Abnormal tyrosine phosphorylation on T-cell receptor in lymphoproliferative disorders

The study of human autoimmune diseases has benefited greatly from analysis of animal models. Mice that are homozygous for either the lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) mutant genes develop a disease characterized by massive lymphadenopathy and autoantibody formation. With age, the lymphoid organs in these mice are replaced with a greatly expanded population of abnormal lymphocytes. Recent work has shown that these cells are likely to be in the T-cell lineage. They rearrange and transcribe the genes for the alpha and beta subunits of the T-cell receptor (TCR) and a third, T-cell receptor-like gene, T gamma. As determined by immunofluorescence with anti-receptor antibodies the cells also express TCR on the cell surface. The murine T-cell receptor consists of the alpha and beta chains, derived from the rearranged alpha and beta genes, in non-covalent association with seven other chains; the delta chain, of relative molecular mass (Mr) 26,000 (26K), the epsilon chain (25K), a glycosylated 21K chain (gp21) which is probably the homologue of the gamma chain of T3 (CD3), a 16K homodimer (zeta) and a 21K dimer (p21). This multichain complex is thought to be the murine analogue of the human T3 complex. After activation of normal T cells by antigen or lectin, p21 is phosphorylated on tyrosine residues and gp21 is phosphorylated on serine residues. In contrast, in the gld and lpr cells, p21 is phosphorylated even in the absence of antigen or lectin, whereas gp21 is not phosphorylated.     

Isolation of cDNA clones encoding the 20K non-glycosylated polypeptide chain of the human T-cell receptor/T3 complex

The antigen receptor on human T lymphocytes consists of two variable immunoglobulin-like glycoproteins, alpha and beta, which occur in association with three invariable T3 membrane proteins. In humans two of these proteins, T3-gamma and T3-delta, are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20,000 (20K), respectively, while the third, T3-epsilon, is a 20K non-glycosylated protein. On the surface of murine T cells, a non-glycosylated protein dimer composed of 17K subunits (T3-zeta) is found associated with the T-cell receptor alpha and beta chains and the three T3-like polypeptide chains. It is generally accepted that major histocompatibility complex-restricted antigen recognition is a function of the alpha-beta heterodimer. This has led to the postulation that the proteins of the T3 complex are involved in the signal transduction that immediately follows antigen recognition via the antigen receptor. Events believed to be involved in early T-cell activation, such as rapid increases in phosphatidylinositol turnover and free intracellular calcium, can be triggered by antibodies directed against either the T3 complex or the clonotypic receptor. We have previously reported our findings on the cloning of the complementary DNA and genomic structure encoding both the human and murine 20K glycoprotein, T3-delta (refs 11-13). We now present our results on the cloning of the cDNA encoding the human 20K non-glycosylated chain, T3-epsilon.     

Isolation and characterization of a cDNA clone encoding the murine homologue of the human 20K T3/T-cell receptor glycoprotein

The antigen receptor on the surface of human T lymphocytes, which consists of a heterodimer of relative molecular mass (Mr) 90,000 (90K) (alpha- and beta-chains), is associated with the T3 antigen (gamma = 25K, delta = 20K and epsilon = 20K). A working model for the mode of action of the T3/T-cell receptor complex is that the clonotypic alpha- and beta-chains are involved in the recognition and binding of antigen in the context of polymorphic major histocompatibility complex (MHC) gene products on the surface of target cells. Antigen binding by the clonotypic receptor probably results in conformational changes in this structure which are recognized by and subsequently trigger the associated T3 complex to transmit signals into the cell, resulting in a proliferative response. The similarity in structure between murine and human clonotypic antigen receptors suggests that such a mechanism of recognition and activation also exists in mouse T lymphocytes, but so far there has been no evidence for the existence of a murine T3 complex. Here we demonstrate the existence of a T3 delta-chain mRNA in murine T lymphocytes. Our sequence data strongly suggest that this mouse mRNA codes for a complete T3 delta polypeptide chain and reveal some interesting properties of the protein.     

