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.     

Abolition of specific immune response defect by immunization with dendritic cells

Murine cytotoxic T (Tc)-cell responses to various antigens are controlled by immune response (Ir) genes mapping in the major histocompatibility complex (H-2). The genes responsible are those encoding the class I and class II H-2 antigens. The H-2 I-Ab mutant mouse strain bm12 differs from its strain of origin, C57BL/6 (H-2b), only in three amino acids in the I-A beta bm12 class II H-2 molecule. As a consequence, female bm12 mice are Tc-cell nonresponders to the male antigen H-Y and do not reject H-Y disparate skin grafts. We now report that bm12 mice generate strong H-Y-specific Tc cells following priming in vivo and restimulation in vitro with male bm12 dendritic cells (DC). Female bm12 mice primed with male DC also reject male skin grafts. Furthermore, we demonstrate that only responder cell populations containing a mixture of L3T4+ (T-helper (Th) phenotype) and Lyt 2+ (Tc phenotype) T lymphocytes generate H-Y-specific Tc cells. These data imply an essential role for Th cells, activated by DC as antigen-presenting cells (APC), in changing H-Y-nonresponder bm12 mice into H-Y responders. Priming and restimulation with DC allows the triggering of a T-cell repertoire not demonstrable by the usual modes of immunization. This principle might be used to overcome other specific immune response defects.     

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.     

Epithelial cells expressing aberrant MHC class II determinants can present antigen to cloned human T cells

The first step in the induction of immune responses, whether humoral or cell mediated, requires the interaction between antigen-presenting cells and T lymphocytes restricted at the major histocompatibility complex (MHC). These cells invariably express MHC class II molecules (HLA-D region in man and Ia in mouse) which are recognized by T cells of the helper/inducer subset in association with antigen fragments. Interestingly, in certain pathological conditions, for example in autoimmune diseases such as thyroiditis and diabetic insulitis, class II molecules may be expressed on epithelial cells that normally do not express them. We speculated that these cells may be able to present their surface autoantigens to T cells, and that this process may be crucial to the induction and maintenance of autoimmunity. A critical test of this hypothesis would be to determine whether epithelial cells bearing MHC class II molecules (class II+ cells) can present antigen to T cells. We report here that class II+ thyroid follicular epithelial cells (thyrocytes) can indeed present viral peptide antigens to cloned human T cells.     

Cytotoxic T-cell response to H-Y in 'non-responder' CBA mice

Murine cytotoxic T-cell (Tc cell) responses to various antigens are controlled by immune response genes (Ir) mapping in the major histocompatibility complex (H-2). Both helper T cells, controlled by I region-coded genes, and Tc cells, controlled by K/D antigens, are necessary for a positive response. An H-2-restricted Tc-cell response to the male specific minor transplantation antigen (H-Y) can be elicited in B10 (H-2b) female mice primed with syngeneic male spleen cells intraperitoneally (i.p.) or intravenously (i.v.), or by skin grafting followed by restimulation in vitro in mixed lymphocyte culture (MLR) with male cells. CBA (H-2k) mice do not respond by these routes of in vivo priming, and this was thought to be due to a lack of permissible Ir genes for helper function. However, we now report that subcutaneous hind-footpad (fp) immunisation of 'non-responder' CBA mice with syngeneic male cells changes them to responders, a result which argues against a generalised Ir gene-controlled helper defect.     

Induction of cytotoxic T-cell responses in vivo in the absence of CD4 helper cells

Cytotoxic T lymphocytes (CTL) seem to provide the major line of defence against many viruses. CTL effector functions are mediated primarily by cells carrying the CD8 (Ly-2) antigen (CD8+ cells) and are triggered by interactions of the T-cell receptor with an antigenic complex, often termed 'self plus X', composed of viral determinants in association with class I molecules of the major histocompatibility complex (MHC). The mechanism(s) of induction of virus-specific CTL in vivo is poorly understood, but data from in vitro experiments suggest that their generation is strictly dependent on functions provided by CD4+ helper T cells (also referred to as L3T4+; or TH) that respond to antigens in the context of class II (Ia) MHC determinants. The prevailing opinion that induction of most functions of CD8+ cells requires help provided by CD4+ cells has recently been challenged by the observation that CD8+ cells alone can mediate a variety of responses to alloantigens in vitro and in vivo; however, the possibility that CTL to self plus X could be generated in vivo in the absence of TH cells has not been evaluated. We report here that C57BL/6J (B6) and AKR/J mice, when functionally depleted of CD4+ cells by in vivo treatment with the CD4+-specific rat monoclonal antibody GK1.5 (refs 8-14) responded to ectromelia virus infection by developing an optimal in vivo virus-specific CTL response, and subsequently recovered from the disease (mousepox) that was lethal for similarly infected nude mice (CD4-, CD8-).     

