Strong xenogeneic HLA response in transgenic mice after introducing an alpha 3 domain into HLA B27.

The pronounced response by mouse T cells to the major histocompatibility complex (MHC) class I antigens of the same species is characterized by a relatively large fraction of responding cells. Responses to MHC class I allelles of other species are, however, generally much weaker. T lymphocytes are positively selected on thymic MHC antigens, resulting in a T-cell repertoire with strong alloreactivity. This has been explained in terms of a mouse T-cell repertoire that is not efficiently selected for recognition of HLA molecules owing to the absence of HLA in mice. Here we show that mice transgenic for HLA mount a T-cell response against allogeneic HLA that is no better than in normal mice. We decided instead to test whether the mouse accessory molecule Lyt-2 on cytotoxic T lymphocytes could interact efficiently with the alpha 3 domain of HLA. To do this, we replaced the alpha 3 domain of HLA-B27 by a murine alpha 3 domain in a gene construct used to produce transgenic mice, and then used the spleen cells from these mice to stimulate normal mouse T cells. Under these conditions cytotoxic T lymphocytes were generated with the same frequency against xenogeneic HLA-B27 determinants as against allogeneic mouse class I antigens. These findings indicate that the normally weak xeno-MHC response is due to the inefficient interaction of the murine Lyt-2 accessory molecule with HLA class I, and not to limitations of the mouse T-cell repertoire.     

Self-tolerance alters T-cell receptor expression in an antigen-specific MHC restricted immune response

The influence of major histocompatibility complex (MHC) gene products on the T-lymphocyte alpha beta receptor (TCR) repertoire is well documented, but how specificity is also generated for a diverse array of foreign peptide antigens is unknown. One proposed mechanism is that the TCR repertoire is selected by the recognition of processed self-antigens bound to MHC molecules. Here, we examine the influence of non-MHC-encoded self-antigens on the TCR repertoire expressed in an antigen-specific immune response. Most pigeon cytochrome c-specific, Ek alpha Ek beta (Ek) Ia-restricted T cells from B10.A mice express a product of the V alpha 11 gene family in association with a V beta 3 gene-encoded protein. We therefore examined V alpha 11 and V beta 3 gene expression in cytochrome c-specific T-cell lines derived from various mouse strains with different non-MHC genetic backgrounds. T cells from several strains failed to express any V beta 3 due to tolerance induced by Mlsc-encoded self-antigens. Variable levels of V alpha 11 messenger RNA (mRNA) were expressed by antigen-specific T cells from all the strains. In one strain V beta 3 was expressed in the relative absence of V alpha 11. These results directly demonstrate that self-tolerance alters TCR gene usage in the immune response to a foreign antigen, and indicate that TCR V alpha and V beta proteins may, in part, be independently selected.     

Qa-1 restricted recognition of foreign antigen by a gamma delta T-cell hybridoma

Distinct T-lymphocyte subsets recognize antigens in conjunction with different classes of major histocompatibility complex (MHC) glycoproteins using the T-cell receptor (TCR), a disulphide-linked heterodimer associated with the CD3 complex on the cell surface. In general, class I and class II MHC products provide a context for the recognition of foreign antigens by CD8+ and CD4+ T cells, respectively. This recognition seems to be largely dependent on alpha beta TCR heterodimers, whereas the function of the second gamma delta TCR, present on a minor subpopulation of cells, is still unknown. In the mouse, the existence of six cell-surface MHC class I products (K, D, L, Qa-1, Qa-2 and Tla) has been firmly established by serological, biochemical and genetic evidence. So far, only the most polymorphic of them, K, D and L ('classical' class I) have been reported as restriction elements for T-cell recognition of foreign antigens. The function of the relatively invariant Qa and Tla molecules remains unknown. We have made a T-helper cell hybridoma clone (DGT3) that recognizes synthetic copolymer poly(Glu50Tyr50) in the context of Qa-1 cell surface product, and has a CD4-CD8- phenotype. Our studies indicate that DGT3 cells express the gamma delta TCR on the cell surface, implicating its role in Qa-1-restricted antigen recognition. This is the first evidence that T cells can recognize foreign antigen in association with self Qa product, confirming that Qa molecules not only topologically, but also functionally, belong to the MHC.     