A T3-like protein complex associated with the antigen receptor on murine T cells

Antigen recognition by human T lymphocytes and initiation of T-cell activation are mediated by a group of integral membrane proteins, the T-cell antigen receptor (TCR) and the T3 complex. The polypeptides which comprise T3 (a gamma-chain of relative molecular mass (Mr) 25,000 (25K), and delta and epsilon chains of 20K each) are physically associated with the TCR chains. Surface expression of the complex requires the presence of all the component T3 and TCR proteins. In contrast to the human system, murine T3 has not been identified using antibodies. Here we describe a murine T3-like protein complex. It appears to be more complicated than human T3, containing three monomeric glycoproteins (21-28K), two of which have N-linked carbohydrate side chains and a novel family of TCR-associated homo- and heterodimers. The 28K protein is identified as the murine T3 delta-chain. The 21K protein is phosphorylated on cell activation with concanavalin A (Con A).     

T-cell receptors of human suppressor cells

Cells which can suppress the immune response to an antigen (TS cells) appear to be essential for regulation of the immune system. But the characterization of the TS lineage has not been extensive and many are sceptical of studies using uncloned or hybrid T-cell lines. The nature of the antigen receptor on these cells is unclear. T cells of the helper or cytotoxic lineages appear to recognize their targets using the T-cell receptor (TCR) alpha beta-CD3 complex. TCR beta-gene rearrangements are also found in some murine and human suppressor cell lines but others have been shown not to rearrange or express the beta-chain or alpha-chain genes. We previously established TS clones derived from lepromatous leprosy patients which carry the CD8 antigen and recognize antigen in the context of the major histocompatibility complex (MHC) class II molecules in vitro. We here report the characterization of additional MHC-restricted TS clones which rearrange TCR beta genes, express messenger RNA for the alpha and beta chains of the TCR and express clonally unique CD3-associated TCR alpha beta structures on their cell surface but do not express the gamma chain of the gamma delta TCR on the cell surface. We conclude that antigen recognition by at least some human CD8+ suppressor cells is likely to be mediated by TCR alpha beta heterodimers.     

T-cell antigen receptor genes and T-cell recognition

The four distinct T-cell antigen receptor polypeptides (alpha, beta, gamma, delta) form two different heterodimers (alpha:beta and gamma:delta) that are very similar to immunoglobulins in primary sequence, gene organization and modes of rearrangement. Whereas antibodies have both soluble and membrane forms that can bind to antigens alone, T-cell receptors exist only on cell surfaces and recognize antigen fragments only when they are embedded in major histocompatibility complex (MHC) molecules. Patterns of diversity in T-cell receptor genes together with structural features of immunoglobulin and MHC molecules suggest a model for how this recognition might occur. This view of T-cell recognition has implications for how the receptors might be selected in the thymus and how they (and immunoglobulins) may have arisen during evolution.     

Predictable acquisition of a new MHC recognition specificity following expression of a transfected T-cell receptor beta-chain gene

Activation of mature T lymphocytes requires specific corecognition of antigen together with membrane-associated glycoprotein products of the major histocompatibility complex (MHC). This dual specificity is determined by a single receptor structure consisting of a clone-specific alpha beta heterodimer. Because both the alpha and beta subunits possess unique combining-site-containing V regions, it remains an open issue as to what contribution each of the two chains of the receptor makes to the antigen versus MHC recognition specificities of the complete dimer present on any given T cell or in the T-cell pool as a whole. In the present work, we have used DNA-mediated gene transfer to express a new alpha or beta chain in a recipient murine T-cell hybridoma possessing a related antigen but distinct MHC specificity compared to the receptor-gene donor. Our results demonstrate that a beta-gene transfected hybridoma expresses new receptors with a predictable hybrid specificity, establishing that the beta chain has the predominant role in MHC molecule recognition in this model.     

Association of phosphorylation of the T3 antigen with immune activation of T lymphocytes

In human T lymphocytes the antigen receptor (Ti) is associated non-covalently on the cell surface with the invariant T3 antigen which comprises 3 chains: two glycosylated polypeptides of relative molecular mass 26,000 (Mr 26K) and 21K (gamma and delta) and one non-N-glycosylated polypeptide of Mr 19K (epsilon). The proposed function of T3 is to transduce the activation signals delivered via the antigen receptor. Recently we have shown that phorbol esters, which stimulate protein kinase C, can induce phosphorylation of the gamma subunit of the T3 antigen. But the critical question is whether T3 phosphorylation occurs as a normal consequence of immune activation of T lymphocytes. In this respect, it has been shown that immune stimulation of murine T cells results in phosphorylation of Ti-associated polypeptides that may be the functional analogues of the human T3 antigen. We have therefore monitored T3 phosphorylation after exposure of human T cells to antigen or phytohaemagglutinin (PHA). The data show that both stimuli initiate phosphorylation of the gamma subunit of the T3 antigen which indicates that T3 phosphorylation is a physiological response to immune activation.     

