A role for clonal inactivation in T cell tolerance to Mls-1a

Clonal deletion plays a major part in the maintenance of natural self-tolerance in both normal and transgenic mice. Self antigens that are expressed in the thymus result in the physical elimination of autoreactive thymocytes at a particular stage in their development. For example, the majority V beta 6- and V beta 8.1-bearing T cells that recognize the minor lymphocyte-stimulating antigen, Mls-1a (ref. 10) , are clonally deleted in the thymuses of normal mice and transgenic mice expressing Mls-1a (refs 2, 3, 9). In contrast, a very different mechanism of tolerance involving the functional inactivation, but not elimination, of autoreactive cells, termed clonal inactivation or clonal anergy, has been implicated in some experimentally manipulated systems of tolerance. To test further the mechanisms involved in self-tolerance, we have generated transgenic mice expressing a V beta 8.1 beta chain on greater than 95% of peripheral T cells and have tested tolerance to Mls-1a in these mice. Surprisingly, a significant fraction of the CD4+ peripheral cells that survived deletion were non-responsive in vitro to any stimulus tested. Naturally occurring tolerance to a self antigen expressed in the thymus can thus be mediated by clonal anergy, as well as by clonal deletion.     

Deletion of self-reactive thymocytes occurs at a CD4+8+ precursor stage

As T cells develop in the thymus, they become tolerant of self-antigens. A major advance in the understanding of how this process occurs was the direct demonstration that cells bearing autoreactive T-cell receptors (TCRs) are physically eliminated from the population of functionally mature T cells present in both the thymus and periphery. We have sought to determine the developmental stage at which autoreactive T cells are eliminated by examining the expression of V beta 17a anti-I-E TCRs under various experimental conditions. In vivo antibody blockage of the CD4 molecule on developing thymocytes of I-E+ C57BR mice was found to inhibit the deletion of V beta 17a-bearing cells from the CD4-8+ single positive thymocyte subset. This result provides strong evidence that deletion of potentially autoreactive T cells occurs at a CD4+8+ precursor stage, that the non-clonally distributed accessory molecules (CD4, CD8) are significant participants in the self-recognition process that leads to clonal elimination, and that thymic class II major histocompatibility complex (MHC) molecules can influence the repertoire of CD4-8+ cells.     

Deletion of self-reactive T cells before entry into the thymus medulla

The thymus is important in the differentiation of bone marrow-derived precursor cells into functional T cells; humoral factors, as well as physical interactions with nurse cells, dendritic cells and epithelial cells, are thought to be instrumental in this process. Thymic lymphocytes mature during their migration from the cortical to the medullary region of the thymus, when they undergo phenotypic changes that include the acquisitions of T-cell antigen receptors, hormone receptors and differentiation antigens. Cortical T cells are thus mostly CD4+CD8+, whereas medullary T cells are either CD4+CD8- or CD4-CD8+. During this period T cells are subjected to two types of repertoire selection: all T cells recognizing self-MHC with low affinity may be preferentially amplified (positive selection), and in a second step T cells with high-affinity receptors for self-MHC determinants plus self antigens are eliminated (negative selection). We have described two monoclonal antibodies specific for the V beta 6 gene segment of the alpha/beta heterodimeric T-cell antigen receptor and have shown that most CD4+/V beta 6+ T cell recognize the Mlsa antigenic determinant but not Mlsb; similar results have been reported for V beta 8.1 and Mlsa. In both situations, tolerance to Mlsa correlated in an MHC-dependent fashion with absence of V beta 6 or V beta 8.1 T-cell antigen receptor expressing T cells in the periphery. We show here by immunostaining of thymus cryosections and cytofluorometric analysis that V beta 6-expressing cortical T cells are present at high density in both Mlsa and Mlsb mice, but do not enter the medullary region of Mlsa animals.     

Positive selection of CD4-CD8+ T cells in the thymus of normal mice

The diversification of the repertoire of T-cell antigen receptor (TCR) specificities is influenced by at least two selection processes which occur in the thymus. One of these, termed 'negative selection', is required to install a state of tolerance to self-antigens in the T-cell repertoire and is often achieved by clonal deletion. The second type of selection operating in the thymus results in preferential differentiation of T cells that have restriction specificity for thymic major histocompatibility complex glycoproteins, but the mechanisms leading to this selective process are not yet clear. One model used to describe this 'positive selection' proposes that only those T cells with sufficient avidity for the MHC glycoproteins expressed in the thymus are allowed to acquire functional competence. Here we directly investigate the generation of TCR specificities by following the fate of developing V beta 17+ CD4-CD8+ T cells under conditions where one of the main class I-MHC molecules, either H-2K or H-2D, was specifically blocked by in vitro monoclonal antibody treatment. The results show that development of V beta 17+ CD4-CD8+ T cells in the SJL H-2s mouse strain is selectively abrogated by blocking class I-Ks molecules but is unaffected by blocking class I-Ds molecules. These data directly demonstrate that generation of CD4-CD8+ T cells expressing a particular TCR V beta segment can be correlated with the expression of a particular class I-MHC molecule, thereby providing evidence for positive selection.     

