Ia Loci Research Articles

Expression of HLA class I genes in meiotic and post-meiotic human spermatogenic cells.

In human spermatogenic cells, in contrast to somatic cells, expression of major histocompatibility complex (MHC) class I molecules is undetectable. This lack of expression may contribute to the absence of female immune reaction against spermatozoa and may be necessary for gamete fusion. Among the molecular repressor mechanisms that may be used at the DNA level, we investigated 5' CpG methylation of the different class Ia and class Ib loci in meiotic pachytene spermatocytes and postmeiotic round spermatids, which had been purified from human testes by centrifugal elutriation. These results were compared with those obtained with mature spermatozoa and peripheral blood mononuclear cells. Using methylation-sensitive restriction enzymes and DNA locus-specific probes, we found that HLA-A, HLA-B/C, and HLA-E loci were similarly unmethylated in the germ and somatic cells tested, whereas HLA-F and HLA-G were even less methylated in the former cells. Together with the observation that spermatozoon DNA contains class I genes that are transfectable and able to direct transcription and protein synthesis in murine L cells, these data suggest that HLA class I genes are in an active conformation in male germ cells. We indeed found that both spermatocytes and spermatids contained low levels of class Ia and class Ib mRNA. Using reverse transcriptase-polymerase chain reaction, followed by DNA sequencing, we also detected three HLA-G transcriptional isoforms, resulting from alternative splicings, which suggested that this class Ib gene may have a potential function in these germ cells. Although intracellular expression of beta2-microglobulin (the light chain that associates with HLA class I heavy chains) was found in spermatocytes but not in round spermatids, no membrane-bound nor intracellular translated HLA class I heavy chain was detected in either germ cell type, when monomorphic anti-HLA class I monoclonal antibodies were used. Thus, lack of expression of HLA class I proteins in the male germ line is likely to involve post-transcriptional mechanisms of regulation.

Class I and class II regions of the major histocompatibility complex both contribute to individual odors in congenic inbred strains of rats

The major histocompatibility complex (MHC) of the rat has three regions--A (class I), B/D (class II), and C/E (class I)--and congenic strains are available which differ in each of these regions. We used the habituation-dishabituation procedure to examine the ability of PVG-RT1u male rats to discriminate between the urinary odors of congenic rat strains which differ genetically only at certain individual regions of the MHC. The results of five experiments indicate that discrimination can be made between urine from rats which differ in all three regions of the MHC (PVG vs. PVG-RT1av1 donors), only in the class I A region (PVG vs. PVG.R1 donors), only in the class I C/E region (PVG.R19 vs. PVG-RT1av1 donors), only in the class II B/D region (PVG.R1 vs. PVG.R19 donors), and in all regions except the classical class IA locus (PVG-RT1av1 vs. PVG.R1 donors). These results indicate that all of the MHC regions may contribute to the individual odors of rats.

The conundrum of immune response: Two Ia loci specifically control multiple immune responses

The conundrum of immune response: Two Ia loci specifically control multiple immune responses

Establishment and functional analysis of a cloned, antigen-specific suppressor effector T cell line.

A long-term cultured suppressor T cell line (3D10) was established from the spleen cells of KLH hyperimmunized C3H mice. The cells of 3D10 suppressed the secondary antibody response against dinitrophenylated KLH both in vitro and in vivo. The 3D10 cells were capable of directly suppressing the antibody response mounted by B cells and Lyt-1+2-helper T cells without participation of Lyt-1+2+ cells, which indicates that 3D10 is an effector rather than an inducer-type suppressor T cell. The cell line was unable to suppress the response against DNP-FGG even in the presence of free KLH. By analysis with a fluorescence-activated cell sorter, 3D10 was found to bear determinants detectable by conventional anti-Ia as well as rabbit anti-VH. The Ia locus was mapped in the I-J subregion and was detected by either conventional or monoclonal anti-I-J antibodies. The I-J determinants detected by monoclonal antibodies were different from those expressed on inducer-type suppressor T cells. The cell line also bears an antigenic determinant detected by a monoclonal SJL anti-SJA/9 antibody (HA16) that recognizes an allotypic determinant on T cells linked to the immunoglobulin heavy chain locus. The results indicate that the cell line 3D10 represents a suppressor effector T cell that acts directly on carrier-specific helper T cells.

