A potential donor gene for the bm1 gene conversion event in the C57BL mouse

The mammalian major histocompatibility complex (MHC; H-2 complex in mouse) is a large multigene complex which encodes cell-surface antigens involved in the cellular immune response to foreign antigens. Class I polypeptides expressed at the H-2K and H-2D loci of numerous mouse strains exhibit an unusually high degree of genetic polymorphism, which is assumed to be related to their function as primary recognition elements in the immune response. We suggested that this H-2 polymorphism may arise by gene conversion-like events between non-allelic class I genes. This is supported by our recent comparison of the DNA sequences of the normal H-2Kb gene sequence, from the C57BL/10 mouse, and a mutant form of this gene called H-2Kbm1: the mutant allele differs from the H-2Kb gene in seven bases out of a region of 13 bases in exon 3 of the class I gene (which encodes alpha 2 (C1) the second highly polymorphic protein domain), suggesting that this region of new sequence had been introduced into the H-2Kb sequence following unequal pairing of two class I genes in the genome of the C57BL mouse. Schulze et al. have obtained similar results. Here we report work identifying a potential donor gene in our library of 26 class I genes cloned from the C57BL/10 mouse.     

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.     

SB-restricted presentation of influenza and herpes simplex virus antigens to human T-lymphocyte clones

The HLA-D region of the human major histocompatibility complex (MHC) has been shown to be homologous to the murine I region in terms of both structure and function. Both regions encode class II MHC molecules which restrict T-lymphocyte interactions with antigen-presenting cells. We have recently described the MHC restriction and antigen specificities of human T-lymphocyte clones directed at strain A influenza virus. The majority of T-lymphocyte clones recognized antigen in the context of cell surface interaction products encoded by HLA-D/DR genes. However, a few clones recognized antigen presented by cells histoincompatible for D/DR antigens. We report here that some of these clones recognized viral antigens in association with antigens encoded by genes identical with or closely linked to the recently described secondary B-cell (SB) locus of the MHC. This is the first report that SB-restricted antigen recognition may form an integral part of normal, human immune responses.     

Susceptibility to murine lymphocytic choriomeningitis maps to class I MHC genes--a model for MHC/disease associations

Susceptibility to some human diseases is linked, albeit weakly, to major transplantation antigens (HLA) encoded by the major histocompatibility gene complex (MHC). Here we have studied MHC/disease association in inbred strains of mice after intracerebral (i.c.) injection of lymphocytic choriomeningitis virus (LCMV). This route of infection leads to a lymphocytic choriomeningitis (LCM) which is not the result of direct cytopathic effects of the virus but is caused by the induced T-cell immune response: immunocompetent mice die whereas T-cell-deficient mice survive. By using two plaque variants of LCMV strain UBC (refs 7,8), we found that susceptibility to LCM was dependent on the LCMV strain used ('aggressive' versus 'docile' UBC-LCMV) and on the various genes of the host mouse strains. In addition, susceptibility to LCM caused by docile UBC-LCMV was clearly linked to the murine major histocompatibility locus H-2D: in MHC-congeneic C57BL/10 mice, susceptibility correlated with early onset and high activity of measurable LCMV-specific cytotoxic T cells in meninges and spleens and could be mapped to H-2D. This model shows that a severe immunopathologically mediated clinical disease in mice can be regulated directly by MHC genes of class I type and supports the notion that many MHC/disease associations directly reflect MHC-restricted and MHC-regulated T-cell reactivity.     

Homology of proteasome subunits to a major histocompatibility complex-linked LMP gene.

The class II region of the major histocompatibility complex (MHC) contains genes encoding at least two subunits of a large, intracellular protein complex (the low molecular mass polypeptide, or LMP, complex). This complex is biochemically similar to the proteasome, an abundant and well conserved protein complex having multiple proteolytic activities. Here we report the isolation of a complementary DNA corresponding to one of the subunits of the LMP complex, LMP-2. The protein predicted from this cDNA sequence closely matches the amino-terminal peptide sequence of a rat proteasome subunit, confirming that the proteasome and the LMP complex share polypeptide subunits. The LMP-2 gene is tightly linked to HAM1, a gene thought to be required for translocating peptide fragments of endogenous antigens into the endoplasmic reticulum for association with MHC class I molecules. These observations suggest that the LMP complex may be responsible for generating peptides from cytoplasmic antigen during antigen processing.     

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.     

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.     

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.     

HLA-restricted recognition of viral antigens in HLA transgenic mice

Cytotoxic T lymphocytes (CTL) recognize antigen in the context of the class-I products of the major histocompatibility complex (MHC). The extensive polymorphism of class-I molecules is thought to be linked to their capacity to present a large variety of foreign antigens. Whether a single T-cell receptor (TCR) recognizes two separate epitopes (the foreign antigen and an epitope on MHC molecules), or a single epitope resulting from the combination of a foreign antigen and an MHC molecule, has not yet been resolved. In view of the differences between species in primary structure of histocompatibility antigens, it might be predicted that the TCR repertoire would evolve in concert with the diversity of MHC antigens. The mouse and human TCR repertoire would be optimally adapted to engage in productive interactions only with mouse (H-2) and human (HLA) MHC antigens respectively, especially if the more conserved features of histocompatibility antigens, in addition to foreign antigen, were seen by the TCR. Alternatively, only the most variable segments of MHC antigens might be engaged in antigen presentation and thus in interaction with the TCR. In that case, interaction between MHC plus antigen and the TCR might not necessarily be limited by species-specific features. By analysis of the T-cell response against virus-infected cells in HLA-B27/human beta 2-microglobulin double transgenic mice, we report here that the mouse T-cell repertoire is perfectly capable of using the human HLA-B27 antigen as a restriction element.     