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.     

Infection breaks T-cell tolerance.

Clonal deletion or clonal anergy establish tolerance in T cells that bear potentially autoreactive antigen receptors. Here we report that concomitant infection with the nematode Nippostrongylus brasiliensis breaks an established T-cell tolerance induced by injection of mice with Staphylococcus enterotoxin B (SEB). CD4+ T cells from SEB-tolerant mice did not produce either interleukin-2 or interleukin-4 when challenged in vitro with SEB. N. brasiliensis infection of SEB-primed animals resulted in a normal expansion of SEB-tolerant CD4+V beta 8+ T cells in vivo as well as an equivalent increase of SEB-reactive, interleukin-4-producing CD4+V beta 8+ T cells both in SEB-tolerant and in normal animals. Thus, infection with N. brasiliensis circumvented the tolerance established with SEB. Activation of anergic, potentially autoreactive CD4+ T cells by infectious agents seems to be a major pathway for the initiation of autoimmune diseases. Our results suggest that infectious agents may break tolerance in potentially autoreactive CD4+ T cells by activation of alternative reaction pathways.     

Phenotypic differences between alpha beta versus beta T-cell receptor transgenic mice undergoing negative selection

T-cell differentiation in the thymus is thought to involve a progression from the CD4-CD8- phenotype through CD4+CD8+ intermediates to mature CD4+ or CD8+ cells. There is evidence that during this process T cells bearing receptors potentially reactive to 'self' are deleted by a process termed 'negative selection' One example of this process occurs in mice carrying polymorphic Mls antigens, against which a detectable proportion of T cells are autoreactive. These mice show clonal deletion of thymic and peripheral T-cell subsets that express the autoreactive V beta 3 segment of the T-cell antigen receptor, but at most a two-fold depletion of thymic cells at the CD4+CD8+ stage. By contrast, transgenic mice bearing both alpha and beta chain genes encoding autoreactive receptors recognizing other ligands, show severe depletion of CD4+CD8+ thymocytes as well, suggesting that negative selection occurs much earlier. We report here the Mls 2a/3a mediated elimination of T cells expressing a transgene encoded V beta 3-segment, in T-cell receptor alpha/beta and beta-transgenic mice. Severe depletion of CD4+CD8+ thymocytes is seen only in the alpha/beta chain transgenic mice, whereas both strains delete mature V beta 3 bearing CD4+ and CD8+ T cells efficiently. We conclude that severe CD4+CD8+ thymocyte deletion in alpha/beta transgenic mice results from the premature expression of both receptor chains, and does not reflect a difference in the timing or mechanism of negative selection for Mls antigens as against the allo- and MHC class 1-restricted antigens used in the other studies.     

Self-tolerance eliminates T cells specific for Mls-modified products of the major histocompatibility complex

In mice the product of the Mlsa locus is an unusual antigen capable of interaction with certain products of the major histocompatibility locus (MHC) to form a ligand for a large portion of the T-cell alpha/beta receptor repertoire, including nearly all receptors that use V beta 8.1. The presence of Mlsa/MHC during T-cell development results in the deletion of T cells that express V beta 8.1, documenting the importance of clonal deletion in establishing tolerance to self antigens.     

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.     

Genes encoding ligands for deletion of V beta 11 T cells cosegregate with mammary tumour virus genomes.

The T-cell receptor (TCR) repertoire is selected in the thymus after rearrangement of genes encoding TCR alpha and beta chains. Selection is based on the recognition by newly emergent T cells of self-ligands associated with molecules of the major histocompatibility complex: some combinations result in positive selection, others in negative selection. Negative selection, or clonal deletion, is an important mechanism for eliminating autoreactive T cells. A group of self-ligands involved in clonal deletion was identified because they, like exogenous superantigens, were recognized by almost all T cells expressing particular TCR V beta genes. V beta 17a T cells are deleted by a tissue-specific ligand; V beta 6, V beta 7, V beta 8.1 and V beta 9 T cells are deleted by the minor lymphocyte-stimulating (Mls) determinant Mls-1a; V beta 3 T cells by Mls-2a and Mls-3a; V beta 11 T cells by ligands encoded by independently segregating genes; and V beta 5 T cells by ligands encoded by two genes. Chromosome mapping using recombinant inbred strains of mice and classic backcrosses show that Mls-1a in DBA/2 mice is encoded on chromosome 1, that one of the two ligand genes for deletion of V beta 5 T cells maps to chromosome 12 and that a ligand gene for V beta 11 deletion is linked to the CD8 locus on chromosome 6. Here we present evidence from three sets of backcross mice for concordance between V beta 11 deletion ligand genes on chromosomes 6, 12 and 14 and endogenous mouse mammary tumour virus integrant (Mtv) genomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

