Major histocompatibility complex-linked specificity of gamma delta receptor-bearing T lymphocytes

Several recent studies have identified a distinct subset of CD3(T3)+CD4-CD8-T lymphocytes that express a CD3-associated heterodimer made up of the protein encoded by the T-cell receptor (TCR) gamma-gene and a second glycoprotein termed TCR delta (refs 1-4). TCR gamma delta is expressed on CD3+ thymocytes during fetal ontogeny before the appearance of TCR alpha-beta (alpha beta) (refs 5-7), on CD3+CD4-CD8- adult thymocytes, and on a subset (1-10%) of CD3+ cells in adult peripheral lymphoid organs and the peripheral blood. TCR gamma delta-expressing T cells probably represent a distinct mature T-cell lineage with the capacity to proliferate in response to receptor-mediated signals, and to display non-major histocompatibility complex (MHC)-restricted cytolysis. Critical to understanding the function of this T-cell subset is the identification of the ligand(s) recognized by TCR gamma delta. Here we describe an alloreactive CD3+CD4-CD8-TCR gamma delta-expressing, TCR alpha beta-negative, T-cell line that manifests MHC-linked recognition specificity for both proliferation and cytotoxicity. Our results suggest that T cells expressing TCR gamma delta are capable of self-non-self MHC discrimination and that they can undergo MHC-influenced selection during differentiation like TCR alpha beta-expressing T cells.     

Self-reactive gamma delta T cells are eliminated in the thymus

The genes encoding a gamma delta T-cell receptor specific for a major histocompatibility complex class I molecule encoded by the TIa locus have been inserted into the mouse germ line. In mice that do not express the TIa-encoded determinant, transgenic gamma delta T cells are a functional component of the CD4-CD8- 'double-negative' T cells in the thymus and peripheral lymphoid organs. In mice that express the TIa-encoded determinant, there are no transgenic gamma delta T cells in peripheral lymphoid organs, and there are no thymocytes expressing normal levels of the transgenic gamma delta T-cell receptor.     

Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen

The crucial role of the thymus in immunological tolerance has been demonstrated by establishing that T cells are positively selected to express a specificity for self major histocompatibility complex (MHC), and that those T cells bearing receptors potentially reactive to self antigen fragments, presumably presented by thymic MHC, are selected against. The precise mechanism by which tolerance is induced and the stage of T-cell development at which it occurs are not known. We have now studied T-cell tolerance in transgenic mice expressing a T-cell receptor with double specificities for lymphocytic choriomeningitis virus (LCMV)-H-2Db and for the mixed-lymphocyte stimulatory (MIsa) antigen. We report that alpha beta TCR transgenic mice tolerant to LCMV have drastically reduced numbers of CD4+CD8+ thymocytes and of peripheral T cells carrying the CD8 antigen. By contrast, tolerance to MIsa antigen in the same alpha beta TCR transgenic MIsa mice leads to deletion of only mature thymocytes and peripheral T cells and does not affect CD4+CD8+ thymocytes. Thus the same transgenic TCR-expressing T cells may be tolerized at different stages of their maturation and at different locations in the thymus depending on the antigen involved.     

Differential expression of two distinct T-cell receptors during thymocyte development

The product of the T-cell receptor (TCR) gamma-gene has recently been found to be expressed on a subset of both peripheral cells and thymocytes. As an initial approach to understanding the role of this gamma-chain of TCR (TCR gamma) in T-cell development, we have studied the ontogeny of TCR expression at the protein level in the developing murine thymus. We show here that the first T3-associated TCR to be expressed in the developing thymus is a disulphide-linked heterodimer composed of a gamma-chain of relative molecular mass 35,000 (Mr 35K) and a 45K partner (termed TCR delta). This TCR gamma delta is first detected approximately two days before the appearance of cell-surface TCR alpha beta heterodimers. We report that N-glycosidase digestions reveal that all of the gamma-protein expressed on fetal thymocytes, as in adult CD4-8-(L3T4-, Lyt2-) thymocytes, bear N-linked carbohydrate side chains. The major gamma-gene transcribed in mature, alpha beta-bearing T cells (V gamma 1.2C gamma 2)encodes no N-linked glycosylation site so these results suggest that the fetal gamma delta receptor defines a distinct T-cell lineage whose development in the thymus precedes classical alpha beta-bearing cells.     

