Specificity for molecules of the major histocompatibility complex mediated by a hybrid immunoglobulin-T cell receptor.

A chimeric T-cell receptor (TCR) alpha-chain gene was produced by shuffling the immunoglobulin VDJH from a 40-140 digoxin-specific hybridoma onto alpha-chain constant region (C alpha) exons. This hybrid immunoglobulin-TCR gene was used to produce transgenic mice. Previous results indicated that this chimeric gene encoded a polypeptide that associated with endogenously encoded beta chains to form a hybrid TCR. T cells expressing this receptor could be stimulated with antibodies specific for CD3 or the 40-140 idiotype (Id40-140), and also with digoxin coupled to bovine serum albumin (digoxin-BSA). We were interested in determining whether a hybrid receptor such as this could also recognize the natural ligand of T cells, namely allelic variants of major histocompatibility complex (MHC) molecules. A T-cell hybridoma was produced that expressed a hybrid receptor with specificity for an IAk-encoded determinant, digoxin-BSA, or staphyloccocal enterotoxin B. Transfection experiments showed that the specificity for MHC determinants was dependent on both the hybrid alpha chain and a particular beta chain. These results indicate that a V beta domain combined with a VH domain can produce a receptor capable of reacting with MHC molecules, and at the same time retain specificities mediated by the beta chain and alpha chain alone. A conclusion is that the pervasive MHC specificity of the TCR is not unique to the family of TCR heterodimers, but is selected, and can be mediated by immunoglobulin domains.     

TCR beta and TCR alpha gene enhancers confer tissue- and stage-specificity on V(D)J recombination events.

We describe transgenic mice carrying germline variable gene segments associated with either the T cell receptor (TCR) beta or alpha gene enhancers (E beta or E alpha). Transgenic constructs underwent high rates of site-specific rearrangements predominantly in T cells from independent mice. Rearrangements of the E beta-containing transgenes began at different stages of T cell differentiation in embryonic and adult thymus than did the E alpha-containing ones, with a pattern superimposable upon the patterns of TCR beta or TCR alpha gene expression, respectively. We demonstrate that sequences within the TCR beta and TCR alpha gene enhancers confer tissue- and stage-specificity upon the V(D)J recombination events affecting adjacent gene segments. The patterns of transgene expression also gave information on developmental events and lineage relationships (gamma delta versus alpha beta) during T cell development.     

In vivo overexpression of Dad1, the defender against apoptotic death-1, enhances T cell proliferation but does not protect against apoptosis.

The Dad1 protein has been shown to play a role in prevention of apoptosis in certain cell types. Dad1 is also a subunit of the oligosaccharyltransferase enzyme complex that initiates N-linked glycosylation. It is encoded by a gene located adjacent to the TCR alpha and delta genes on mouse chromosome 14. We have investigated the role of Dad1 during T cell development and activation. We observe that endogenous Dad1 levels are modulated during T cell development to reach maximal expression in mature thymocytes. Transgenic mice that overexpress Dad1 in both the thymus and peripheral immune system have been generated. Apoptosis of thymocytes from such mice is largely unaffected, but peripheral T cells display hyperproliferation in response to stimuli. Therefore, the linkage between the TCR and Dad1 genes may have important consequences for T cell function.     

T cell receptor genes and T cell development in virus-transformed early T cell lines

We have derived T cell lines from mice inoculated with Gross leukemia virus, which appear to represent early T cell developmental stages and to reflect normal T cell development. These cell lines may provide a breakthrough in the study of T cell development as Abelson transformants have done for the study of B cell development. Analysis of the TCR gene expression in these cell lines reveals that the sequence of rearrangement and expression of each TCR gene is not strictly ordered. Expression of RNA for the TCR alpha and -beta genes appears to be coordinated with rearrangement at the alpha and beta loci. This is not the case for gamma gene expression. Availability of the homogeneous populations of cells represented in these cells lines allows for a more detailed molecular analysis of T cell development than was previously possible.     