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.     

Self-recognition promotes the foreign antigen sensitivity of naive T lymphocytes

Major histocompatibility complex (MHC) class I and II molecules are highly polymorphic proteins that bind and present foreign peptides to the clonally distributed alphabeta receptors (TCR) of T lymphocytes. As a population, the immature T lymphocytes generated in the thymus express a very diverse set of TCR specificities. A process of positive selection filters this broad repertoire to optimize peripheral T cells for antigen recognition in the context of available MHC products. Only those precursor T cells whose TCRs generate an adequate but not excessive signalling response to self-peptides bound to the expressed MHC proteins undergo successful maturation. Here we show that post-thymic self-recognition facilitates the antigen reactivity of mature T cells. Both experimental and physiological interruption of T-cell contact with self-peptide MHC ligands leads to a rapid decline in signalling and response sensitivity to foreign stimuli. Because the adaptive immune system must be recruited early in an infectious process when antigen is limiting, these findings suggest that positive selection ensures predictable T-cell recognition of available self-ligands, which in turn promotes efficient responses to pathogens.     

HLA-A2 peptides can regulate cytolysis by human allogeneic T lymphocytes

The class-I and class-II molecules encoded by the major histocompatibility complex (MHC) are homologous proteins which allow cytotoxic and helper T cells to recognize foreign antigens. Recent studies have shown that the form of the antigen recognized by T cells is generally not a native protein but rather a short peptide fragment and that class-II molecules specifically bind antigenic peptides. Furthermore, the three-dimensional structure of the human MHC class-I molecule, HLA-A2, is consistent with a peptide-binding function for MHC class-I molecules. An outstanding question concerns the molecular nature and involvement of MHC-bound peptides in antigens recognized by alloreactive T cells. In this study the effects of peptides derived from HLA-A2 on cytolysis of alloreactive cytotoxic T cells (TC) cells are presented. Peptides can inhibit lysis by binding to the T cell or sensitize to lysis by binding an HLA-A2-related class-I molecule (HLA-Aw69) on the target cell. Thus, allospecific TC cells can recognize HLA-derived peptides in the context of the MHC.     

Preferential expression of the T-cell receptor V beta 3 gene by Mlsc reactive T cells

The precursor frequency of T cells specific for any given foreign antigen is, in general, extremely low. Prominent exceptions to this rule are the T cells that are specific for foreign major histocompatibility complex (MHC) products or for products of the minor lymphocyte stimulatory (Mls) genes in the mouse which are present at high frequencies. Here, we report a striking overlap or cross-reactivity between the T cells specific for the protein antigen pigeon cytochrome c in association with Ek alpha Ek beta and the set of T cells specific for Mlsc products. In addition, we demonstrate that the basis for this overlap is the predominant expression of one T-cell receptor (TCR) V beta gene, V beta 3, by T cells that recognize Mlsc products. These results indicate the importance of specific TCR alpha beta dimers in the recognition of Mlsc products and that positive or negative selection of T cells specific for Mls self-determinants may selectively alter the repertoire of T cells available for MHC-restricted recognition of foreign antigens.     

A nucleoprotein peptide of influenza A virus stimulates assembly of HLA-B27 class I heavy chains and beta 2-microglobulin translated in vitro

Most cytotoxic T lymphocytes (CTL) recognize epitopes of foreign viral proteins in association with class I major histocompatibility complex (MHC) molecules. Viral proteins synthesized in the cytoplasm require intracellular fragmentation and exposure to the class I antigens for the development of CTL responses. Although indirect evidence for binding of peptides to class I antigens has accumulated, direct binding has only been shown recently. The formation of complexes between peptide and class I antigen may occur in the endoplasmic reticulum (ER) and peptides have been shown to induce assembly of the class I complex. We have translated the messenger RNAs encoding HLA-B27 (subtype 2705) and beta 2-microglobulin in a rabbit reticulocyte lysate supplemented with human microsomal membranes (to mimic ER membranes), in the absence and presence of a peptide derived from the nucleoprotein (residues 384-394) of influenza A virus. This peptide induces CTL activity against target cells expressing the HLA-B27 antigen. Here we report direct evidence that the nucleoprotein peptide promotes assembly of the HLA-B27 heavy chain and beta 2-microglobulin, and that this can occur in the ER immediately after synthesis of the two proteins.     