Specific recognition of staphylococcal enterotoxin A by human T cells bearing receptors with the V gamma 9 region

T cells bearing the alpha beta receptor can specifically react with target cells coated with staphylococcal enterotoxin and expressing major histocompatibility complex class II molecules; these responses depend on which variable region (V) of the receptor's beta-subunit is used. We have now examined whether a similar situation exists for human T cells bearing the gamma delta receptor. We found that reactivity to staphylococcal enterotoxin A is strictly dependent on the presence of the V gamma 9 variable region in the gamma delta T-cell receptor (TCR). These cytotoxic responses required the expression of HLA class II molecules by the target cell and could be inhibited by anti-gamma delta TCR and by anti-HLA-class-II monoclonal antibodies. In contrast to alpha beta TCR+ cell clones, no proliferative response of V gamma 9+ T-cell clones towards stimulator cells coated with enterotoxin A was observed in vitro. These results indicate that the gamma delta TCR repertoire might be influenced by enterotoxin A produced during staphylococcal infections in vivo. This could provide a molecular basis for the observation that V gamma 9+ T cells form the large majority of peripheral gamma delta TCR+ cells but only a small proportion of thymic gamma delta TCR+ cells.     

Signal for T-cell differentiation to a CD4 cell lineage is delivered by CD4 transmembrane region and/or cytoplasmic tail.

Mature T cells express either CD4 or CD8 on their surface. Most helper T cells express CD4, which binds to class II major histocompatibility complex (MHC) proteins, and most cytotoxic T cells express CD8, which binds to class I MHC proteins. In the thymus, mature CD4+CD8- and CD4-CD8+ T cells expressing alpha beta T-cell antigen receptors (TCR) develop from immature thymocytes through CD4+CD8+ alpha beta TCR+ intermediates. Experiments using mice transgenic for alpha beta TCR suggest that the specificity of the TCR determines the CD4/CD8 phenotype of mature T cells. These results, however, do not indicate how a T cell differentiates into the CD4 or CD8 lineage. Here we show that the CD4 transmembrane region and/or cytoplasmic tail mediates the delivery of a specific signal that directs differentiation of T cells to a CD4 lineage. We generated transgenic mice expressing a hybrid molecule composed of the CD8 alpha extracellular domains linked to the CD4 transmembrane region and cytoplasmic tail. We predicted that this hybrid molecule would bind to class I MHC proteins through the extracellular domains but deliver the intracellular signals characteristic of CD4. By crossing our transgenic mice with mice expressing a transgenic alpha beta TCR specific for a particular antigen plus class I MHC protein, we were able to express the hybrid molecule in developing thymocytes expressing the class I MHC-restricted TCR. Our results show that the signal transduced by the hybrid molecule results in the differentiation of immature thymocytes expressing a class I-restricted TCR into mature T cells expressing CD4.     

T-cell-specific deletion of T-cell receptor transgenes allows functional rearrangement of endogenous alpha- and beta-genes

