in normal mouse tissues except testis. A recurrent, public T cell response can thus be observed to an antigen derived from a self protein expressed by a tumor.     

Granulocyte-macrophage colony-stimulating factor abrogates transforming growth factor-beta 1-mediated cell cycle arrest by up-regulating cyclin D2/Cdk6

Beh?et's disease (BD) is a chronic multisystemic inflammatory disorder characterized mainly by recurrent oral and genital aphthous ulcerations and uveitis. Etiology and pathogenesis of BD remain unknown. T cell receptor (TCR) V alpha/V beta gene product expression as well as Jbeta gene segment expression in peripheral blood of BD patients were analysed to investigate the possible role of T lymphocytes in the etiopathogenesis of BD. Flow cytometry with 12 TCR V-specific MoAbs was used for TCRV analyses. Jbeta gene segment usage by T cell populations expressing certain V betas was determined by polymerase chain reaction (PCR) technique with V beta- and C beta-specific primers, Southern blotting of PCR products, and subsequent hybridization with radiolabelled Jbeta gene segment-specific probes. Although 13 of the 23 BD patients exhibited increases in expression of one or more TCR V-gene products, only expansions among the CD4+ T cell subset were significantly more frequent in BD patients (7/23) compared with healthy controls (0/15) (P = 0.019). Six out of eight cases followed for up to 20 months had at least one expansion correlated with disease activity. A strict preference for particular Jbeta gene segments implicating clonality was apparent in all analysed T cell expansions and correlated well with disease activity. These results suggest a possible involvement of antigen-specific T lymphocytes in the pathogenesis of BD.     

The British Medical Bulletin 1943-1993. A guide to medical science and thought in Britain

CD4-CD8- (double negative [DN]) alpha/beta T cells are a largely uncharacterized subpopulation of unknown function. To investigate whether these cells are selected to recognize particular antigens or antigen-presenting molecules, DN alpha/beta T cells were purified from the peripheral blood of five normal donors and their T cell receptor (TCR) alpha and beta chains were examined. Random cloning of TCR alpha chains by single-sided polymerase chain reaction (PCR) amplification identified an invariant rearrangement between V alpha 24 and J alpha Q, with no N region diversity, which was expressed preferentially by DN alpha/beta T cells from all donors. Random cloning also identified a precise V alpha 7.2-J alpha (IGRJa14) rearrangement, with two variable amino acids encoded in the V-J junction, which was enriched in the DN alpha/beta T cell preparations from some, but not all, donors. Analysis of TCR beta chains by quantitative PCR amplification demonstrated that the expression of four V beta gene families, V beta 2, 8, 11, and 13, was markedly increased in these DN alpha/beta T cell preparations. The expression of particular TCRs by DN alpha/beta T cells from multiple donors indicates that these cells, or at least a subpopulation of cells with this phenotype, recognize a limited spectrum of antigens and suggests that they may use nonpolymorphic antigen-presenting molecules.     

Evidence for superantigen involvement in insulin-dependent diabetes mellitus aetiology

Insulin-dependent diabetes mellitus (IDDM) is a T-cell-mediated autoimmune disease whose onset is believed to be triggered by unknown environmental factors acting on a predisposing genetic background. Islet-infiltrating T (IIT) cells from two IDDM patients, who had died at the onset of the disease from brain swelling as a complication of ketoacidosis, were analysed. The results provided evidence for the involvement of a pancreatic islet cell membrane-bound superantigen as a diabetes aetiopathogenetic factor. There was a selective expansion of a T-cell receptor (TCR) variable segment of the beta-chain (V beta 7) in these IIT cells in association with unselected V alpha-chain segments; extensive junctional diversity of the TCR V beta 7 chains; and evidence of positive selection, after exposure to diabetic islet cell membrane preparations, of V beta 7+ T-cell clones among peripheral blood lymphocytes from non-diabetic individuals.     

T cell receptor beta chain dimers on immature thymocytes from normal mice

During T cell development the T cell receptor (TCR) beta chain is expressed before the TCR alpha chain. Experiments in TCR beta transgenic severe combined immune deficiency (SCID) mice have shown that the TCR beta protein can be expressed on the cell surface of immature thymocytes in the absence of the TCR alpha chain and that the TCR beta protein controls T cell development with regard to cell number, CD4/CD8 expression and allelic exclusion of the TCR beta chain. Subsequent experiments have shown that on the surface of thymocytes from TCR beta transgenic SCID mice the TCR beta protein can be expressed in a monomeric and dimeric form whereas only the dimeric form was found on the surface of a TCR beta-transfected, immature T cell line. The results presented here show that normal thymocytes from 16-day-old fetuses likewise express only the dimeric form and that the monomeric form on the surface of thymocytes from transgenic mice results from glycosyl phosphatidylinositol linkage. Our results show for the first time that under physiological conditions a TCR beta dimer can be expressed on the cell surface without the TCR alpha chain.     

T cell receptor heterogeneity in gamma delta T cell clones from intestinal biopsies of patients with celiac disease

In celiac disease large numbers of gamma delta T lymphocytes infiltrate the intestinal epithelia. We have isolated intestinal gamma delta T cell clones from patients with celiac disease and have analyzed their T cell receptor repertoire. T cell lines and clones were obtained from jejunal biopsies of 14 celiac patients and 12 individuals without celiac disease. These were analyzed by staining with monoclonal antibodies against CD3, alpha beta and gamma delta T cell receptor, by Southern blot with gamma- and delta-specific probes and by polymerase chain reaction using V delta-specific oligonucleotides. Intestinal gamma delta cells from patients with celiac disease differed from those of controls with normal jejunal histology in that V delta 1+ cells and V delta 1-V delta 2- cells were significantly increased. There was no evidence of the expansion of one or more clones expressing particular types of gamma delta T cell receptor.