Extrathymic positive selection of alpha beta T-cell precursors in nude mice.

T lymphocytes expressing alpha beta T-cell receptors with sufficient affinity to major histocompatibility complex (MHC) molecules expressed on thymus epithelial cells are positively selected and mature to functional T cells. But several studies have demonstrated that athymic nude mice grafted with MHC-incompatible thymuses developed T cells specific for nude host rather than thymic MHC. We examined this paradox by analysing the specificity of T lymphocytes derived from nude mice. We report here that nude T lymphocyte precursors transferred to allogeneic SCID (severe combined immunodeficiency) mice with a functioning thymus (but lacking T or B cells) generated host MHC-restricted effector T cells but also contained T cells restricted to donor MHC. If nude T cells were depleted from nude lymphohaemopoietic donor cells before or after transfer, only host MHC-specific T cells matured. The results may explain the unusual MHC specificities of nude T lymphocytes described in earlier studies and demonstrate two separate differentiation steps: in nude mice, T cells may be positively selected for self-MHC restriction specificity extrathymically; then a functional thymus is required for efficient T cell maturation.     

A cell line that can induce thymocyte positive selection.

The thymus positively selects thymocytes that bear T-cell receptors which recognize antigen presented by self major histocompatibility complex (MHC) proteins. Positive selection is usually driven by MHC products on radiation-resistant cortical epithelial cells. It is unknown whether positive selection is mediated by all thymic epithelial cells or by some specialized subsets. Here we introduce an H-2b-expressing thymic epithelial cell line into the thymuses of lethally irradiated H-2k animals reconstituted with H-2b/k F1 BM or fetal liver cells. I-Ab-restricted T cells are found in these animals, demonstrating that selection occurs on the introduced epithelial cells.     

Positive selection of T-lymphocytes induced by intrathymic injection of a thymic epithelial cell line.

T lymphocytes recognize antigens as peptide fragments associated with molecules encoded by the major histocompatibility complex (MHC) and expressed on the surface of antigen-presenting cells. In the thymus, T cells bearing alpha beta receptors that react with the MHC molecules expressed by radioresistant stromal elements are positively selected for maturation. In (A x B-->A) bone marrow chimaeras, T cells restricted to the MHC-A haplotype are positively selected, whereas MHC-B-reactive thymocytes are not. We investigated whether the introduction of particular thymic stromal elements bearing MHC-B molecules could alter the fate of B-reactive T cells in these (A x B-->A) chimaeras. Thymic epithelial cell (TEC) lines expressing H-2b were introduced by intrathymic injection into (H-2b/s-->H2s) bone marrow chimaeras and we measured their ability to generate H-2b-restricted cytotoxic T-lymphocytes (CTLs). We report here that one TEC line, 427.1, was able positively to select CTLs specific for influenza and vesicular stomatitis virus antigens in association with class I H-2b molecules. In addition, line 427.1 can process cytoplasmic proteins for presentation to H-2Kb- and H-2Db-restricted CTLs. Thus, a TEC line capable of normal class I MHC antigen processing and presentation in vitro can induce positive selection after intrathymic injection.     

Tolerance of class I histocompatibility antigens expressed extrathymically

Although convincing evidence has been obtained for the imposition of self-tolerance by the intrathymic deletion of self-reactive T cells, the development of tolerance to antigens which are expressed only in the periphery is not so well understood. We have approached this question by creating transgenic mice which carry a class I major histocompatibility complex (MHC) gene (H-2Kb) linked to the rat insulin promoter. Mice expressing the transgene develop diabetes, but do not appear to mount an immune response against the transgene-expressing pancreatic beta-cells, even when the transgene is allogeneic with respect to the endogenous host H-2 antigens. We have now explored the mechanism of this tolerance further. We find that spleen cells from pre-diabetic transgenic (RIP-Kb) mice do not kill targets bearing H-2Kb, whereas thymus cells from the same mice do. The unresponsiveness of these spleen cells can be reversed in vitro by providing recombinant interleukin-2 (rIL-2). In older, diabetic mice, responsiveness develops as the pancreatic beta-cells are lost. Our results point to an extrathymic mechanism of tolerance induction, dependent on the continuous presence of antigen and the lack of IL-2 in the local environment of potentially reactive T cells.     

Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells.

