Restoration of early thymocyte differentiation in T-cell receptor beta-chain-deficient mutant mice by transmembrane signaling through CD3 epsilon.

Thymic repertoire selection requires the expression of the alpha beta CD3 T-cell receptor (TCR) together with the coreceptors CD4 and CD8. The appearance of CD4 and CD8 on thymocytes is the hallmark of a complex maturation step, accompanied by downregulation of the interleukin 2 receptor (IL-2R) alpha chain, arrest of rearrangement (i.e., allelic exclusion) of the TCR beta-chain locus, a burst of cell divisions, and reduction in cell size. This maturation step is inhibited in TCR beta-chain-deficient mouse strains and may depend on surface expression of an immature TCR complex containing CD3 and TCR beta chains but no TCR alpha chain. Here we show that the CD4+8+ double-positive (DP) stage can be induced by treatment of fetal thymic organ cultures with anti-CD3 epsilon monoclonal antibodies in several TCR beta-chain-deficient mouse strains: severe combined immunodeficient (scid) mice, mice carrying a mutation in the recombination activating gene 1 (Rag-1), or mice carrying a deletion in the TCR beta-chain locus itself. These findings suggest that CD3 epsilon is expressed on the thymocyte surface independent of and prior to the TCR beta chain. The data are consistent with the notion that in wild-type mice the DP stage is induced by transmembrane signaling through an immature CD3-TCR beta-chain complex, which can be bypassed by crosslinking of CD3 epsilon alone.     

Positive and negative selection of T cells in T-cell receptor transgenic mice expressing a bcl-2 transgene.

To explore the role of bcl-2 in T-cell development, a bcl-2 transgene was introduced into mice expressing a T-cell receptor (TCR) transgene encoding reactivity for the mouse male antigen HY presented by the H-2Db class I antigen of the major histocompatibility complex (MHC). Normal thymic development is contingent on the ability of immature thymocytes to interact with self-MHC molecules presented by thymic stroma (positive selection). Thus, thymocyte numbers are low in female anti-HY TCR transgenic mice with a nonselecting (H-2Dd) background. Expression of bcl-2 inhibited the death of nonselectable thymocytes since, strikingly, female H-2Dd bcl-2/TCR transgenic mice developed normal numbers of CD4+CD8+ thymocytes, although these did not mature further into functional T cells. Hence, TCR-MHC interaction may induce positive selection through two signals, one which saves cells from death by increasing Bcl-2 synthesis and another which promotes maturation. Male H-2Db anti-HY TCR transgenic mice normally have a very small thymus, due to deletion of the self-reactive T cells. Expression of bcl-2 reduced the efficiency of deletion, since bcl-2/TCR transgenic male mice accumulated 4- to 6-fold more thymocytes than did TCR transgenic male littermates. Anti-HY TCR-expressing cells were also more numerous in the peripheral lymphoid tissues, but these cells expressed abnormally low levels of CD8 co-receptor and were not responsive to the HY antigen. Thus, although bcl-2 expression hampers the deletion of immature self-reactive cells in the thymus, self-tolerance is maintained.     

Positive selection of an MHC class-I restricted TCR in the absence of classical MHC class I molecules

The H-2Ld alloreactive 2C T cell receptor (TCR) is commonly considered as being positively selected on the H-2Kb molecule. Surprisingly, 2C TCR+ CD8+ single-positive T cells emerge in massive numbers in fetal thymic organ culture originating from 2C transgenic, H-2KbD(b-/-) (2C+KbD(b-/-)) but not in fetal thymic organ culture from beta2-microglobulin(-/-) 2C transgenic animals. Mature CD8+ T cells are observed in newborn but not in adult 2C+KbD(b-/-) mice. These CD8+ T cells express the alpha4beta7 integrin, which allows them to populate the intestine, a pattern of migration visualized by intrathymic injection of FITC and subsequent accrual of FITC-labeled lymphocytes in the gut. We conclude that the 2C TCR is reactive not only with H-2Ld and H-2Kb, but also with nonclassical MHC class I products to enable positive selection of 2C+ T cells in the fetal and newborn thymus and to support their maintenance in the intestine.     

Selection of intraepithelial lymphocytes with CD8 alpha/alpha co-receptors by self-antigen in the murine gut.