Multiple mRNA species with distinct 3' termini are transcribed from the beta 2-microglobulin gene

beta 2-Microglobulin is the small, relatively invariant subunit of a family of cell-surface glycoproteins encoded within the major histocompatibility complex (MHC). Proteins associated with beta 2-microglobulin in the mouse include the classical transplantation antigens (H-2K, D and L), the thymus leukaemia antigen (TL) and certain haematopoietic cell differentiation antigens (Qa-1 and Qa-2). The genes encoding these proteins are members of a large, multigene family. In contrast, beta 2-microglobulin is encoded by a single copy gene on mouse chromosome 2 (refs 5, 6). We have shown that this gene consists of four coding blocks separated by three intervening sequences. We now demonstrate that the single beta 2-microglobulin gene is transcribed into at least two different size classes of mRNA that differ in the lengths of their 3' untranslated regions. We further show that three polyadenylation signals and a poly (A) tail are encoded at the 3' end of the gene.     

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.     

Igh-V or closely linked gene(s) control immunological memory to a thymus-independent antigen

Mice mount a normal primary antibody response on stimulation with the thymic-independent antigen trinitrophenylated lipopolysaccharide (TNP-LPS). Although we have previously reported the generation of functional B-memory lymphocytes to TNP-LPS, this memory response was only observed in few mouse strains. Here we have used congeneic mouse strains in an attempt to locate the genetic regions involved in the memory response. We show that genes of the major histocompatibility complex (MHC) do not have a critical role but that genes coding for the variable region of immunoglobulin heavy chains or gene(s) closely linked to them are required for memory cell induction by TNP-LPS.     

Thymic major histocompatibility complex antigens and the alpha beta T-cell receptor determine the CD4/CD8 phenotype of T cells

T-cell receptors and T-cell subsets were analysed in T-cell receptor transgenic mice expressing alpha and beta T-cell receptor genes isolated from a male-specific, H-2Db-restricted CD4-8+ T-cell clone. The results indicate that the specific interaction of the T-cell receptor on immature thymocytes with thymic major histocompatibility complex antigens determines the differentiation of CD4+8+ thymocytes into either CD4+8- or CD4-8+ mature 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.     

The gene encoding the Ia antigen-associated invariant chain (Ii) is not linked to the H-2 complex

The invariant (Ii) chain of murine Ia antigens is associated with the intracellular but not the cell-surface forms of the A alpha:A beta and E alpha:E beta Ia complexes. Due to its unique subcellular localization, Ii has been postulated to play a part in the assembly or intracellular transport of the Ia alpha:beta complexes, which function in immune recognition. A more general role for Ii in the transport of other cell proteins has also been suggested. Because of the unusual subunit composition of Ia antigens and because the synthesis of alpha, beta and Ii chains is coordinately regulated, it was of interest to determine whether, like the alpha and beta chains, Ii is encoded by a gene in the I region of the H-2 histocompatibility complex. We report here the use of an Ii chain polymorphism present in Mus spretus to demonstrate that the gene for Ii is not linked to the H-2 complex. Thus, intracellular Ia antigens consist of the products of two linked genes and one unlinked gene.     

Exon shuffling: mapping polymorphic determinants on hybrid mouse transplantation antigens

The mouse major transplantation antigens H-2K, H-2D and H-2L are highly polymorphic cell-surface glycoproteins which may serve as recognition elements in cell-cell interactions. Each antigen possesses a number of alloantigenic determinants defined by antisera of various specificities. Recently, monoclonal antibodies have been produced which redefine and extend our knowledge of these determinants2,3, but structural information has not yet been correlated with the serological definition of the antigens. We have previously reported the molecular cloning of genes for H-2Ld and H-2Dd transplantation antigens from the BALB/c mouse and the expression of these genes in mouse L cells4,5. To localize the serological determinants to discrete regions of the H-2 protein, we have now constructed new H-2 antigen genes by joining together fragments of the H-2Ld and H-2Dd genes. In L cells, these genes direct the synthesis of hybrid H-2 proteins and by using monoclonal antibodies of defined specificities, we have mapped classically defined serological specificities to structurally defined domains of the transplantation antigen protein. We conclude that polymorphic determinants recognized by monoclonal antibodies are located in functionally distinct portions of the protein.     

New class II-like genes in the murine MHC

Major histocompatibility complex (MHC) class I molecules present endogenous antigens to CD8+ (cytotoxic) T cells. MHC class II molecules present primarily exogenously derived antigens to CD4+ T cells. Three new genes (Ma, Mb1 and Mb2) located between the Pb and Ob genes of the murine MHC have properties indicating that they are members of the MHC class II gene family, but they are the most divergent class II members so far identified and are almost as closely related in sequence to class I genes as they are to the known class II genes.     

Identification of susceptibility loci for insulin-dependent diabetes mellitus by trans-racial gene mapping

INSULIN-dependent (type I) diabetes mellitus (IDDM) follows an autoimmune destruction of the insulin-producing beta-cells of the pancreas. Family and population studies indicate that predisposition is probably polygenic. At least one susceptibility gene lies within the major histocompatibility complex and is closely linked to the genes encoding the class II antigens, HLA-DR and HLA-DQ (refs 3, 4). Fine mapping of susceptibility genes by linkage analysis in families is not feasible because of infrequent recombination (linkage disequilibrium) between the DR and DQ genes. Recombination events in the past, however, have occurred and generated distinct DR-DQ haplotypes, whose frequencies vary between races. DNA sequencing and oligonucleotide dot-blot analysis of class II genes from two race-specific haplotypes indicate that susceptibility to IDDM is closely linked to the DQA1 locus and suggest that both the DQB1 (ref. 7) and DQA1 genes contribute to disease predisposition.     

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.