Primary structure of human T-cell receptor alpha-chain

The T-cell receptor has been studied intensely over the past 10 years in an effort to understand the molecular basis for major histocompatibility complex (MHC) restricted antigen recognition. The use of anti-receptor monoclonal antibodies to isolate and characterize the receptor from human and murine T-cell clones has shown that the protein consists of two disulphide-linked glycopeptides, alpha and beta, distinct from known immunoglobulin light and heavy chains. Like immunoglobulin light and heavy chains, however, both the alpha- and beta-chains are composed of variable and constant regions. Molecular cloning has revealed that the beta-chain is evolutionarily related to immunoglobulins, and is encoded in separate V (variable), D (diversity), J (joining) and C (constant) segments that are rearranged in T cells to produce a functional gene. We report here cDNA clones encoding the alpha-chain of the receptor of the human T-cell leukaemia line HPB-MLT. Using these cDNA probes, we find that expression of alpha-chain mRNA and rearrangement of an alpha-chain V-gene segment occur only in T cells. The protein sequence predicted by these cDNAs is homologous to T-cell receptor beta-chains and to immunoglobulin heavy and light chains, particularly in the V and J segments.     

Specific antigen-Ia activation of transfected human T cells expressing murine Ti alpha beta-human T3 receptor complexes

The genes encoding the alpha and beta chains of the T-cell receptor (Ti) of an antigen-specific, Ia-restricted murine T-cell hybridoma were introduced into T3-positive or T3-negative human T cells. The resultant transfectants express either mouse-human or mouse-mouse Ti alpha beta molecules functionally associated with the human T3 complex. Only the complete murine Ti alpha beta dimer mediates specific functional corecognition of the appropriate antigen-Ia pair.     

Delta is the Cx-gene product in the gamma/delta antigen receptor of dendritic epidermal cells

Most T cells bear an antigen receptor that is a protein of a disulphide-linked heterodimer composed of an alpha chain and a beta chain associated with the non-polymorphic CD3 (T3) complex. A small subpopulation of thymic and peripheral T cells, as well as Thy-1+dendritic epidermal cells (dEC), express an alternative CD3-associated dimeric receptor composed of the product of the T-cell antigen receptor (TCR) gamma gene and a fourth chain, designated delta. Recently a new murine TCR constant-region gene, designated Cx, has been cloned and proposed as a candidate for the C delta gene. We have previously demonstrated that murine Thy-1+ dEC cell lines express a CD3-associated disulphide-linked heterodimer composed of a relative molecular mass Mr 41,000 (41K) gamma chain and a 50K delta chain. We have further analysed the receptor of one of these cloned dEC lines, 7-17.1, by endoglycosidase treatment of the isolated gamma and delta chains. The gamma chain was found to contain two N-linked oligosaccharide residues, consistent with the expression of a chain encoded by the V gamma 3 and C gamma 1 gene segments. The delta chain contains at least three N-linked oligosaccharides and has a core size of 38K. Northern blot analysis indicated the presence of abundant Cx messenger RNA in 7-17.1 cells. Immunoprecipitation with two antisera to peptides comprising distinct regions of the Cx sequence indicates that the delta chain is encoded by the Cx gene.     

Isolation of CD4- CD8- mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovial fluid

The majority of peripheral T cells express a heterodimeric, alpha/beta T-cell receptor, which recognizes specific antigenic peptides bound to self major histocompatibility complex (MHC) molecules, and either the CD4 or CD8 surface markers. An additional subset of T cells, whose physiological function is unknown, express a distinct CD3-associated receptor composed of gamma and delta chains. This subset includes cells lacking both CD4 and CD8 surface markers, which may be involved in autoimmunity. The recognition specificity of the gamma/delta receptors is not well characterized and has been defined in only one case to date, a murine cell line which shows MHC-linked specificity. In this report, we describe the isolation of CD4- CD8-, gamma/delta TCR bearing T cell clones from the synovial fluid of a rheumatoid arthritis patient. These T cell clones respond specifically to mycobacterial antigens without MHC restriction.     