We have studied T-cell receptor (TCR) and alpha/alpha CD8 expression in thymus-independent intraepithelial lymphocytes (TI IELs) from the gut of mice bearing transgenic (TG) TCR alpha beta specific for the male antigen, presented by H-2Db class I major histocompatibility complex (MHC) molecules. In contrast to TCR+ alpha beta cells differentiating in the thymus (from CD4+CD8+ precursors to CD4+CD8- or CD4-CD8+ progeny), TI IELs are not deleted by self-antigens, nor are they positively selected in the absence of the specific peptide. On the contrary, recognition of the antigen in the context of self-MHC is required for selection and granular differentiation of CD8+ TI IELs. Our results also show that, in contrast to the thymus, expression of the beta TG does not block expression of endogenous TCR gamma delta genes in TI IELs. The size of this gut IEL subpopulation and its difference in mechanisms of repertoire selection demonstrate the existence of a major extrathymic pathway of T-cell differentiation, the role of which remains to be elucidated.     

Development of CD4+ T cells expressing a nominally MHC class I-restricted T cell receptor by two different mechanisms

Differences in T cell receptor (TCR) signaling initiated by interactions among TCRs, coreceptors, and self-peptide-MHC complexes determine the outcome of CD4 versus CD8 lineage of T cell differentiation. The H-2Ld and Kbm3 alloreactive 2C TCR is positively selected by MHC class I Kb and a yet-to-be identified nonclassical class I molecule to differentiate into CD8+ T cells. Here we describe two mechanisms by which CD4+ 2C T cells can be generated in 2C TCR-transgenic mice. In the RAG-/- background, development of CD4+ 2C T cells requires the expression of both I-Ab and the TAP genes, indicating that both MHC class I and II molecules are required for positive selection of these T cells. Notably, only some of the 2C+ RAG-/- mice (approximately 30%) develop CD4+ 2C T cells, with frequencies in individual mice varying from 0.5% to as high as approximately 50%. In the RAG+ background, where endogenous TCRalpha genes are rearranged and expressed, CD4+ 2C T cells are generated because these cells express the 2C TCR as well as additional TCRs, consisting of the 2C TCRbeta and endogenous TCRalpha chains. Similarly, T cells expressing the OT-1 TCR, which is nominally MHC class I-restricted, can also develop into CD4+ T cells through the same two mechanisms. Thus, expression of two TCRs by a single thymocyte, TCR recognition of multiple MHC molecules, and heterogeneity of TCR, coreceptors, and peptide-MHC interactions in the thymus all contribute to the outcome of CD4 versus CD8 lineage development.     

Essential requirement of an invariant V alpha 14 T cell antigen receptor expression in the development of natural killer T cells.

NK1.1+ T [natural killer (NK) T] cells express an invariant T cell antigen receptor alpha chain (TCR alpha) encoded by V alpha 14 and J alpha 281 segments in association with a limited number of V betas, predominantly V beta 8.2. Expression of the invariant V alpha 14/J alpha 281, but not V alpha 1, TCR in transgenic mice lacking endogenous TCR alpha expression blocks the development of conventional T alpha beta cells and leads to the preferential development of V alpha 14 NK T cells, suggesting a prerequisite role of invariant V alpha 14 TCR in NK T cell development. In V beta 8.2 but not B beta 3 transgenic mice, two NK T cells with different CD3 epsilon expressions, CD3 epsilon(dim) and CD3 epsilon(high), can be identified. CD3 epsilon(high) NK T cells express surface V alpha 14/V beta 8 TCR, indicating a mature cell type, whereas CD3 epsilon(dim) NK T cells express V beta 8 without V alpha 14 TCR and no significant CD3 epsilon expression (CD3 epsilon(dim)) on the cell surface. However, the latter are positive for recombination activating gene (RAG-1 and RAG-2) mRNA, which are only expressed in the precursor or immature T cell lineage, and also possess CD3 epsilon mRNA in their cytoplasm, suggesting that CD3 epsilon(dim) NK T cells are the precursor of V alpha 14 NK T cells.     

The T-cell-receptor repertoire in the synovial fluid of a patient with rheumatoid arthritis is polyclonal.

