A monoclonal antibody against antigen-specific helper factor augments T-cell help

Antigen-specific molecules, commonly termed 'factors', have been shown to be released from helper and suppressor T cells. These factors mimic the activity of the cells that secrete them and there is much speculation about the relationship of antigen-specific factors to T-cell receptors for antigen. We have raised a variety of antisera in rabbits which were shown to react against conserved 'constant' determinants on either helper or suppressor factors independently of antigenic specificity or mouse strain of origin of the factor. In contrast, syngeneic mouse antisera were found to react with 'variable' factor determinants in an antigen-specific and mouse strain-dependent manner. These antisera thus define two regions on factor molecules, one 'variable' (related to antigen specificity) and the other 'constant' (related to function). However, potential contaminants in these antisera have limited their usefulness. Thus, we are now generating monoclonal antibodies against T-cell factors and report here the properties of a monoclonal antibody (AF3.44.4) which reacts with antigen-specific helper factors. This antibody also binds to helper T cells and, in the presence of antigen, augments helper cell induction in vitro, which, in turn, leads to enhanced antibody production in vitro. These characteristics suggest that AF3.44.4 recognizes a determinant shared by helper factor and the antigen receptor on helper T cells.     

Rearrangement and transcription of the beta-chain genes of the T-cell antigen receptor in different types of murine lymphocytes

Rearrangements of T-cell receptor beta-chain genes are usually found on both chromosomal homologues, occurring by both deletional and non-deletional mechanisms. Two constant-region (C beta) genes have been identified previously and at least one is transcribed in every helper or cytotoxic T cell tested, but the choice of C beta gene expression is not correlated with the specialized functions of these T lymphocytes. By contrast, four of five suppressor T-cell hybridomas examined have deleted all known joining (J beta) gene segments and C beta genes and therefore may have antigen receptors encoded by different T-cell receptor gene families.     

Induction of regulatory T-lymphocyte responses by liposomes carrying major histocompatibility complex molecules and foreign antigen

Regulatory (helper and suppressor) T lymphocytes become activated only when foreign antigen is presented to them on the surface of antigen-presenting cells (APC), together with class II major histocompatibility complex (MHC) molecules (heterodimers of polypeptides of 28,000 and 35,000 relative molecular mass). Once activated by a certain foreign antigen--MHC combination, T cells react to the same antigen only in combination with the same MHC molecule, a phenomenon termed MHC restriction of T-cell recognition (reviewed in refs 1,5). Studies of the mechanisms involved in antigen presentation and MHC restriction have been hampered mainly by the virtual impossibility of inducing T-cell responses in the absence of APC. We describe here the production of synthetic lipid vesicles with inserted class II MHC molecules and a protein antigen coupled covalently to the lipid. These liposomes are shown to stimulate cloned helper T cells and T-cell hybridomas in an antigen-specific, MHC-restricted manner in the absence of APC. Thus, the recognition of foreign antigen together with class II MHC molecules seems to be the only signal required for the activation of antigen-primed regulatory T cells. Furthermore, 'processing' of antigen by APC is not essential for its recognition by T cells.     

A late-differentiation antigen associated with the helper inducer function of human T cells

T lymphocytes possessing helper function produce soluble factors that greatly augment B-cell proliferation and differentiation into antibody-secreting cells. In humans the subset of T lymphocytes bearing the T4 surface antigen comprises most of the cells that display helper activity and recognize class II antigens of the major histocompatibility complex (MHC), while the subset bearing the T8 antigen comprises T cells recognizing class I MHC antigens and exhibiting cytotoxic or suppressor function. Monoclonal antibodies to T4 or T8 greatly inhibit the cognitive and effector function of cells with the corresponding phenotype. This function/phenotype correlation is not absolute, however, for there are many examples of T8-positive clones that recognize MHC class II antigens and have helper activity, as well as of T4-positive clones with suppressor or cytotoxic function. Recently a family of cell-surface neoantigens, which might be relevant to T-cell function and which are present on activated but not on resting T lymphocytes, has been identified in mouse and humans using monoclonal antibodies. Some of these antibodies block the cytolytic activity of alloreactive T-cell clones, suggesting the possible involvement of such molecules in the activation of cytotoxic T-cell clones or in the lytic process itself. We now describe a similar late-differentiation antigen (LDA1) that is expressed by human T lymphocytes only following activation and is recognized by a monoclonal antibody that inhibits the antibody-inducing helper function of T lymphocytes.     