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.     

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.     

Immunomodulation of experimental allergic encephalomyelitis by antibodies to the antigen-Ia complex.

Autoimmune diseases occur when T lymphocytes become activated on recognizing self antigen linked to the autologous class II molecule of the major histocompatibility complex (MHC). The resulting complex of antigen MHC T-cell receptor could be a target for treatment of autoimmune diseases. Studies in which each component is blocked separately might be limited by interference in non-relevant immune responses that either use the same set of T-cell-receptor V gene segments or are linked to the same MHC. We report here an attack by a specific antibody on the unique antigenic site formed by the binding of two components of the trimolecular complex, the autoantigen bound to the self MHC. We tested its effect in experimental allergic encephalomyelitis, an acute neurological autoimmune disease which is widely regarded as a model for autoimmune disorders and which is mediated by CD4+ T cells recognizing myelin basic protein (BP), or its peptides, in association with self Ia. We made monoclonal antibodies which bound only the complex of BP and I-As. These antibodies blocked the proliferative response in vitro to the encephalitogenic determinant of BP and reduced the response to intact BP, without affecting the response to a nonrelevant antigen-purified protein derivative of tuberculin presented on syngeneic macrophages. They also inhibited experimental allergic encephalomyelitis in H-2s mice. Hence, antibodies directed specifically to the autoantigen-Ia complex, may offer a highly selective and effective treatment in autoimmune diseases.     

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.     

T-cell recognition of a chimaeric class II/class I MHC molecule and the role of L3T4

In addition to expressing clonally distributed antigen-specific and major histocompatibility complex (MHC)-restricted receptors, T cells also express non-clonally distributed surface molecules that are involved in T-cell function. Among the most intriguing of the latter are L3T4 and Lyt 2, which are expressed on individual T lymphocytes in striking, though not absolute, concordance with their restriction by either class II or class I MHC determinants, and which are thought to contribute to the overall avidity of T-cell interactions by binding to monomorphic determinants on class II and class I MHC molecules, respectively. To examine the ability of T cells to recognize a single class II domain in the absence of the remainder of the Ia molecule, as well as to evaluate the structural basis for the putative interaction of L3T4 with Ia, a recombinant class II/class I murine MHC gene was constructed and introduced into mouse L cells. Here we demonstrate that a subset of class II allospecific cytotoxic T lymphocytes (CTL) can specifically recognize and lyse L-cell transfectants expressing an isolated polymorphic A beta 1 domain, and that anti-L3T4 antibody can block such killing, a result inconsistent with the highly conserved membrane-proximal domains of Ia acting as unique target sites for L3T4 binding.     

The class I major histocompatibility antigen gene activated in a line of SV40-transformed mouse cells is H-2Dd, not Qa/Tla

We have previously described several complementary DNA clones isolated because they correspond to messenger RNAs present at higher levels in the simian virus 40 (SV40)-transformed BALB/c 3T3 cell line SV3T3 Cl38 than in the normal, parental BALB/c 3T3 line. One of these clones, pAG64, hybridizes to RNAs which, while present in BALB/c 3T3 cells, are 10-20-fold more abundant in SV3T3 Cl38 and are found at high levels in a wide variety of transformed cell lines. Nucleotide sequence analysis showed that pAG64 encodes a class I antigen of the major histocompatibility complex. To ascertain the identity of pAG64, we compared its sequence with the available sequences of d haplotype class I antigen genes [K locus, L locus, D locus and the Qa gene defined by genomic clone 27.1] and found that it showed multiple clustered differences from each of these sequences. We therefore concluded that it was not derived from the H-2Kd, H-2Ld or H-2Dd genes and thus must correspond to one of the other class I antigen genes, namely those of the Qa/Tla complex, although it was clearly not the Qa gene defined by the genomic clone 27.1. We now report subsequent findings which indicate that pAG64 in fact corresponds to the H-2Dd gene and not to a Qa/Tla gene.     