Virus-infected cells can be eliminated by cytotoxic T lymphocytes (CTL), which recognize virus-derived peptides bound to major histocompatibility complex (MHC) class I molecules on the cell surface. Until now, this notion has relied on overwhelming but indirect evidence, as the existence of naturally processed viral peptides has not been previously reported. Here we show that such peptides can be extracted from virus-infected cells by acid elution. Both the naturally processed H-2-Db-restricted and H-2-Kd-restricted peptides from influenza nucleoprotein are smaller than the corresponding synthetic peptides, which have first been used to determine the respective CTL epitopes. As with minor histocompatibility antigens, occurrence of viral peptides seems to be heavily dependent on MHC class I molecules, because infected H-2d cells do not contain the H-2-Db-restricted peptide, and infected H-2b cells do not contain the H-2-Kd-restricted peptide. Our data provide direct experimental proof for the above notion on MHC-associated viral peptides on virus-infected cells.     

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.     

Positive and negative selection of an antigen receptor on T cells in transgenic mice

The T-cell repertoire found in the periphery is thought to be shaped by two developmental events in the thymus that involve the antigen receptors of T lymphocytes. First, interactions between T cells and major histocompatibility complex (MHC) molecules select a T-cell repertoire skewed towards recognition of antigens in the context of self-MHC molecules. In addition, T cells that react strongly to self-MHC molecules are eliminated by a process called self-tolerance. We have recently described transgenic mice expressing the alpha beta T-cell receptor from the cytotoxic T lymphocyte 2C (ref. 11). The clone 2C was derived from a BALB.B (H-2b) anti-BALB/c (H-2d) mixed lymphocyte culture and is specific for the Ld class I MHC antigen. In transgenic H-2b mice, a large fraction of T cells in the periphery expressed the 2C T-cell receptor. These T cells were predominantly CD4-CD8+ and were able to specifically lyse target cells bearing Ld. We now report that in the periphery of transgenic mice expressing Ld, functional T cells bearing the 2C T-cell receptor were deleted. This elimination of autoreactive T cells appears to take place at or before the CD4+CD8+ stage in thymocyte development. In addition, we report that in H-2s mice, a non-autoreactive target haplotype, large numbers of CD8+ T cells bearing the 2C T-cell receptor were not found, providing strong evidence for the positive selection of the 2C T-cell receptor specificity by H-2b molecules.     

Recognition of H-2 domains by cytotoxic T lymphocytes

The polymorphic major histocompatibility antigens (H-2) have a crucial role in the activation of antigen-specific T lymphocytes. Thus, H-2 antigens are not only recognized by allogenic lymphocytes leading to generation of cytotoxic T lymphocytes (CTLs), but it has also been demonstrated that in syngeneic systems most T cells are only able to recognize foreign antigens in conjunction with their own MHC (major histocompatibility complex) antigens. This phenomenon, termed H-2 restriction, may be the key to our understanding to the biological function of MHC antigens. It is not clear whether recognition by T cells of H-2 on a molecular level is confined to particular domains on the H-2 molecule, nor whether the same polymorphic H-2 sites, which are characterized by antibodies, are recognized by allogeneic as well as by H-2 restricted syngeneic CTLs. Previous findings indicate the existence of at least two major polymorphic domains on the H-2Kk molecule as defined by antibodies. Here we show the existence of CTLs with specifity for these polymorphic domains, and the preferential recognition of a particular domain by both alloreactive as well as H-2 restricted CTLs.     

Identity of cells that imprint H-2-restricted T-cell specificity in the thymus

The thymus has two important roles in controlling the specificity of T lymphocytes. First, T cells differentiating in the thymus are rendered tolerant of 'self' antigens, particularly antigens encoded by the major histocompatibility complex, the H-2 complex in mice. Second, the thymus imbues T cells with the property of H-2-restricted recognition of antigen, that is, the capacity of T cells to react with foreign antigens presented in association with self H-2 gene products. Until recently it has generally been assumed that self-tolerance and H-2-restricted specificity both reflect early T-cell contact with self H-2 determinants expressed on thymic epithelial cells. Recent evidence suggests, however, that intrathymic cells of the macrophage/dendritic cell (Mphi/DC) lineage also have a role in shaping T-cell specificity. In particular, it has been found that the tolerance to graft-type H-2 determinants which normally ensues when T cells differentiate in an H-2-different thymus fails to occur when the thymus is pretreated with deoxyguanosine (dGuo), a procedure that selectively destroys Mphi/DC but spares epithelial cells. In contrast to these findings on tolerance induction, evidence is presented here that dGuo-treated thymus grafts do imprint T cells with H--2-restricted specificity for antigen. It appears, therefore, that induction of tolerance and H--2 restriction are controlled by different cells in the thymus.     