Antigen-specific interaction between T and B cells

It is well known that B cells require T-cell help to produce specific antibody. Classic experiments suggested that antigen-specific helper T cells interact with antigen-specific B cells via an antigen 'bridge', the B cells binding to one determinant on an antigen molecule (the 'hapten'), while the T cells at the same time recognize another determinant (the 'carrier'). T-helper cells bind specifically to antigen-presenting cells (APC), which have picked up and processed the appropriate antigen, and this interaction, like the interaction of T-helper cells with specific B cells, is restricted by products encoded by the major histocompatibility complex (MHC). Whereas conventional APC such as macrophages display no binding specificity for antigen, B cells have clonally distributed antigen-specific surface immunoglobulin receptors which would be expected to enhance their capacity to present antigen to T cells. These findings are difficult to reconcile with the simple 'antigen bridge' mechanism of interaction, because it is hard to visualize how the bimolecular complex (processed antigen plus MHC molecule) on the APC surface can resemble the trimolecular complex (antigen bound to surface immunoglobulin plus MHC molecule) on the B-cell surface. To address this problem, we have cloned and immortalized human antigen-specific B cells with Epstein-Barr virus (EBV) and analysed their interaction with T-cell clones specific for the same antigen. We report here that surface immunoglobulin is indeed involved in the uptake and concentration of antigen, allowing specific B cells to present antigen to T cells with very high efficiency. However, the antigen must first be internalized and processed by specific B cells and it is then presented to T cells in an MHC-restricted manner indistinguishable from that characteristic of conventional APC.     

Functionally distinct subsites on a class II major histocompatibility complex molecule

Mature T lymphocytes are activated by recognition of the combination of foreign protein antigen and membrane products of the major histocompatibility complex (MHC). Studies of peptide antigen binding to detergent-solubilized class II MHC molecules (Ia) have established that peptide-Ia interaction occurs in the absence of the T-cell receptor and varies according to allele-specific features of Ia molecules. The residues of immunogenic peptides thus contribute to two largely independent functions--the control of association with Ia molecules and the determination of the specificity of T-cell receptor binding. Two analogous and potentially independent functional sites have been postulated for Ia molecules--a region that controls binding to peptides and a region that interacts with T-cell receptors. Here we present evidence from functional analysis of recombinant class II molecules that these two postulated functional regions of Ia molecules do exist and can be independently manipulated, consistent with our recent demonstration of the segmental nature of Ia molecule structure-function relationships.     

Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen

Mature T cells segregate phenotypically into one of two classes: those that express the surface glycoprotein CD4, and those that express the glycoprotein CD8. The CD4 molecule is expressed primarily on helper T cells whereas CD8 is found on cytotoxic and suppressor cells. A more stringent association exists, however, between these T-cell subsets and the major histocompatibility complex (MHC) gene products recognized by their T-cell receptors (TCRs). CD8+ lymphocytes interact with targets expressing class I MHC gene products, whereas CD4+ cells interact with class II MHC-bearing targets. To explain this association, it has been proposed that these 'accessory' molecules bind to monomorphic regions of the MHC proteins on the target cell, CD4 to class II and CD8 to class I products. This binding could hold the T cell and its target together, thus improving the probability of the formation of the trimolecular antigen: MHC: TCR complex. Because the TCR on CD4+ cells binds antigen in association with class II MHC, it has been difficult to design experiments to detect the association of CD4 with a class II molecule. To address this issue, we devised a xenogeneic system in which human CD4 complementary DNA was transfected into the murine CD4-, CD8- T-cell hybridoma 3DT-52.5.8, the TCR of which recognizes the murine class I molecule H-2Dd. The murine H-2Dd-bearing target cell line, P815, was cotransfected with human class II HLA-DR alpha, beta and invariant chain cDNAs. Co-culture of the parental T-cell and P815 lines, or of one parental and one transfected line resulted in a low baseline response. In contrast, a substantial increase in response was observed when CD4+ 3DT-52.5.8 cells were co-cultured with HLA-DR+ P815 cells. This result strongly indicates that CD4:HLA-DR binding occurs in this system and that this interaction augments T-cell activation.     