We have analyzed the T-cell-receptor repertoire expressed in the synovial fluid of a patient with rheumatoid arthritis by using an inverse polymerase chain reaction. Total RNA was isolated from Ficoll-purified mononuclear cells and converted into circularized double-stranded cDNA. Specific amplification of alpha- and beta-chain variable regions (V alpha and V beta) was achieved with inverted alpha- and beta-chain constant region (C alpha and C beta) primer pairs, and the amplification products were cloned into phage vectors. A total of 78 alpha and 76 beta clones were sequenced, and 67 and 72 productively rearranged alpha and beta genes were identified, respectively. Thirty-one V alpha, 33 alpha-chain joining region (J alpha), 29 V beta, and 12 beta-chain joining region (J beta) gene segments were found in the productively rearranged clones, indicating that the T-cell repertoire expressed in the synovial fluid of this RA patient is highly heterogenous and polyclonal. Comparison of peripheral blood and synovial fluid repertoires showed that the most abundant V beta sequences, V beta 2.1 and V beta 3.1, were enriched in the inflamed joint by a factor of 2 to 3. It is possible that T cells expressing these V beta gene segments, which recognize bacterial superantigens, play a role in the disease.     

Early expression of a T-cell receptor beta-chain transgene suppresses rearrangement of the V gamma 4 gene segment.

beta transgenic mice have a T-cell receptor beta-chain gene that is prematurely expressed on the surface of CD4- CD8- thymocytes and paired with an uncharacterized non-T-cell receptor alpha-chain polypeptide. The rearrangement of the T-cell receptor variable region gamma chain gene segment V gamma 4, a component of the gamma-chain gene that is rearranged and expressed preferentially on thymocytes of normal adult mice, is severely repressed in beta transgenic mice. Consequently no gamma delta T-cell receptor heterodimers are detectable on the surface of adult thymocytes or splenic T cells. These results indicate that cells expressing alpha beta or gamma (V gamma 4)-delta TCRs originate from a common precursor in which the first productive rearrangement of either the beta or gamma locus determines the further differentiation pathway into either alpha beta or gamma delta T cells. The repression of V gamma 4 rearrangement by a preexisting beta-chain gene may be indicative of one of several mechanisms which ensure that gamma delta and alpha beta receptors do not as a rule appear on the surface of the same cell.     

Staphylococcal exotoxins deliver activation signals to human T-cell clones via major histocompatibility complex class II molecules

We investigated whether staphylococcal exotoxins (SEs), in addition to their capacity to induce T-cell activation restricted by the T-cell receptor (TCR) beta-chain variable region, can deliver an activation signal to human T-cell clones through major histocompatibility complex (MHC) class II molecules. Eleven human T-cell clones (9 alpha beta TCR and 2 gamma delta TCR clones) of different antigenic specificities were tested for their capacity to proliferate in response to toxic shock syndrome toxin 1 (TSST-1) and two SEs, SEA and SEB. In the absence of accessory cells, only 4 alpha beta TCR clones were stimulated to proliferate, each by a single SE, and to mobilize intracellular free Ca2+ in response to that SE, events indicative of TCR engagement and, presumably, recognition restricted by the beta-chain variable region. In the presence of accessory cells, each of the 11 T-cell clones was stimulated to proliferate by any one of the three SEs tested. This apparently TCR-unrestricted SE-mediated polyclonal proliferation of T-cell clones occurred in the absence of an increase in intracellular free Ca2+ and was not dependent on the presence of MHC class II expression on accessory cells. In contrast, SE-mediated polyclonal proliferation did not occur in 3 alpha beta TCR clones derived from an MHC class II-deficient patient. Furthermore, all of the three SEs induced the proliferation of 4 natural-killer-cell clones, suggesting that expression of TCR/CD3 complex is not essential for SE-mediated polyclonal proliferation of activated lymphocytes. These results indicate that MHC class II molecules transduce activation signals to human T- and natural-killer-cell clones.     

Direct binding of secreted T-cell receptor beta chain to superantigen associated with class II major histocompatibility complex protein.

The interaction of the T-cell receptor (TCR) with peptide antigen plus major histocompatibility complex (MHC) protein requires both alpha and beta chains of the TCR. The "superantigens" are a group of molecules that are recognized in association with MHC class II but that do not appear to conform to this pattern. Superantigens are defined as such because they cause the activation or thymic deletion of many or all T cells bearing specific TCR beta-chain variable region (V beta) elements. The strong association of particular V beta S with T-cell responses to superantigens suggests that their interaction with the TCR is fundamentally different from that of most antigens. We have directly investigated the involvement of the beta chain in recognition of a superantigen by using a secreted, truncated TCR beta chain and the bacterial superantigen staphylococcal enterotoxin A complexed to cell-surface MHC class II. We demonstrate that this interaction is specific for the enterotoxin and is dependent on MHC class II expression by the cell. The reaction can be inhibited by antibodies against the three components of the reaction: V beta, enterotoxin, and class II. This shows that the TCR beta chain is sufficient to mediate the interaction with a superantigen-class II complex. The TCR alpha chain and co-receptors such as CD4 are not required.     