The conundrum of immune response: Two Ia loci specifically control multiple immune responses

The conundrum of immune response: Two Ia loci specifically control multiple immune responses

The distribution of DR5, MT2, and MB3 specificities on human Ia subsets.

Human Ia molecules were isolated from cells of LG-38, an HLA-homozygous lymphoid cell line of DR5 specificity. Three Ia subsets could be distinguished and separated by using specific alloantisera and a monoclonal antibody with polymorphic reactivity. These subsets carried the specificities DR5 and MT2, MT2 alone and MB3 alone. The structure of the three molecular species was analyzed by microfingerprinting. The subset carrying only MT2 was similar, in both the component alpha and beta chains, to the major subset carrying DR5 and MT2, whereas the subset carrying MB3 was distinct in both chains from the other two subsets. These data are compatible with our previous findings obtained for the products of two Ia loci closely linked to the DR locus and provisionally called DC and BR; they also support the conclusion that the subset carrying only MT2 is an allelic product of the BR locus, whereas the MB3 subset is an allelic product of the DC locus. MT2 appears to be a shared specificity of DR and BR loci products.

A new wild-derived H-2 haplotype enhancing K-IA recombination.

It is generally accepted that the overall recombination frequency between the H–2K and H–2D loci of the mouse major histocompatibility complex (H–2) is at a low level of about 0.4%1. However, the value fluctuates depending upon the combinations of H–2 haplotypes in the heterozygote1. We re-examined the intra-H–2 recombination frequency, using three B10.W congeneic strains which carry the H–2 complex of Japanese wild mice. Of the three, one strain, B10.MOL-SGR(H–2wm7), showed an extremely high incidence of recombination (about 3%) in all combinations with three different inbred B10 congeneic strains. The serological typing of these recombinants revealed that most of the crossing-over points had occurred between the H–2K and IA loci. These results suggest that the wm7 haplotype enhances specific recombination irrespective of H–2 partners in heterozygous parents.

Regulation of immune responses by I-J gene products. II. Presence of Both I-Jb and I-Jk suppressor factors in (nonsuppressor x nonsuppressor) F1 mice.

Antigen-specific suppression to poly(Glu50-Tyr50) (GT) is under the control of two complementary immune suppressor (Is) genes located in the major histocompatibility (H-2) complex of the mouse. Suppressor strains of mice produce both suppressor T (Ts) cells and Ts-derived suppressor factors (TsF) that bear antigenic determinants of the I-J subregion of the H-2 complex. Nonsuppressor strains of mice, on the other hand, are not suppressed by GT preimmunization. These nonsuppressor mice, however, can be classified according to those that lack the ability to make GT-specific T cell-derived suppressor factor (GT-TsF) after GT injection (i.e., H-2a, I-Jk mice) and those that lack the ability to be suppressed by the appropriate GT-TsF (i.e., H-2b,g2, I-Jb mice). In the present study, we demonstrate that (H-2a x H-2b,g2)F1 hybrid mice produce distinct GT-specific suppressor factors of both parental I-J haplotypes. Moreover, only the I-Jb-bearing GT-TsF derived from these F1 hybrid mice is able to induce second-order suppressor cells (Ts2). This is consistent with the observation that injection of GT-TsF1 derived from C57BL/6 (I-Jb) mice into A/J (I-Jk) mice leads to the production of an antigen-specific I-Jk GT-TsF2. Our results suggest that Is gene complementation occurs through a different cellular mechanism that was previously observed for Ir gene complementation. Further, we show that complementing (non-suppressor X nonsuppressor)F1 hybrid mice produce an I-Jb (and not an I-Jk) GT-TsF1 and an I-Jk (not an I-Jb) GT-TsF2, thus suggesting a heterogeneity of Ia loci within the I-J subregion. Data presented in the present study suggest that there may be even more heterogeneity within the I-J subregion than has has been heretofore reported with regard to I-J expression on Ts.