Characterization of murine cytolytic-helper hybrid T cell clones

L3T4, Lyt-2 and the T-cell receptor for antigen are cell-surface molecules involved in antigen specific T cell activation. We have constructed functional murine cytolytic-helper T-cell hybrid clones to study the link between expression of cell-surface molecules and specific cell function. Three of the clones express two antigen receptors and both Lyt-2 and L3T4, normally expressed on mutually exclusive subsets of mature T lymphocytes. The pattern of lymphokines produced by the hybrid cells in response to antigen was not controlled by the specific antigen receptor; both T-cell growth factor, produced only by the helper T-cell partner, and gamma-interferon, produced only by the cytolytic T-cell partner, were secreted when either antigen receptor was stimulated. However, cytolytic activity appeared to be restricted to the recognition of antigen by the T-cell receptor of the cytolytic partner. Thus cytolysis appears to be rightly linked to the antigen receptor of the cytolytic parent but lymphokine release is not tightly linked.     

Lysozyme-induced T-suppressor cells and antibodies have a predominant idiotype.

The existence of shared idiotypic determinants on the surfaces of T and B cells is now firmly established, suggesting that on both these cell types immunoglobulin variable regions are expressed which presumably function as antigen receptors. In most systems this has been inferred through the use of anti-idiotypic antibody instead of antigen to induce either helper or suppressor T cells. Recent evidence demonstrates that antigen-specific suppressor or helper factors can also bear idiotypic determinants. It is possible that these factors represent released receptors or portions of receptors. We show here the direct elimination of an antigen-induced T-suppressor population by an anti-idiotypic serum and complement. These suppressor T cells as well as the idiotypic population used to generate the antiserum are each specific for the same limited portion of the multi-determinant antigen, lysozyme. Apparently, these suppressor cells are restricted in specificity as well as share idiotypy with antibodies of the same specificity.     

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.     

Accessory cell-dependent selection of specific T-cell functions

Activation of many T-cell functions depends on their interaction with antigen-presenting accessory cells which express I region associated (Ia) products. Cells expressing accessory cell function include those of the monocyte/macrophage lineage and dendritic cells. More recently, B cells and a number of tumour cell lines of macrophage or B-cell origin were shown to act as accessory cells in certain assays. We showed previously that normal peritoneal exudate macrophages (PEC) induced both T-cell proliferation as well as T-cell help, whereas various Ia+ tumour lines of macrophage or B-cell origin, although stimulating antigen-specific T-cell proliferation, did not significantly activate T-cell help. We report here that during the initial T-cell activation in vitro accessory cells (PEC or Ia+ tumour cells) select particular T cells to express previously determined functions. Moreover, some tumour cell lines induce suppressor T cells which inhibit helper activity.     

Positive selection of antigen-specific T cells in thymus by restricting MHC molecules

Thymus-derived lymphocytes (T cells) recognize antigen in the context of class I or class II molecules encoded by the major histocompatibility complex (MHC) by virtue of the heterodimeric alpha beta T-cell receptor (TCR). CD4 and CD8 molecules expressed on the surface of T cells bind to nonpolymorphic portions of class II and class I MHC molecules and assist the TCR in binding and possibly in signalling. The analysis of T-cell development in TCR transgenic mice has shown that the CD4/CD8 phenotype of T cells is determined by the interaction of the alpha beta TCR expressed on immature CD4+8+ thymocytes with polymorphic domains of thymic MHC molecules in the absence of nominal antigen. Here we provide direct evidence that positive selection of antigen-specific, class I MHC-restricted CD4-8+ T cells in the thymus requires the specific interaction of the alpha beta TCR with the restricting class I MHC molecule.     