In B cells the loci encoding immunoglobulin chains usually show allelic exclusion; a given B cell transcribes and translates only one productively rearranged allele of the heavy and light chain loci. This ensures that each B cell expresses only one antigen receptor. The loci encoding T-cell receptor (TCR) alpha- and beta-genes may behave similarly. We have previously reported that the expression of a transgenic TCR beta-chain prevents functional and nonfunctional V beta rearrangements in the endogenous beta-chain loci but not D beta J beta rearrangements. We have also been unable to detect the expression of the TCR gamma-chain locus in thymocytes of these mice (unpublished observations). To study the mechanisms involved in forming a mature T-cell repertoire further, we have constructed mice expressing alpha- and beta-TCR transgenes derived from a cytotoxic T-cell clone that is specific for the male antigen H-Y in the context of H-2Db MHC molecules. Here we show that in these mice rearrangement of endogenous alpha-chain loci is also suppressed, although to a lesser extent than rearrangement of beta-chain loci. In addition, in male alpha beta TCR transgenic mice we observed T-cell clones which had deleted both transgenic alpha- and beta-chain genes and expressed endogenous alpha- and beta-chain TCR genes. These cells are presumably derived from rare thymocytes that leave the male thymus because their TCR no longer recognizes self antigen. The vast majority of CD4+8+ nonmature thymocytes expressing alpha- and beta-transgenes are deleted in the male thymus.     

The generation of mature T cells requires interaction of the alpha beta T-cell receptor with major histocompatibility antigens

THE T-cell repertoire within an individual is biased to recognize antigen in the context of self major histocompatibility complex (MHC) antigens. This is thought to depend on a process of positive selection during development. Support for this notion has recently been obtained in experiments using transgenic mice bearing genes for T-cell receptors (TCR) of defined specificity: T cells expressing the introduced genes form the main part of the mature T-cell population only in mice that express the appropriate MHC product. We have now extended these observations using TCR transgenic mice homozygous for the severe combined immunodeficiency (SCID) mutation which are defective in the rearrangement of both TCR and immunoglobulin genes. In this case mature thymocytes develop only in transgenic mice that express the MHC product which restricts the specificity of the transgenic TCR. This shows that the interaction of the alpha beta TCR with thymic MHC antigen is essential for the development of mature T cells. Furthermore, the peripheral lymph nodes of such mice are underdeveloped, suggesting that the peripheral expansion of mature T cells may require interactions with other lymphocytes expressing a range of receptors.     

Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.     

Abnormal differentiation of thymocytes in mice treated with cyclosporin A

Cyclosporin A (CsA) acts as a powerful immunosuppressive agent, and also, when given in repeated doses, can cause T-cell-dependent graft-versus-host disease and organ-specific autoimmune disease in rodents. This suggests that CsA interferes with the processes governing self-tolerance, either by nullifying the activity of T suppressor cells or by preventing the deletion of autoreactive T cells during ontogeny in the thymus. We report here that irradiated mice given repeated injections of CsA show striking dysfunction of the thymus. There are two different effects, the first of which is that CsA seems to block the differentiation of immature CD4+CD8+ thymocytes into mature CD4+CD8- and CD4-CD8+ cells expressing a high density of T-cell receptors and CD3 molecules. Second, CsA-treated mice show incomplete deletion of T cells expressing T-cell receptor molecules reactive to self H-2 I-E molecules.     

Negative selection of murine medullary-type single positive thymocytes

Negative selection depletes self-reactive T cells, thus ensuring self-tolerance. It is usually considered that negative selection imposed on double-positive (DP) thymocytes that reside at the cortico-medullary junction. Negative selection model was set up by injecting mice with anti-T cell receptor (TCR) monoclonal antibody (mAb) intraperitoneally in this work. As shown in phenotypic analysis of thymocytes, negative selection destroys not only cortical-type DP thymocytes, but also medullary-type CD3+TCRαβ+CD4SP and CD3+TCRαβ+CD8SP thymocytes. Negative selection of medullary-type single positive (SP) are more susceptible to apoptosis, while with development of the cells, their resistance to apoptosis increases. Therefore, negative selection does not operate on functionally mature thymocytes at the late stage. This result is a supplement to the traditional theory of negative selection. Negative selection of medullary-type thymocytes is probably to further deplete self-reactive T cells, thus producing precise TCR repertoire and inducing self-tolerance.     