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

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

Binding of immunogenic peptides to Ia histocompatibility molecules

Most cellular interactions essential for the development of an immune response involve the membrane glycoproteins encoded in the major histocompatibility gene complex. The products of the I region, the class II histocompatibility molecules (Ia molecules), are essential for accessory cells such as macrophages to present polypeptide antigens to helper T cells. This interaction, antigen presentation, is needed for T-cell recognition of the antigen and its consequent activation. How the Ia molecules regulate the immune response during antigen presentation is not known, although it is commonly thought to result from their association with the presented antigen. Recent studies, including the elucidation of the structure of the T-cell receptor, favour recognition of a single structure, an antigen-Ia complex. Here we report attempts to determine whether purified Ia glycoproteins have an affinity for polypeptide antigens presented by intact cells in an Ia-restricted manner. We first identified the epitope of a peptide antigen involved in presentation. Several laboratories have shown that globular proteins are altered (processed) in intracellular vesicles of the antigen-presenting cell before antigen presentation. A major component of the T-cell response is directed toward determinants found in the unfolded or denatured molecule, and our laboratory has shown that the determinant of the hen-egg lysozyme protein (HEL), presented in H-2k mice to T cells, is a sequence of only 10 amino acids. This portion resides in an area of the native molecule partially buried inside the molecule, in a beta-sheet conformation. To be presented, intact or native HEL must first be processed in acidic intracellular vesicles. Having isolated the peptide responsible for T-cell recognition of HEL, we sought a physical association of this peptide with purified, detergent-solubilized I-Ak molecules from B-hybridoma cells. We have found such an association, which may explain the role of the Ia glycoproteins in cellular interactions.     

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.     

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.     

Expression of interleukin-2 receptors as a differentiation marker on intrathymic stem cells

The thymus is regarded as the primary site for T-cell lymphopoiesis, but very little is known about the lineage inter-relationships of cells within that organ. At least four subpopulations of mouse thymocytes can be defined on the basis of staining with monoclonal antibodies directed against the T-cell differentiation antigens Lyt-2 and L3T4 (ref. 2). Thus immunocompetent (medullary) thymocytes, like peripheral T cells, express either Lyt-2 (cytotoxic phenotype) or L3T4 (helper phenotype) but not both, whereas non-functional (cortical) thymocytes express both markers. In addition, a small subpopulation comprising 2-3% of cells in the thymus and expressing neither Lyt-2 nor L3T4 has recently been described. The latter cells have the properties of intrathymic 'stem cells' in that they are the first to appear in the embryonic thymus and at least some can be shown to give rise, both in vivo (ref. 4. and our unpublished data) and in vitro, to other thymocyte subpopulations. We show here that 50% of Lyt-2-/L3T4- cells in the adult thymus express receptors for the polypeptide growth hormone interleukin-2 (IL-2) whereas other cells in the thymus do not. Furthermore, immunohistochemical localization studies on frozen sections indicate a disperse distribution of IL-2 receptor-positive cells in both the cortex and medulla. These novel findings have potential implications in the context of current models of differentiation pathways within the thymus.     

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.     

Abrogation of oral tolerance by contrasuppressor T cells suggests the presence of regulatory T-cell networks in the mucosal immune system

Continuous ingestion of a thymus-dependent (TD) antigen differentially affects two compartments of the immune system. A secretory IgA antibody response is induced in mucosal tissues, concurrent with a state of antigen-specific systemic unresponsiveness to parenteral challenge, termed oral tolerance. The precise mechanisms whereby gut antigenic exposure induces oral tolerance are unknown, although T-suppressor cells, anti-idiotypic networks and immune complex formation have all been proposed. Here we show that the systemic unresponsiveness of mice made orally tolerant to the TD antigen sheep red blood cells (SRBC) is reversed by the adoptive transfer of Lyt-1+,2-, Vicia villosa lectin-adherent and I-J+ T cells derived from mice which are genetically resistant to the induction of oral tolerance to SRBC. This T-cell subpopulation has the characteristics of contrasuppressor effector T cells (Tcs). Small numbers of these Tcs cells reverse SRBC-specific tolerance both in vivo and in vitro. This finding offers new insight into the mechanisms of oral tolerance induction and maintenance, and suggests that a network of T cells are involved in the regulation of host responses to ingested antigens.     

Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes

The mechanism of self-tolerance is studied in T-cell-receptor transgenic mice expressing a receptor in many of their T cells for the male (H-Y) antigen in the context of class I H-2Db MHC antigens. Autospecific T cells are deleted in male mice. The deletion affects only transgene-expressing cells with a relatively high surface-density of CD8 molecules, including nonmature CD4+ CD8+ thymocytes, and is not caused by anti-idiotype cells.