The unfolding story of T cell receptor gamma

Antigen-specific, major histocompatibility complex-restricted recognition by classical T cells is mediated by a T cell receptor (TCR) consisting of a disulfide-linked alpha beta heterodimer. During the search for the genes encoding the alpha and beta proteins, a third immunoglobulin-like gene, termed gamma, was uncovered. Like the TCR alpha and beta genes, the TCR gamma gene consists of variable and constant segments that rearrange during T cell development in the thymus. Although the physiological role of TCR gamma remains an enigma, much has been learned with the recent identification of the protein products of this gene family in both mice and humans. The gamma chain is associated with a partner chain, termed delta. The gamma delta heterodimer is associated with an invariant T3 complex, very similar to that associated with the alpha beta heterodimer, and appears predominantly, if not exclusively, on cells with a CD4-, CD8- phenotype both in the thymus and in the periphery. TCR gamma delta is the first T3-associated receptor to appear during thymocyte development and defines a separate T cell lineage distinct from alpha beta-bearing cells. Although TCR alpha beta-bearing cells and TCR gamma delta-bearing cells follow parallel developmental pathways, the diversity of expressed gamma delta receptors is extremely limited relative to that of alpha beta receptors.     

CD3 delta establishes a functional link between the T cell receptor and CD8

T cells expressing T cell receptor (TCR) complexes that lack CD3 delta, either due to deletion of the CD3 delta gene, or by replacement of the connecting peptide of the TCR alpha chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8 beta or tailless CD8 beta, we examined whether CD3 delta serves to couple TCR.CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252-260 peptide in the context of H-2K(d). We report that, in thymocytes and hybridomas expressing the T1 TCR.CD3 complex, CD8 alpha beta associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8 alpha alpha or a CD3 delta(-) variant of the T1 TCR. CD3 delta was selectively co-immunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3 delta. Because CD8 alpha beta is a raft constituent, due to this association a fraction of TCR.CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3 delta couples TCR.CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8(+) T cells.     

Monte Carlo simulations of voltage-driven translocation of a signal sequence

CD1d-deficient (CD1d-/-) mouse lymphocytes were analyzed to classify the natural killer T (NKT) cells without reactivity to CD1d. The cells bearing a V(alpha)19.1-J(alpha)26 (AV19-AJ33) invariant TCR alpha chain, originally found in the peripheral blood lymphocytes, were demonstrated to be abundant in the NK1.1+ but not NK1.1- T cell population isolated from CD1d-/- mice. Moreover, more than half (11/21) of the hybrid cell lines established from CD1d-/- NKT cells expressed the V(alpha)19.1-J(alpha)26 invariant TCR alpha chain. The expression of the invariant V(alpha)19.1-J(alpha)26 mRNA was absent in beta2-microglobulin-deficient mice. Collectively, the present findings suggest the presence of a second NKT cell repertoire characterized by an invariant TCR alpha chain (V(alpha)19.1-J(alpha)26) that is selected by an MHC class I-like molecule other than CD1d.     

Generation of CD4(+)CD25(+) regulatory T cells from autoreactive T cells simultaneously with their negative selection in the thymus and from nonautoreactive T cells by endogenous TCR expression