Analysis of T-cell subsets in rejection of Kb mutant skin allografts differing at class I MHC

The T-cell subpopulations which initiate and mediate tissue allograft rejection remain controversial. In the present study we attempted to identify the phenotype and function of the T-cell subset(s) primarily responsible for the rejection of skin allografts differing at a single class I locus in the major histocompatibility complex (MHC). We found that the rejection rates by B6 mice (H-2b) of four different class I mutant (Kbm) skin allografts form a distinct hierarchy. This hierarchy correlates strikingly and uniquely with the relative precursor frequencies of Lyt2+ interleukin-2-secreting T-helper cells reactive against the various Kbm mutants. To investigate the role of Lyt2+ T cells in the rejection of class I-disparate skin allografts directly, H-2b nude mice were engrafted with Kbm skin allografts and then reconstituted with L3T4+ or Lyt2+ T-cell subpopulations from syngeneic H-2b mice. Lyt2+ T cells were observed to be both necessary and sufficient for the rejection of class I-disparate Kbm skin allografts, whereas L3T4+ T cells were neither necessary nor sufficient. These results identify the Lyt2+ interleukin-2-secreting T-cell subset as the critical cell type determining the rejection rate of class I-disparate Kbm skin allografts.     

MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells.

The molecular basis of target cell recognition by CD3- natural killer (NK) cells is poorly understood, despite the ability of NK cells to lyse specific tumour cells. In general, target cell major histocompatibility complex (MHC) class I antigen expression correlates with resistance to NK cell-mediated lysis, possibly because NK cell-surface molecules engage MHC class I antigens and consequently deliver inhibitory signals. Natural killer cell allospecificity involves the MHC class I peptide-binding cleft, and further understanding of this allospecificity should provide insight into the molecular mechanisms of NK cell recognition. The Ly-49 cell surface molecular mechanisms of NK cell recognition. The Ly-49 cell surface molecule is expressed by 20% of CD3- NK cells in C57BL/6 mice (H-2b). Here we show that C57BL/6-derived, interleukin-2-activated NK cells expressing Ly-49 do not lyse target cells displaying H-2d or H-2k despite efficient spontaneous lysis by Ly-49- effector cells. This preferential resistance correlates with expression of target cell MHC class I antigens. Transfection and expression of H-2Dd, but not H-2Kd or H-2Ld, renders a susceptible target (H-2b) resistant to Ly-49+ effector cells. The transfected resistance is abrogated by monoclonal antibodies directed against Ly-49 or the alpha 1/alpha 2 domains of H-2Dd, suggesting that Ly-49 specifically interacts with the peptide-binding domains of the MHC class I alloantigen, H-2Dd. Inasmuch as Ly-49+ effector cells cannot be stimulated to lyse H-2Dd targets, our results indicate that NK cells may possess inhibitory receptors that specifically recognize MHC class I antigens.     

Inefficient positive selection of T cells directed by haematopoietic cells.

Intrathymic differentiation of alpha beta TCR+ T cells depends on positive selection of CD4+CD8+ thymocytes by thymic major histocompatibility complex (MHC) molecules. Positive selection allows the maturation of only those T cells capable of restricted antigen recognition in the context of the hosts' MHC alleles. Studies of normal or T-cell receptor-transgenic mice engrafted with MHC-different bone marrow or thymuses support the conclusion that positive selection is directed by MHC molecules expressed on non-haematopoietic cells, presumably thymic epithelial cells. Here we, present contrary evidence that class I MHC molecules expressed by haematopoietic cell types direct positive selection of CD8+ T cells, though at a reduced rate compared with positive selection directed by thymic epithelial cells. The identity of cell types that direct positive selection bears directly on mechanistic models of the process, including the idea that thymic epithelial cell MHC molecules uniquely present specialized peptides that mediate positive selection, and the notion that thymic epithelial cells express unique differentiation-inducing cell surface molecules.     

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.     

Rejection of transplantable AKR leukaemia cells following MHC DNA-mediated cell transformation

Major histocompatibility complex (MHC) class I molecules can function as specific target antigens in T-cell-mediated cytotoxity. In addition, T cells can kill target cells through non-MHC antigens, for example, virally infected cells, if the target and effector cells express the same MHC class I antigens. Consequently, quantitative and/or qualitative variations in the expression of the H-2/HLA antigens on the target cells could interfere with MHC-restricted immune reactions. We have reported that the AKR leukaemia cell line K36.16, a subline of K36 (ref. 3), on which the H-2Kk antigen cannot be detected, is resistant to T-cell lysis and grows very easily in AKR mice. Other AKR tumour cell lines, like 369, which have a relatively large amount of H-2Kk on their surface, are easily killed by T cells in vitro and require a much larger inoculum to grow in vivo. Monoclonal antibodies against H-2Kk, but not against H-2Dk, prevented the killing by T cells. This suggests that some tumour cells grow in vivo because tumour-associated antigen(s) cannot be recognized efficiently by the host's immune system, due to the absence of MHC molecules which would function as restriction elements for T-cell cytotoxicity. We have tested this hypothesis by introducing the H-2Kk gene into the H-2Kk-deficient AKR tumour cell line K36.16 and have now demonstrated directly the biological relevance of H-2Kk antigen expression in the regulation of the in vivo growth of this tumour cell line.     