An explanation for the protective effect of the MHC class II I-E molecule in murine diabetes

Insulin-dependent diabetes mellitus is widely believed to be an autoimmune disease. Recent onset diabetics show destruction of insulin-secreting pancreatic beta-cells associated with a lymphocytic infiltrate (insulitis), with autoantibodies to beta-cells being found even before the onset of symptoms. Susceptibility to the disease is strongly influenced by major histocompatibility complex (MHC) class II polymorphism in both man and experimental animal models such as the non-obese diabetic (NOD) mouse. As MHC class II molecules are usually associated with dominant immune responsiveness, it was surprising that introduction of a transgenic class II molecule, I-E, protected NOD mice from insulitis and diabetes. This could be explained by a change either in the target tissue or in the T cells presumed to be involved in beta-cell destruction. Recently, several studies have shown that I-E molecules are associated with ontogenetic deletion of T cells bearing antigen/MHC receptors encoded in part by certain T-cell receptor V beta gene segments. To determine the mechanism of the protective effect of I-E, we have produced cloned CD4+ and CD8+ T-cell lines from islets of recently diabetic NOD mice. These cloned lines are islet-specific and pathogenic in both I-E- and I-E+ mice. Both CD4+ and CD8+ cloned T cells bear receptors encoded by a V beta 5 gene segment, known to be deleted during development in I-E expressing mice. Our data provide, therefore, an explanation for the puzzling effect of I-E on susceptibility to diabetes in NOD mice.     

Intrathymic deletion of self-reactive cells prevented by neonatal anti-CD4 antibody treatment

T-cell differentiation in the thymus involves the coordinate expression of genes encoding the alpha and beta chains of the major histocompatibility complex-restricted heterodimeric antigen receptor (TCR) complex, as well as other functionally important molecules such as CD4 and CD8. The repertoire of TCR expressed by T cells is generally thought to be influenced by positive and/or negative selection events occurring when TCRs on developing T cells interact with self-antigens and major histocompatibility complex components. Using a model system in which specific antigen-reactive cells can be monitored by virtue of their preferential expression of certain TCR beta-chain variable (V beta) domains, it has been shown that self-reactive T cells are clonally deleted during development. We report here that clonal deletion of V+ beta 6 cells in Mlsa mice can be prevented by in vivo neonatal administration of monoclonal antibodies directed against CD4. Furthermore, as anti-CD4 monoclonal antibody treatment resulted in the reappearance of V+ beta 6 cells in the mature CD8+ T-cell subset, it is likely that clonal deletion acts on the CD4+CD8+ thymocyte subset and that this subset is an intermediate stage in the differentiation pathway of both CD4+ and CD8+ T-cell lineages.     

T cells can distinguish between allogeneic major histocompatibility complex products on different cell types

In the response of T cells to foreign antigens, the ligand for the T cell alpha/beta receptor is presented on a cell surface as a fragment of antigen complexed to one of the membrane molecules encoded in the major histocompatibility complex (MHC). The receptor apparently interacts via its variable elements (V beta, D beta, J beta, V alpha and J alpha) with residues within both the antigen and MHC portion of the ligand. The frequency of T cells responding to a conventional antigen plus self MHC is usually quite low, presumably reflecting the relative rarity of receptors with the particular combination of variable elements to match the antigen/MHC ligand. T cells also respond to allogeneic forms of MHC molecules in the absence of added antigen. In this case the frequency of responding T cells is very high. One hypothesis to explain this observation is that, in the absence of foreign antigen, MHC molecules are complexed to a large array of peptides derived from self-proteins. In this case the combination of the polymorphic MHC amino acid residues and many different self peptides presents so many possible ligands that the likelihood of recognition by a given T cell receptor is quite high. The recent crystallography experiments which revealed a dramatic binding cleft on the face of a human MHC molecule have given impetus to this view, but as yet there is no direct supporting evidence. We have recently described a close association between murine T cell receptors utilizing the V beta 17a element and reactivity to various allogeneic forms of the murine MHC molecule, I-E (ref. 8). In this paper, we show that this I-E ligand is detected on B cells, but not on I-E+ macrophages or fibroblasts expressing a transfected I-E gene. These results strongly suggest a B cell specific product combines with I-E to form the allogeneic ligand for V beta 17a+ receptors and thus support the concept of alloreactivity described above.     