Class II-positive hematopoietic cells cannot mediate positive selection of CD4+ T lymphocytes in class II-deficient mice.

Generation of immunocompetent alpha/beta T-cell receptor-positive T cells from CD4+CD8+ thymocytes depends upon their interaction with thymic major histocompatibility complex (MHC) molecules. This process of positive selection provides mature T cells that can recognize antigens in the context of self-MHC proteins. Previous studies investigating haplotype restriction in thymic and bone-marrow chimeras concluded that radioresistant thymic cortical epithelium directs the positive selection of thymocytes. There is controversy, however, as to whether intra- or extrathymic radiosensitive bone marrow-derived macrophage and dendritic cells also can mediate positive selection. To determine whether CD4+ T cells can be positively selected by hematopoietic cells, we generated chimeric animals expressing MHC class II molecules on either bone marrow-derived or thymic stromal cells by using a recently produced strain of MHC class II-deficient mice. CD4+ T cells developed only when class II MHC molecules were expressed on radioresistant thymic cells. In contrast to what recently has been observed for the selection of CD8+ T lymphocytes, MHC class II-positive bone marrow-derived cells were unable to mediate the selection of CD4+ T cells when the thymic epithelium lacked MHC class II expression. These data suggest that CD4+ and CD8+ T cells may be generated by overlapping, but not identical, mechanisms.     

Both alpha-helices along the major histocompatibility complex binding cleft are required for staphylococcal enterotoxin A function.

The superantigen staphylococcal enterotoxin A (SEA) requires interaction with class II major histocompatibility complex (MHC) molecules to activate T cells. We have previously used the synthetic peptide approach to establish one side of the hypothetical class II foreign-antigen binding cleft, alpha-helical region 65-85 of the beta chain, as a binding site involved in accessory cell presentation of SEA to T cells. To further characterize the structural basis for MHC-SEA interaction we have examined the role of the alpha-helical regions of the class II alpha and beta chains in SEA function. Using the synthetic peptide approach, we have found that both alpha-helical regions are required for SEA-induced proliferation. Their corresponding peptides directly bound SEA. Although the beta-chain peptides were able to inhibit SEA binding to human and mouse cells, the alpha-chain peptides were not. The data suggest that the alpha-helices along both sides of the hypothetical class II MHC molecule binding cleft are required for SEA-induced function, whereas the beta-chain alpha-helix is sufficient for SEA binding. A model of superantigen presentation is proposed wherein the MHC beta chain, possibly region 70-80, interacts with SEA region 1-45, whereas another region of SEA binds region 51-80 of the alpha chain.     

Structure of human T-cell receptors specific for an immunodominant myelin basic protein peptide: positioning of T-cell receptors on HLA-DR2/peptide complexes.

T-cell receptors (TCRs) recognize peptide bound within the relatively conserved structural framework of major histocompatibility complex (MHC) class I or class II molecules but can discriminate between closely related MHC molecules. The structural basis for the specificity of ternary complex formation by the TCR and MHC/peptide complexes was examined for myelin basic protein (MBP)-specific T-cell clones restricted by different DR2 subtypes. Conserved features of this system allowed a model for positioning of the TCR on DR2/peptide complexes to be developed: (i) The DR2 subtypes that presented the immunodominant MBP peptide differed only at a few polymorphic positions of the DR beta chain. (ii) TCR recognition of a polymorphic residue on the helical portion of the DR beta chain (position DR beta 67) was important in determining the MHC restriction. (iii) The TCR variable region (V) alpha 3.1 gene segment was used by all of the T-cell clones. TCR V beta usage was more diverse but correlated with the MHC restriction--i.e., with the polymorphic DR beta chains. (iv) Two clones with conserved TCR alpha chains but different TCR beta chains had a different MHC restriction but a similar peptide specificity. The difference in MHC restriction between these T-cell clones appeared due to recognition of a cluster of polymorphic DR beta-chain residues (DR beta 67-71). MBP-(85-99)-specific TCRs therefore appeared to be positioned on the DR2/peptide complex such that the TCR beta chain contacted the polymorphic DR beta-chain helix while the conserved TCR alpha chain contacted the nonpolymorphic DR alpha chain.     