Responses to the H-2Kba mutant involve recognition of syngeneic Ia molecules

Conventional antigens appear to be recognized by T lymphocytes only when associated with major histocompatibility complex (MHC) antigens. Using antigen-specific proliferation as a model for helper T lymphocytes, it has been demonstrated that Ly1+T cells recognize antigen presented in association with syngeneic Ia molecules. In contrast to responses to conventional antigens, however, a large number of studies have suggested that the stimulation of alloreactive Ly1+T cells, and helper T cells specific for allogeneic cytotoxic T lymphocyte (CTL) responses, involve the direct recognition of Ia alloantigens. For the generation of optimal allogeneic CTL activity it has been proposed that Ly1+T cells recognize allo-Ia antigens directly and provide help to pre-CTLs that respond to allo-H-2K and/or D determinants. Thus, the B6.C.H-2bm1 mutant (bm1, formerly referred to as Hz1), which is believed to consist of a substitution of two amino acids in the H-2Kb antigen, has presented a paradox, for it can stimulate strong mixed lymphocyte culture (MLC), graft versus host and CTL responses by T cells of H-2b haplotype mice in the apparent absence of any alloantigenic differences in the I region. We now present evidence that the stimulation of proliferative and helper T cells by the mutant B6.C.H-2bm1 results from the H-2Kba antigen being recognized in the context of syngeneic Ia determinants. Thus responses to both conventional antigens and allogeneic MHC gene products may proceed via the recognition of antigen in the context of self Ia molecules.     

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.     

T3-Ti receptor triggering of T8+ suppressor T cells leads to unresponsiveness to interleukin-2

T3-associated disulphide linked heterodimers (Tin) comprised of clonally unique alpha-chains of molecular weight (MW) 49,000-54,000 and beta chains of MW 43,000 have been identified as the antigen receptors on human cytotoxic effector and inducer T-lymphocytes. Crosslinking of Ti molecules by either the appropriate nominal antigen/MHC specificity or anti-clonotypic monoclonal antibody results in clonal expansion of such cells via induction of IL-2 receptor expression, endogenous IL-2 release and IL-2-IL-2 receptor interaction. To determine whether analogous antigen receptor molecules and autocrine growth mechanisms are utilized by suppressor T-cells, we produced an anti-clonotypic monoclonal antibody against a non-cytotoxic T8+ suppressor T-cell, T8AC6, which defines a T3-associated disulphide-linked heterodimer of similar molecular weight to the above clonotypes. We find that Te-Ti triggering of suppressor clones (T8AC6, T8AC7 or T8RW) does not result in IL-2 production or T-cell proliferation and in contrast to inducer clones, also leads to a transient IL-2 unresponsive state. We suggest that such T3-Ti receptor mediated autoregulation of suppressor T-cell growth is necessary in the facilitation of initial inducer T-cell activation following antigenic perturbation.     

The CD2 antigen associates with the T-cell antigen receptor CD3 antigen complex on the surface of human T lymphocytes

T lymphocytes can be activated by various stimuli directed either against the T-cell antigen receptor-CD3 antigen complex (Ti-CD3) or the CD2 molecule; see ref. 1 for a review. Activation signals generated by antigen binding to the antigen-specific alpha/beta heterodimer (Ti) are thought to be transduced via the invariant CD3 gamma, epsilon and delta chains, and the associated zeta and eta subunits. The physiological role of the interaction of CD2 with its homologous cell-surface associated ligand LFA-3 remains to be fully elucidated. It has been suggested that CD2 regulates an antigen-independent pathway of activation or that signals delivered via CD2 are an integral part of the antigen-specific pathway. Several recent studies have indicated a requirement for the Ti-CD3 complex in CD2 signalling. Thus, mutant T-cell lines expressing CD2, but not Ti-CD3, on the cell surface cannot be activated via the CD2 molecules. Functional interaction between the Ti-CD3 complex and the CD2 antigen suggests that these T-lymphocyte cell-surface structures are physically associated. Here we use a digitonin-based solubilization procedure to explore this possibility and show that 40% of the cell-surface CD2 molecules can be specifically co-precipitated in association with the Ti-CD3 complex.     