Participation of CD4 coreceptor molecules in T-cell repertoire selection.

During thymocyte development, progenitor cells bearing both CD4 and CD8 coreceptor molecules mature into functional T lymphocytes that express these proteins in a mutually exclusive way. Although T-cell specificity is determined primarily by the structure of the T-cell antigen receptor (TCR) heterodimer, a developmentally regulated process acts to ensure that cells bearing class II-restricted TCRs are CD4+ and those bearing class I-restricted TCRs express only CD8. To investigate this maturation process, we have engineered transgenic mice in which CD4 is expressed in all thymocyte subsets and in all peripheral T cells. Peripheral CD4+8+ T lymphocytes from these mice react with both class I and class II alloantigens. Moreover, expression of the CD4 transgene disrupts the positive selection of doubly transgenic thymocytes bearing a class I-restricted TCR specific for the male (H-Y) antigen. Hence the CD4 coreceptor participates directly in T-cell repertoire selection.     

Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages.

Analysis of mice carrying mutant T-cell antigen receptor (TCR) genes indicates that TCR-beta gene rearrangement or expression is critical for the differentiation of CD4-CD8- thymocytes to CD4+CD8+ thymocytes, as well as for the expansion of the pool of CD4+CD8+ cells. TCR-alpha is irrelevant in these developmental processes. The development of gamma delta T cells does not depend on either TCR-alpha or TCR-beta.     

Resident pulmonary lymphocytes expressing the gamma/delta T-cell receptor

The biological role of cells bearing the gamma delta T-cell antigen receptor (TCR) is as yet unclear. Although there are indications that some gamma delta+ cells can mediate cytotoxicity, their antigen-related functions have not yet been defined. In the mouse, gamma delta+ cells constitute 1-3% of T cells in lymphoid organs. Intestinal intraepithelial lymphocytes (IELs) and dendritic epidermal cells (DECs) also appear to carry the gamma delta TCR. The strategic locations of DECs and IELs have led to the suggestion that gamma delta+ cells could constitute a first line of defence in the vicinity of large surfaces of contact with the environment. We report here that an estimated 8-20% of resident pulmonary lymphocytes (RPLs) are CD3+ alpha beta TCR-, and presumably gamma delta TCR+. Furthermore, mice exposed to aerosols containing a Mycobacterium tuberculosis extract have an increased number of activated CD3+ alpha beta-TCR- pulmonary T cells which can be propagated in vitro.     

A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family

Recent studies have demonstrated that CD3 is expressed on a subset of thymocytes with a CD4-CD8- (double negative) phenotype. At least some of these cells bear the CD3-associated gamma delta T-cell receptor (TCR gamma delta). Here we describe a second subset of double negative thymocytes which expresses CD3-associated alpha beta receptors (TCR alpha beta). Surprisingly, these cells express predominantly the products of a single V beta gene family (V beta 8). These CD4-CD8-, TCR alpha beta+ cells appear relatively late in ontogeny (between birth and day 5 of life) and thus are unlikely to be the precursors to the TCR alpha beta-bearing cells (CD4+CD8- and CD4-CD8+) already present at birth. They can be selectively expanded in vitro by stimulation with a monoclonal antibody to V beta 8 (F23.1) in the presence of interleukin I (IL-1). We propose that this cell type is a unique T-cell population distinguishable from typical TCR alpha beta+ T cells by its CD4-CD8- phenotype and a restricted TCR V beta repertoire. Analysis of the unique phenotype of these cells suggests that they may represent the normal counterpart of the defective CD4-CD8- T cells found in the lpr autoimmune mouse.     

Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes

Human cluster-of-differentiation 1 (CD1) is a family of cell surface glycoproteins of unknown function expressed on immature thymocytes, epidermal Langerhans cells and a subset of B lymphocytes. Three homologous proteins, CD1a, b and c, have been defined serologically, and the CD1 gene locus on human chromosome 1 contains five potential CD1 genes. Analysis of the predicted amino-acid sequences of CD1 molecules reveals a low but significant level of homology to major histocompatibility complex (MHC) class I and class II molecules, and, like MHC class I molecules, CD1 molecules are associated non-covalently with beta 2-microglobulin. These structural similarities to known antigen-presenting molecules, together with the expression of CD1 on cells capable of antigen presentation, suggest a role for CD1 molecules in antigen recognition by T cells. Here we demonstrate the specific recognition of CD1a by a CD4-CD8- alpha beta T-cell receptor (TCR) expressing cytolytic T lymphocyte (CTL) line and the specific recognition of CD1c by a CD4-CD8- gamma delta TCR CTL line. The interaction of CD1-specific CTLs with CD1+ target cells appeared to involve the CD3-TCR complex, and did not show evidence of MHC restriction. These results suggest that for a subset of T cells, CD1 molecules serve a function analogous to that of MHC class I and II molecules.     

Diversity of gamma delta T-cell receptors on murine intestinal intra-epithelial lymphocytes

The search for the genes encoding the T-cell receptor (TCR) alpha- and beta-subunits revealed a third gene gamma which shares with the alpha- and beta-genes several properties including somatic rearrangement. This gene, together with a fourth rearranging gene delta, encodes a second type of T-cell receptor, TCR gamma delta. Although TCR gamma delta-bearing T cells constitute a relatively minor subpopulation in the thymus and in peripheral lymphoid organs, they are the major lymphocytes of epidermis (dendritic epidermal cells or DEC) and of intestinal epithelium (intestinal intraepithelial lymphocytes or IEL) in mice, suggesting that at least some gamma delta T cells are important in the surveillance of a variety of epithelia. It was recently reported, however, that the TCR gamma delta on DEC has essentially no structural diversity, implying that the putative ligand is monomorphic. As this finding, if generally applicable, poses severe restrictions on the origin of the ligand, we investigated the diversity of the TCR on the second major epithelium-associated gamma delta T cells, namely IEL from mice. We report here that by contrast with the DEC gamma delta, the IEL gamma delta TCR are structurally diverse.     

Importance of IL-2 receptors in intra-thymic generation of cells expressing T-cell receptors

During development, lymphoid stem cells migrate into the thymic rudiment where they proliferate, rearrange their antigen receptor genes and become differentiated into functionally mature T cells. At present, the regulation of these processes is poorly understood, although recent studies have shown that early fetal and adult immature thymocytes express receptors for the T-cell growth factor, interleukin-2 (IL-2). We now present direct evidence that IL-2 receptors have a function in intra-thymic development by demonstrating that proliferation and the generation of cells expressing the T-cell antigen receptor (alpha beta TCR), which is responsible for the recognition of antigens in the context of MHC, are inhibited when antibodies to IL-2 receptors are added to fetal thymus organ cultures. The inhibition is specific in that it does not affect pre-thymic stem cells and can be partially reversed by addition of exogenous recombinant IL-2.     

Homing of a gamma delta thymocyte subset with homogeneous T-cell receptors to mucosal epithelia

In mice gamma delta T-cell populations with distinct T-cell receptor (TCR) repertoires and homing properties have been identified. Diversified populations are found in lymphoid organs and intestinal epithelia. By contrast, the gamma delta T-cells that have been found in the murine skin are homogeneous. They express a TCR consisting of one particular V gamma 5 and one particular V delta 1 chain and seem to originate from early fetal thymocytes. We have now systematically analysed many tissues by immunohistochemistry and TCR gene sequencing aided by the polymerase chain reaction. These studies revealed a second homogeneous gamma delta T-cell subset in epithelia not of the intestine and skin, but of the vagina, uterus and tongue. The TCR expressed by this gamma delta T-cell subset consists of the same V delta 1 chain. Cells that express this particular TCR have previously been shown to be positively selected in the late fetal thymus.