Role of self-peptides in positively selecting the T-cell repertoire

The fate of an immature thymocyte is determined by the specificity of its alpha beta T-cell receptor. Only cells expressing receptors that interact with sufficient affinity with major histocompatibility complex (MHC) molecules expressed on thymus epithelial cells are positively selected and go on to mature and seed the peripheral lymphoid organs. The H-2Kb class-I MHC molecule positively selects for the maturation of cytotoxic T lymphocytes that will respond in the periphery to H-2Kb cells presenting a foreign peptide. We have now analysed the ability of variant H-2Kb molecules to positively select T-cells that respond to H-2Kb with ovalbumin. Our results indicate that self-peptides, presented in the groove of the class-I molecule on thymus epithelial cells, are critically involved in positive selection of the T-cell repertoire. Furthermore, the ability of four different H-2Kb variants to select this response in the thymus correlates with their ability to present the ovalbumin peptide, indicating that a self-peptide mimic of the foreign peptide could be involved in positive selection.     

Evidence from in vitro studies that tolerance to self antigens is MHC-restricted

Mature T cells respond to foreign antigens in the context of self major histocompatibility complex (MHC)-encoded products: T helper cells recognize antigen in the context of class II molecules, while cytotoxic T cells (CTL) recognize antigen plus class I molecules. Recent evidence suggests that the MHC-restricted T cell is unable to recognize either the foreign antigen or the self-MHC product alone, but only a complex of the two. Unresponsiveness to self antigens--self tolerance--implies the deletion or suppression of clones of T cells having reactivity to self antigens. Here we demonstrate the presence in normal mice of T cells which recognize self antigens together with allogeneic MHC products. This finding suggests the MHC restriction of T-cell recognition during the entire process of T-cell ontogeny, that is, MHC restriction of self tolerance.     

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.     

Autophagy in thymic epithelium shapes the T-cell repertoire and is essential for tolerance

Recognition of self-antigen-derived epitopes presented by major histocompatibility complex class II (MHC II) molecules on thymic epithelial cells (TECs) is critical for the generation of a functional and self-tolerant CD4 T-cell repertoire. Whereas haematopoietic antigen-presenting cells generate MHC-II-peptide complexes predominantly through the processing of endocytosed polypeptides, it remains unknown if and how TECs use unconventional pathways of antigen presentation. Here we address the role of macroautophagy, a process that has recently been shown to allow for endogenous MHC II loading, in T-cell repertoire selection in the mouse thymus. In contrast to most other tissues, TECs had a high constitutive level of autophagy. Genetic interference with autophagy specifically in TECs led to altered selection of certain MHC-II-restricted T-cell specificities and resulted in severe colitis and multi-organ inflammation. Our findings indicate that autophagy focuses the MHC-II-peptide repertoire of TECs on their intracellular milieu, which notably comprises a wide array of otherwise strictly 'tissue-specific' self antigens. In doing so, it contributes to T-cell selection and is essential for the generation of a self-tolerant T-cell repertoire.     