Immunoglobulin-like nature of the alpha-chain of a human T-cell antigen/MHC receptor

Although the receptor with which T cells bind specific antigen can, like immunoglobulin, distinguish between antigens which differ only slightly in structure, it is unique in recognizing antigen only in conjunction with one of the self proteins of the major histocompatibility complex (MHC restriction). The receptor was identified and characterized in mouse and man by using monoclonal antibodies to receptor idiotypes, and consists of two disulphide-linked polypeptides, and acidic alpha-chain and a neutral to slightly basic beta-chain. Peptide maps have shown that, like immunoglobulin, both chains vary for receptors of different specificities. T-cell-derived cDNA clones have recently been identified in mouse and man encoding immunoglobulin-like molecules. These were identified as derived from beta-chain genes through a partial N-terminal protein sequence of the beta-chain isolated from a human T-cell tumour. We have now purified the alpha- and beta-chains of the receptor of the human T-cell leukaemia line HPB-MLT, and have determined the amino acid sequence of several tryptic peptides derived from each chain. Our results further confirm that the previously reported cDNA clones encode beta-chains. The sequence of the alpha-chain peptides identify this as another immunoglobulin-like polypeptide chain. Particularly striking was an alpha-chain peptide with high homology to the conserved portion of the immunoglobulin J segment and T-cell receptor beta-chains. Surprisingly, the alpha-chain peptides show little similarity to the sequence predicted by two overlapping putative murine alpha-chain cDNA clones.     

Predominant use of a V alpha gene segment in mouse T-cell receptors for cytochrome c

The T-cell receptor is a cell surface heterodimer consisting of an alpha and a beta chain that binds foreign antigen in the context of a cell surface molecule encoded by the major histocompatibility complex (MHC), thus restricting the T-cell response to the surface of antigen presenting cells. The variable (V) domain of the receptor binds antigen and MHC molecules and is composed of distinct regions encoded by separate gene elements--variable (V alpha and V beta), diversity (D beta) and joining (J alpha and J beta)--rearranged and joined during T-cell differentiation to generate contiguous V alpha and V beta genes. T-helper cells, which facilitate T and B cell responses, bind antigen in the context of a class II MHC molecule. The helper T-cell response to cytochrome c in mice is a well-defined model for studying the T-cell response to restricted antigen and MHC determinants. Only mice expressing certain class II molecules can respond to this antigen (Ek alpha Ek beta, Ek alpha Eb beta, Ev alpha Ev beta and Ek alpha Es beta). Most T cells appear to recognize the C-terminal peptide of cytochrome c (residues 81-104 in pigeon cytochrome c). We have raised helper T cells to pigeon cytochrome c or its C-terminal peptide analogues in four different MHC congenic strains of mice encoding each of the four responding class II molecules. We have isolated and sequenced seven V alpha genes and six V beta genes and analysed seven additional helper T cells by Northern blot to compare the structure of the V alpha and V beta gene segments with their antigen and MHC specificities. We have added five examples taken from the literature. These data show that a single V alpha gene segment is responsible for a large part of the response of mice to cytochrome c but there is no simple correlation of MHC restriction with gene segment use.     

Human gamma delta+ T cells respond to mycobacterial heat-shock protein

Most T cells recognize antigen through the T-cell antigen receptor (TCR)alpha beta-CD3 complex on the T-cell surface. A small percentage of T cells, however, do not express alpha beta but a second type of TCR complex designated gamma delta (ref. 2). Unlike alpha beta+ lymphocytes, gamma delta+ lymphocytes do not generally express CD4 or CD8 molecules, and the nature of antigen recognition by these cells is unknown. To study antigen recognition by gamma delta+ lymphocytes we raised a gamma delta+ alpha beta- -CD4-CD8- line from an individual immune to PPD (purified protein derivative). This line showed a specific proliferative response to PPD and to a recombinant mycobacterial heat-shock protein (HSP) of relative molecular mass 65,000 (65K). The gamma delta+ line was shown to exhibit a major response to HSP in the presence of autologous antigen-presenting cells (APCs). Minor responses occurred, however, with APCs matched for some HLA class I or II antigens, whereas no response occurred with HLA-mismatched APCs. These findings, therefore, document the requirement of HSP-reactive gamma delta+ lymphocytes for histocompatible APCs.     