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.     

Positive selection of CD4+ thymocytes controlled by MHC class II gene products

The mature T-cell antigen receptor repertoire is characterized by lack of reactivity to self-components as well as by preferential reactivity to foreign antigens in the context of polymorphic self-proteins encoded within the major histocompatibility complex. Whereas the former characteristic (referred to as negative selection or tolerance) is associated with intrathymic deletion of T cells expressing T-cell antigen receptor beta-chain variable (V beta) domains, which confer a preferential reactivity to self antigens, the existence of the latter (referred to as positive selection or MHC restriction) has so far only been inferred indirectly from functional studies. We show here that intrathymic deletion of V+beta 6 T cells (reactive with a self-antigen encoded by the Mlsa locus) is controlled by polymorphic MHC class II determinants. Furthermore, in mice lacking expression of Mlsa, the same class II MHC loci control the frequency of occurrence of V+beta 6 cells among mature CD4+ T lymphocytes. These data are direct evidence for positive selection by MHC determinants in the thymus in unmanipulated animals.     

Positive selection of CD4+ T cells mediated by MHC class II-bearing stromal cell in the thymic cortex

T lymphocytes differentiate in the thymus, where functionally immature, CD4+CD8+ (double positive) thymocytes develop into functionally mature CD4+ helper cells and CD8+ cytotoxic (single positive) T cells. The thymus is the site where self-reactive T cells are negatively selected (clonally deleted) and where T cells with the capacity to recognize foreign antigens in association with self-proteins encoded by the major histocompatibility complex (MHC) are positively selected. The net result of these developmental pathways is a T-cell repertoire that is both self-tolerant and self-restricted. One unresolved issue is the identity of the thymic stromal cells that mediate the negative and positive selection of the T-cell repertoire. Previous work has pointed to a bone-marrow-derived macrophage or dendritic cell as the inducer of tolerance, whereas a radiation-resistant, deoxyguanosine-resistant thymic cell seems to mediate the positive selection of self-MHC restricted T cells. Thymic stromal cells in the cortex interact with the T-cell antigen receptor on thymocytes. Using several strains of transgenic mice that express the class II MHC molecule I-E in specific regions of the thymus, we show directly that the positive selection of T cells is mediated by an I-E-bearing cell in the thymic cortex.     

Clonal anergy induced in mature V beta 6+ T lymphocytes on immunizing Mls-1b mice with Mls-1a expressing cells

Tolerance to self-antigens has been shown to develop during ontogeny as a result of the clonal deletion of self-reactive T cells. Tolerance, or better 'nonresponsiveness', to specific antigens can also be induced in adult animals but the mechanism(s) involved are not well understood. Most murine T-helper cells that express the V beta 6 T-cell receptor gene segment are specific for Mls-1a antigens. We have therefore been able to use an anti-V beta 6 monoclonal antibody to follow the fate of Mls-1a specific T cells in adult Mls-1b mice made specifically unresponsive to Mls-1a. We show that the induced unresponsiveness is not due to clonal deletion, but rather to clonal anergy. The anergic V beta 6 T-helper cells express IL-2 receptors and undergo limited blastogenesis in vitro upon stimulation, but do not produce IL-2, in marked contrast to V beta 6 cells from naive mice. Our data appear to represent an in vivo correlate for the induction of anergy that has been observed in T-cell lines in vitro.     

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.     

Clonal deletion of immature CD4+8+ thymocytes in suspension culture by extrathymic antigen-presenting cells

One mechanism ensuring self tolerance of T cells is the clonal deletion of thymocytes bearing alpha beta T-cell receptors. The stage of thymocyte development at which the interaction with antigen-presenting cells (APCs) leads to deletion, however, has not been determined directly. Indirect evidence suggests that intrathymic APCs induce deletion of CD4+8+ thymocytes (which die by apoptosis) but deletion at less and more mature developmental stages has also been implied. It is also not clear if clonal elimination of thymocytes can be triggered by peripheral antigens carried on extrathymic APCs migrating through the thymus. Here we show antigen-specific induction of apoptosis in CD4+8+ thymocytes cultured in suspension, by thymic as well as splenic APCs. Thus the recognition of antigen by CD4+8+ thymocytes may lead to deletion, suggesting that this is the central mechanism of tolerance induction, which is not limited by the antigen-presenting ability of the thymic stroma.