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.     

Differentiation in vitro of Lyt 2+ thymocytes from embryonic Lyt 2- precursors

In mice, the thymus is regarded as being the primary anatomical site for the generation of immunologically competent T lymphocytes. Such cells comprise approximately 20% of the cells in the thymus and share with T lymphocytes from peripheral lymphoid tissues certain phenotypic properties defined by anti-Lyt antibodies. Thus, most immunocompetent T cells are either Lyt 1+2+ or Lyt 1+2- with the former cells being restricted to recognizing antigen in association with class I (H-2K, D) major histocompatability complex (MHC) products and the latter to class II (H-21) MHC products. Although evidence suggests that Lyt 1+2+ cells are generated from Lyt 1+2- precursor the independent development of two separate Lyt-defined lineages of thymocytes could not be ruled out. Here, the acquisition of Lyt 2 antigen by Lyt 2- cells from late embryonic and early postnatal thymuses is directly demonstrated. Furthermore, by combining cell cycle and Lyt phenotype analysis on a flow microfluorometer, the role of cell division in this differentiation process has been investigated.     

Restricted recognition of beta 2-microglobulin by cytotoxic T lymphocytes

Recognition of foreign antigen by cytotoxic T lymphocytes (CTL) is restricted by class I major histocompatibility complex (MHC) products. Class I heavy chains (relative molecular mass (Mr) 45,000-48,000) are reversibly and noncovalently associated with beta 2-microglobulin (beta 2M, Mr = 12,000). Cells expressing human or murine class I heavy chains can exchange their native beta 2M for exogenously added free beta 2M, which is present in serum. Two allelic forms of beta 2M exist among the common laboratory mouse strains, beta 2M-A and beta 2M-B, which are represented in BALB and C57BL mice, respectively. The two forms differ at a single amino acid at position 85, the gene (beta 2m) is located on chromosome 2 linked to a minor histocompatibility (H) region, H-3. It has been proposed that one of the H-3 loci is identical with beta 2m, and that CTL raised across certain H-3 incompatibilities are actually specific for beta 2M. Here we describe CTL raised in such a combination which recognize endogenous as well as exogenous beta 2M-B in the context of H-2Kb. This represents a unique case of CTL recognition, as CTL usually recognize antigens inserted into the membrane, and it is the first molecular identification of the product of a minor H locus.     

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.     

Presentation of antigen, foreign major histocompatibility complex proteins and self by thymus cortical epithelium

In mouse and man most peripheral T cells bear clonally variable receptors made up of alpha- and beta-chains which bind ligands on target cells consisting of peptide fragments of foreign antigens, complexed with cell surface proteins encoded by the major histocompatibility complex (MHC) of the individual. In the thymus, developing T cells are selected to mature only if their receptors will be able to participate in self-MHC plus antigen recognition in the periphery. This positive selection occurs in the presence of self-MHC, but in the apparent absence of antigen, leading to the paradoxical conclusion that developing thymocytes must be positively selected by engagement of their receptors and self-MHC alone, although thymocytes that react too well with self-MHC are eliminated. To account for this, it has been suggested that MHC molecules in the thymus are not identical to those found elsewhere. To test this and other hypotheses, we have examined the ability of the presumed selecting cells, those of the thymus cortical epithelium, to present various MHC complexes to T cells. Our results indicate that MHC molecules on thymus epithelium are not always the same as those found elsewhere.     

Essential requirement for major histocompatibility complex recognition in T-cell tolerance induction

The induction of T-cell responses involves the recognition of extrinsic antigen in association with antigens of the major histocompatibility complex (MHC), in mice and man, with different T cells recognizing antigen in association with either class I (H-2K/D, HLA-A, B, C) or class II (Ia, HLA-D/DR) MHC antigens. However, the requirement of MHC recognition in the induction of immunological tolerance remains ill defined. With human T helper clones recognizing synthetic peptides of influenza haemagglutinin (HA-1), we have investigated the nature of antigen-induced stimulation, and antigen-induced antigen-specific unresponsiveness, immunological tolerance. Tolerance is not due to cell death, as the cells remain responsive to interleukin-2 and is associated with the loss of T3 antigen from the cell surface. Using monoclonal antibodies to the non-polymorphic regions of human class II antigens to inhibit the induction of T-cell tolerance we report here that induction of tolerance requires the recognition of MHC antigens.