Evidence for a physical association of CD4 and the CD3:alpha:beta T-cell receptor

CD4 is a molecule expressed on the surface of T lymphocytes which recognize foreign protein antigens in the context of class II major histocompatibility complex (MHC) molecules. Recognition of antigen:class II MHC complexes by CD4+ T cells can be inhibited by anti-CD4 (ref. 3). Nevertheless, specific recognition of the antigen:Ia complex is clearly a function of the T-cell receptor, which is composed of CD3 and the variable polypeptides alpha and beta. Thus, it has been proposed that CD4 serves an accessory function in the interaction of CD4+ T cells and Ia-bearing antigen-presenting cells by binding to non-polymorphic portions of class II MHC molecules and stabilizing the cell interaction. Based on our observation that anti-CD4 could inhibit activation of a cloned line of CD4+ T cells by antibodies directed at a particular epitope on the variable region of the T-cell receptor, we have recently proposed that CD4 is actually part of the T-cell antigen recognition complex, physically associated with CD3:alpha:beta. But numerous studies showing that CD3 and CD4 are not stably associated on the T-cell surface would appear to contradict this model. Here we show that anti-T-cell-receptor antibodies can co-modulate expression of the T-cell receptor and CD4, and that the monovalent Fab fragment of such an anti-T-cell-receptor antibody can, in conjunction with bivalent anti-CD4 antibody, generate an activating signal for the T cell. These findings provide further evidence for a physical association of the T-cell receptor complex and CD4.     

CD1b restricts the response of human CD4-8- T lymphocytes to a microbial antigen.

Molecules encoded by the human CD1 locus on chromosome 1 (ref. 33) are recognized by selected CD4-8- T-cell clones expressing either alpha beta or gamma delta T-cell antigen receptors. The known structural resemblance of CD1 molecules to antigen-presenting molecules encoded by major histocompatibility complex (MHC) genes on human chromosome 6 (refs 3, 4, 34, 35), suggested that CD1 may represent a family of antigen-presenting molecules separate from those encoded in the MHC. Here we report that the proliferative and cytotoxic responses of human CD4-8- alpha beta TCR+ T cells specific for Mycobacterium tuberculosis can be restricted by CD1b, one of the four identified protein products of the CD1 locus. The responses of these T cells to M. tuberculosis seemed not to involve MHC encoded molecules, but were absolutely dependent on the expression of CD1b by the antigen-presenting cell and involved an antigen processing requirement similar to that seen in MHC class II-restricted antigen presentation. These results provide, to our knowledge, the first direct evidence for the proposed antigen-presenting function of CD1 molecules and suggest that the CD1 family plays a role in cell-mediated immunity to microbial pathogens.     

Analysis of specificity for antigen, Mls, and allogenic MHC by transfer of T-cell receptor alpha- and beta-chain genes

The majority of peripheral T lymphocytes bear cell-surface antigen receptors comprised of a disulphide-linked alpha beta dimer. In an immune response, this receptor endows T cells with specificities for foreign antigenic protein fragments bound to cell surface glycoproteins encoded in the major histocompatibility complex (MHC). At a high frequency (greater than 1%), the same population of T lymphocytes responds to allogeneic MHC glycoproteins, or to differences at other genetic loci termed Mls, in conjunction with MHC. The alpha beta-antigen receptor has been implicated in alloreactivity and Mls reactivity. In fact, many monoclonal T-cell lines recognize a foreign protein fragment bound to self-MHC molecules and, in addition, recognize allogeneic MHC glycoproteins, an Mls-encoded determinant, or both. For at least one T-cell clone, a monoclonal antibody directed against the alpha beta antigen receptor has been shown to block activation induced by either antigen-bound self-MHC or by allogeneic MHC. However, it remains to be demonstrated directly that a single alpha beta receptor can mediate antigen specificity, alloreactivity and Mls reactivity, a prerequisite to understanding the structural basis of these high-frequency cross-reactivities. To address this issue we have performed transfers of receptor chain genes from a multiple-reactive T-cell clone into an unrelated host T lymphocyte. We now demonstrate definitively that the genes encoding a single alpha beta-receptor chain pair can transfer the recognition of self-MHC molecules complexed with fragments of antigen, allogeneic MHC molecules, and an Mls-encoded determinant (presumably in conjunction with MHC). In this case the transfer of antigen specificity and alloreactivity requires a specific alpha beta-receptor chain combination, whereas Mls reactivity can be transferred with the beta-chain gene alone into a recipient expressing a randomly selected alpha-chain.     

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.