Antigen presentation by keratinocytes directs autoimmune skin disease

The antigen-presenting cells that initiate and maintain MHC class II-associated organ-specific autoimmune diseases are poorly defined. We now describe a new T cell antigen receptor (TCR) transgenic (Tg) model of inflammatory skin disease in which keratinocytes activate and are the primary target of autoreactive CD4(+) T cells. We previously generated keratin 14 (K14)-A(beta)b mice expressing MHC class II only on thymic cortical epithelium. CD4(+) T cells from K14-A(beta)b mice fail to undergo negative selection and thus have significant autoreactivity. The TCR genes from an autoreactive K14-A(beta)b CD4 hybridoma were cloned to produce a TCR Tg mouse, 2-2-3. 2-2-3 TCR Tg cells are negatively selected in WT C57BL6 mice but not in 2-2-3K14-A(beta)b mice. Interestingly, a significant number of mice that express both the K14-A(beta)b transgene and the autoreactive 2-2-3 TCR spontaneously develop inflammatory skin disease with mononuclear infiltrates, induction of MHC class II expression on keratinocytes, and T helper 1 cytokines. Disease can be induced by skin inflammation but not solely by activation of T cells. Thus, cutaneous immunopathology can be directed through antigen presentation by tissue-resident keratinocytes to autoreactive TCR Tg CD4(+) cells.     

Fibroblasts can induce thymocyte positive selection in vivo.

During development in the thymus, thymocytes bearing alpha beta T-cell receptors are selected to mature if the receptors they bear are able to interact in some way with major histocompatibility complex (MHC) proteins expressed on thymic stromal cells. It has been shown that thymus cortical epithelial cells are usually the cells presenting the MHC molecules involved in this process of so-called positive selection. Here we tested the ability of fibroblasts to mediate positive selection in vivo. Fibroblasts transfected with the genes for the MHC I-Ab proteins were injected intrathymically into irradiated H-2k animals reconstituted with H-2bxk F1 fetal liver cells. Eight weeks later, the recipient mice were immunized and shown to contain peptide-specific I-Ab-restricted T cells. This demonstrates the ability of I-Ab-transfected fibroblasts to participate in positive selection. Thus a cell type that is not specialized to process and present antigens in the context of MHC class II molecules can mediate positive selection when transfected with an appropriate MHC molecule. The data also support the idea that the ability to mediate positive selection may not be limited to thymic cortical epithelium.     

Evidence for a single-niche model of positive selection.

Thymocyte maturation depends on interactions with thymic stromal elements expressing major histocompatibility complex (MHC) molecules. Mutant mouse strains lacking MHC class I (beta 2-microglobulin-null) or class II (A beta-null) expression fail to generate normal CD8 or CD4 T-cell populations and provide model systems for reconstitution experiments. We have constructed in vitro chimeras between normal and MHC-deficient thymi to evaluate the efficiency of positive selection. Unexpectedly, the generation of mature single-positive thymocytes was proportional to the fraction of wild-type (i.e., MHC-expressing) stroma over a wide range of chimerism. Similar results were obtained for the development of T-cell receptor-transgenic thymocytes in graded chimeras expressing selecting and nonselecting MHC alleles. These findings are best explained by hypothesizing that positive selection involves a rate-limiting step at which each thymocyte can interact with only one stromal cell niche.     

On the role of thymic epithelium vs. bone marrow-derived cells in repertoire selection of T cells

T lymphocytes mature in the thymus to become functional T cells. Studies with chimeric mice and T cell receptor (TCR) transgenic (tg) mice have indicated that the major histocompatibility gene complex (MHC) of thymic radio-resistant (presumed to be epithelial) cells positively select the MHC-restricted T cell repertoire. Surprisingly, mice without a thymus reconstituted with an MHC-incompatible thymus generate effector T cells which are, in general, specific for the host and not for the thymic MHC. The present study reanalyzed this longstanding paradox in nude mice that were reconstituted with an MHC-incompatible thymus plus or minus immunologically defective bone marrow-derived cells or in nude mice expressing a transgenic T cell receptor. A pathway of thymus-dependent but thymic MHC-independent T cell maturation is revealed where expansion of the antiviral T cell repertoire depends on the MHC of bone marrow-derived cells. These results indicate an alternative, if not a general, pathway of T cell maturation and selection: the thymus may function essentially as an organ promoting T cell receptor expression; T cell specificity, however, reflects repertoire expansion plus cell survival and effector T cell induction driven by the MHC of bone marrow-derived cells. Therefore pure thymus defects can be efficiently reconstituted by allo- and xenogeneic thymic grafts.