Normal T cell repertoire contains regulatory T cells that control autoimmune responses in the periphery. One recent study demonstrated that CD4(+)CD25(+) T cells were generated from autoreactive T cells without negative selection. However, it is unclear whether, in general, positive selection and negative selection of autoreactive T cells are mutually exclusive processes in the thymus. To investigate the ontogeny of CD4(+)CD25(+) regulatory T cells, neo-autoantigen-bearing transgenic mice expressing chicken egg OVA systemically in the nuclei (Ld-nOVA) were crossed with transgenic mice expressing an OVA-specific TCR (DO11.10). Ld-nOVA x DO11.10 mice had increased numbers of CD4(+)CD25(+) regulatory T cells in the thymus and the periphery despite clonal deletion. In Ld-nOVA x DO11.10 mice, T cells expressing endogenous TCR alpha beta chains were CD4(+)CD25(-) T cells, whereas T cells expressing autoreactive TCR were selected as CD4(+)CD25(+) T cells, which were exclusively dominant in recombination-activating gene 2-deficient Ld-nOVA x DO11.10 mice. In contrast, in DO11.10 mice, CD4(+)CD25(+) T cells expressed endogenous TCR alpha beta chains, which disappeared in recombination-activating gene 2-deficient DO11.10 mice. These results indicate that part of autoreactive T cells that have a high affinity TCR enough to cause clonal deletion could be positively selected as CD4(+)CD25(+) T cells in the thymus. Furthermore, it is suggested that endogenous TCR gene rearrangement might critically contribute to the generation of CD4(+)CD25(+) T cells from nonautoreactive T cell repertoire, at least under the limited conditions such as TCR-transgenic models, as well as the generation of CD4(+)CD25(-) T cells from autoreactive T cell repertoire.     

Development of the CD4 and CD8 lineage of T cells: instruction versus selection

T cells bearing the alpha beta T cell receptor (TCR) can be divided into CD4+8- and CD4-8+ subsets which develop in the thymus from CD4+8+ precursors. The commitment to the CD4 and CD8 lineage depends on the binding of the alpha beta TCR to thymic major histocompatibility complex (MHC) coded class II and class I molecules, respectively. In an instructive model of lineage commitment, the binding of the alpha beta TCR, for instance to class I MHC molecules, would generate a specific signal instructing the CD4+8+ precursors to switch off the expression of the CD4 gene. In a selective model, the initial commitment, i.e. switching off the expression of either the CD4 or the CD8 gene would be a stochastic event which is then followed by a selective step rescuing only CD4+ class II and CD8+ class I specific T cells while CD4+ class I and CD8+ class II specific cells would have a very short lifespan. The selective model predicts that a CD8 transgene which is expressed in all immature and mature T cells should rescue CD4+ class I MHC specific T cells from cell death. We have performed experiments in CD8 transgenic mice which fail to support a selective model and we present data which show that the binding of the alpha beta TCR to thymic class I MHC molecules results in up-regulation of the TCR in the CD4+8+ population. Therefore, these experiments are consistent with an instructive model of lineage commitment.     

Developmentally regulated rearrangement and expression of genes encoding the T cell receptor-T3 complex

Human leukemic cells corresponding to the earliest identifiable stages of intrathymic T cell differentiation lack cell surface expression of the T cell receptor(TCR alpha/beta)-T3 complex but transcribe TCR beta mRNA from either germ-line configuration (1/13) or partially (DJ) or fully (VDJ) rearranged (12/13) genes. These cells do not produce TCR alpha mRNA, but do contain T3 delta and T3 epsilon mRNA and accumulate T3 polypeptides, primarily in the perinuclear envelope. Equivalent normal T cells isolated from thymus have a predominantly germ-line configuration of TCR beta but contain intracellular T3 proteins. T3 gene expression is therefore a very early event in T cell differentiation. TCR alpha chain production appears to be the limiting maturation-linked event in the transport, assembly, and cell surface membrane insertion of the TCR alpha/beta-T3 complex.     