Striking similarities between antigen receptor J pieces and sequence in the second chain of the murine CD8 antigen

The CD8 antigen is a marker for T-lymphocyte subsets that is absent from helper T cells but expressed on cytotoxic T cells which recognize foreign determinants in association with class I major histocompatibility complex (MHC) antigens. It has been suggested that CD8 plays some part in recognition by CD8+ cytotoxic T cells since anti-CD8 antibodies can block their functions and the human CD8 antigen contains a domain with clear similarities to immunoglobulin and T-cell receptor (TCR) variable-region (V) domains. Human CD8 antigen is thought to be a homodimer but in the mouse and rat the equivalent antigens (alternatively called Lyt2,3 and OX8) are heterodimeric. Rat CD8 contains two chains of relative molecular mass 32,000 (32K) and 37K: the 32K chain is the rat homologue of human CD8 and mouse Lyt2. We describe here the molecular cloning of the rat 37K chain using an oligonucleotide probe predicted from peptide sequence. The full protein sequence is derived from the complementary DNA and matches limited peptide sequence for mouse Lyt3. The new sequence is more like immunoglobulin and T-cell receptor V domains than other T-cell antigens and includes a patch that is almost identical to some joining (J) piece sequences. This suggests that the CD8 and receptor heterodimers may have evolved directly from a common ancestor.     

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.     

Selecting and maintaining a diverse T-cell repertoire

To provide a T-cell population that will respond promptly to foreign antigen, the immune system looks inward, using the variety of self-antigens to select and maintain a diverse repertoire of receptors. A protective immune system must include a T-lymphocyte population that is poised to respond to foreign antigenic peptides presented by self-major histocompatibility complex molecules. As the organism cannot predict the precise pathogen-derived antigens that will be encountered, the system uses the diverse array of self-peptides bound to self-major histocompatibility complex molecules, not only to select a receptor repertoire in the thymus, but also to keep na?ve T cells alive and 'ready for action' in the periphery.     

Development of CD4-CD8+ cytotoxic T cells requires interactions with class I MHC determinants

Differentiation of bone marrow derived precursors into mature T cells takes place in the thymus. During differentiation, T cells develop the receptor repertoire which allows them to recognize antigen in the context of self major histocompatibility complex (MHC) molecules. Mature T helper cells (mostly CD4+ CD8-) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (mostly CD4-CD8+) recognize antigen in the context of class I MHC determinants. Thymic MHC-encoded determinants greatly influence the selection of the T-cell receptor repertoire. In addition to positive selection, a negative selection to eliminate self-reactive T-cell clones is thought to occur in the thymus, but how this 'education' occurs is not well understood. It has been suggested that during differentiation an interaction between the T-cell receptor (TCR) and MHC-encoded determinants occurs, leading to the selection of an MHC-restricted receptor repertoire. In support of this hypothesis, class-II-specific, CD4+ CD8- helper T cells fail to develop in mice neonatally treated with anti-class II monoclonal antibody (mAb). As CD4-CD8+ cells differ from the CD4+ CD8- lineage (in function, MHC-restriction specificity and perhaps site of education) we examined whether interactions with MHC determinants are also necessary for the development of class-I-specific T cells. Here we show that mice chronically treated with anti-class I mAb from birth lack CD4-CD8+ cells and cytotoxic T-cell precursors, indicating that most CD4-CD8+ T cells need interaction with class I MHC molecules during differentiation.     

Homology of human T-cell leukaemia virus envelope gene with class I HLA gene

Human T-cell leukaemia virus (HTLV), first isolated in the United States from a patient with cutaneous T-cell lymphoma, is a unique horizontally transmitted retrovirus which is highly associated with certain adult T-cell malignancies. Also, HTLV can be transmitted in vitro to cord blood T-lymphocytes. In the accompanying paper it was shown that all T cells producing HTLV, whether cultured from infected persons or infected in vitro, bind a monoclonal antibody (4D12) which recognizes an epitope shared by certain cross-reactive class I major histocompatibility antigens. This antigen may account for the extra HLA-A and -B specificities detected in HTLV-infected cells using alloantisera. Because of the unusual findings of apparently inappropriate HLA antigens in HTLV infected cells, we had previously looked for rearrangement of class I-related genes in HTLV infected cells but failed to find any. Here, using molecular clones of HTLV and human major histocompatibility antigen DNA, we have shown homology between the envelope gene region of HTLV and the region of an HLA-B locus gene which codes for the extracellular portion of a class I histocompatibility antigen.     

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.     

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.     

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.