Thymic selection process induced by hybrid antibodies

Thymus-derived (T) lymphocytes using the alpha beta T-cell antigen receptor (TCR) recognize fragmented antigen in conjunction with surface molecules encoded by genes of the major histocompatibility complex (MHC). Peripheral T lymphocytes preferentially see antigen presented by self rather than by foreign MHC molecules, and autoreactive T lymphocytes are deleted. Thus, the peripheral T-lymphocyte repertoire is skewed towards recognition of antigen in the context of self-MHC and towards tolerance to self-antigens. During T-lymphocyte development in the thymus, this repertoire is formed by the interaction of TCR with MHC molecules resulting in positive and negative selection phenomena. Hybrid antibodies (HAbs) that carry binding sites to the TCR and to a surface marker on another cell can engage all T lymphocytes regardless of their specificity. It should be possible to mimic selection processes in normal animals with HAb that specifically link members of a TCR family to MHC molecules on the thymic stroma. We have probed T-lymphocyte development with HAbs linking V beta 8-positive TCR to either class I or class II MHC products in thymic organ culture. Thymocytes exposed to either HAb in an early stage of maturation respond with a significant increase in the frequency of V beta 8-carrying cells. At a later stage of development V beta 8-positive thymocytes are depleted. These results illustrate the succession of positive and negative selection in the developing thymus of normal mice.     

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.     

Cell-cell adhesion mediated by CD8 and MHC class I molecules

CD4 and CD8 are cell-surface glycoproteins expressed on mutually exclusive subsets of peripheral T cells. T cells that express CD4 have T-cell antigen receptors that are specific for antigens presented by major histocompatibility complex class II molecules, whereas T cells that express CD8 have receptors specific for antigens presented by MHC class I molecules (reviewed in ref. 1). Based on this correlation and on the observation that anti-CD4 and anti-CD8 antibodies inhibit T-cell function, it has been suggested that CD4 and CD8 increase the avidity of T cells for their targets by binding to MHC class II or MHC class I molecules respectively. Also, CD4 and CD8 may become physically associated with the T-cell antigen receptor, forming a higher-affinity complex for antigen and MHC molecules, and could be involved in signal transduction. Cell-cell adhesion dependent CD4 and MHC II molecules has recently been demonstrated. To determine whether CD8 can interact with MHC class I molecules in the absence of the T-cell antigen receptor, we have developed a cell-cell binding assay that measures adhesion of human B-cell lines expressing MHC class I molecules to transfected cells expressing high levels of human CD8. In this system, CD8 and class I molecules mediate cell-cell adhesion, showing that CD8 directly binds to MHC class I molecules.     

Negative and positive selection of antigen-specific cytotoxic T lymphocytes affected by the alpha 3 domain of MHC I molecules.

The alpha 1 and alpha 2 domains of major histocompatibility complex (MHC) class I molecules function in the binding and presentation of foreign peptides to the T-cell antigen receptor and control both negative and positive selection of the T-cell repertoire. Although the alpha 3 domain of class I is not involved in peptide binding, it does interact with the T-cell accessory molecule, CD8. CD8 is important in the selection of T cells as anti-CD8 antibody injected into perinatal mice interferes with this process. We previously used a hybrid class I molecule with the alpha 1/alpha 2 domains from Ld and the alpha 3 domain from Q7b and showed that this molecule binds an Ld-restricted peptide but does not interact with CD8-dependent cytotoxic T lymphocytes. Expression of this molecule in transgenic mice fails to negatively select a subpopulation of anti-Ld cytotoxic T lymphocytes. In addition, positive selection of virus-specific Ld-restricted cytotoxic T lymphocytes does not occur. We conclude that besides the alpha 1/alpha 2 domains of class I, the alpha 3 domain plays an important part in both positive and negative selection of antigen-specific cells.