Smad-dependent cooperative regulation of interleukin 2 receptor alpha chain gene expression by T cell receptor and transforming growth factor-beta

The interleukin 2 receptor alpha chain (IL-2Ralpha) is a component of high affinity IL-2 receptors and thus critically regulates T cell growth and other lymphoid functions. Five positive regulatory regions together control lineage-restricted and activation-dependent IL-2Ralpha induction in response to antigen and IL-2. We now show that TGF-beta cooperates with T cell receptor (TCR) signaling to increase IL-2Ralpha gene expression. Moreover, we identify a sixth positive regulatory region that regulates IL-2Ralpha expression in cells treated with anti-CD3 + anti-CD28 as well as TGF-beta and show that this region contains binding sites for Smad3, AP-1, and cAMP-responsive element-binding protein/ATF proteins. The importance of Smad complexes is indicated by impaired IL-2Ralpha induction by TGF-beta in CD4+ T cells from both Smad3-/- and Smad4-/- mice. Thus, we have identified a novel positive regulatory region in the IL-2Ralpha gene that mediates TGF-beta-dependent induction of the gene. These findings have implications related to IL-2Ralpha expression on activated T cells and regulatory T cells.     

Cyclosporin A sensitivity of the NF-kappa B site of the IL2R alpha promoter in untransformed murine T cells.

We have investigated the characteristics of IL2R alpha gene induction in untransformed murine T cells. Induction of IL2R alpha mRNA by TCR/CD3 ligands in a murine T cell clone and in short-term splenic T cell cultures was inhibited by protein synthesis inhibitors and by CsA. This result was contrary to previous observations in JURKAT T leukemia cells and human peripheral blood T cells, suggesting a difference in the mechanisms of IL2R alpha gene induction in these different cell types. The CsA sensitivity of IL2R alpha mRNA induction represented a direct effect on the TCR/CD3 response, and was not due to CsA-sensitive release of the lymphokines IL2 or tumour necrosis factor alpha (TNF alpha) and consequent lymphokine-mediated induction of IL2R alpha mRNA. The NF-kappa B site of the IL2R alpha promoter was essential for gene induction through the TCR/CD3 complex, and the induction of reporter plasmids containing multimers of this site was significantly inhibited by CsA. Northern blotting analysis indicated that while the p65 subunit of NF-kappa B was constitutively expressed and not appreciably induced upon T cell activation, mRNA for the p105 precursor of p50 NF-kappa B was induced in response to TCR/CD3 stimulation and this induction was sensitive to CsA. Electrophoretic mobility shift assays and antiserum against the p50 subunit of NF-kappa B indicated that p50 was a component of the inducible nuclear complex that bound to the IL2R alpha kappa B site. Appearance of the kB-binding proteins was insensitive to CsA at early times after activation (approximately 15 min), but was partially sensitive to CsA at later times. Based on these results, we propose that the NF-kappa B site of the IL2R alpha promoter mediates at least part of the CsA sensitivity of IL2R alpha gene induction in untransformed T cells, possibly because de novo synthesis of p105 NF-kappa B is required for sustained IL2R alpha expression.     

Unusual alpha beta-T cells expanded in autoimmune lpr mice are probably a counterpart of normal T cells in the liver.

Autoimmune MRL-lpr/lpr (lpr) mice develop severe lymphadenopathy, characterized by the accumulation of alpha beta-T cells with CD4-8- double negative (DN) phenotype, at the onset of disease. We previously demonstrated that the liver is a major site for the proliferation of such DN alpha beta-T cells. Herein, we further demonstrate that a large proportion of alpha beta-T cells in the liver and other organs, except the thymus, of lpr mice have unique properties, such as DN phenotype, relatively dull TCR intensity, a preponderance of V beta 8+ cells, and Pgp-1 expression. Interestingly, alpha beta-T cells in the liver of normal mice were found to consist of T cells with intermediate intensity of TCR (i.e., brighter than thymic dull TCR and lower than thymic bright TCR) as well as with bright intensity of TCR in the immunofluorescence test. These hepatic alpha beta-T cells with intermediate TCR in normal mice were found to have properties similar to those of alpha beta-T cells in lpr mice. These results suggest that abnormal alpha beta-T cells in lpr mice are a counterpart of normal T cells in the liver. An abnormal expansion of such T cells in the liver might be fundamental to the pathogenesis involved in these autoimmune mice.