Unique T cell Ia antigen expressed on a hybrid cell line producing antigen-specific augmenting T cell factor.

Hybrid cell lines were established by fusion between keyhole limpet hemocyanin(KLH) binding T cells of A/J mice and an AKR T cell tumor line, BW5147. Hybrids were selected for the presence of Ia antigen and KLH-specific augmenting activity of their extracts in the secondary antibody response. The detailed phenotypic and functional analysis of 1 of these clones, FL10, is reported here. The hybrid was positive for both Thy1.1 and Thy1.2 antigens and possessed the Lyt-1+,2-,3- phenotype. Both VH and Ia determinants were detected on their cell surface. The IA locus was mapped in the I-A subregion, but the Ia specificities were serologically distinct from those of B cell Ia antigen. This was demonstrated by the fact that anti-Ia antiserum preabsorbed with B cells could react with the hybrid cells, whereas none of the monoclonal anti-Ia specific for private and public determinations of Iak could. The extract from the cell line specifically augmented the in vitro secondary antibody response against dinitrophenylated KLH, and this activity was removed by absorption with antigen and conventional anti-Ia antisera. The results indicate that the cell line, FL10, carries Ia antigen unique to the T cell, which is associated with the antigen-specific augmenting molecule.

Serological cross-reactivity between products of different Ia loci.

Antibody prepared in I-E-subregion-incompatible strains (anti-A e s :E /k ) cross-reacts with I-A-subregion controlled A b : A /b and A q A q complexes. This cross-reaction defines a new I-region specificity designated Ia.50. The implications of this finding for the genetic origin of the I region and immuneresponse-gene function are discussed.

Interaction between I region loci influences the expression of a cell surface Ia antigen.

Loci clustered in the I region of the murine H-2 gene complex control the capacity to generate an immune response against foreign antigens (Ir loci) and control differentiation antigens which appear to serve as structures used by cells to interact with and regulate one another (Ia loci). Both genetic and functional studies suggest that Ia antigens may be products of Ir loci. Recent studies have shown that interaction between closely linked Ir loci is required for generating immune responses to certain foreign antigens, and that interaction between H-2-linked loci determines the appearance of an Ia glycoprotein (the Ae chain) on lymphocyte cell surfaces. In this report, we show that one Ia locus regulates the quantitative expression of the product (the E alpha chain) of a second Ia locus. This regulatory locus is dominantly expressed and exerts its effects in either the cis or trans chromosomal position. Thus, the quantitative as well as the qualitative expression of some Ia products is dependent on interaction between tightly linked loci. Our results suggest a possible molecular basis for this regulation: the synthesis and intracellular association of Ae and E alpha chains may be an absolute requirement for the expression of normal levels of either polypeptide chain on the lymphocyte cell surface. The implications these findings have for I region control of immune responses and study of human HLA-D antigens are discussed.

Molecular identification of human Ia antigens coded for by a gene locus closely linked to HLA-DR locus.

Human Ia(-like) specificities controlled by gene loci other than HLA-DR were searched for at the molecular level in cells of human B-cell-type cell lines which carry two established DR specificities. Chevalier cells of DRw3 and 7 and U698M cells of DRw2 and 4 were used. Their Ia molecules were partially purified, radioiodinated and analyzed for Ia specificities by the direct binding and sequential binding assays with a selected panel of human Ia alloantisera. It was possible in both the cell lines to define a third subset of Ia molecules carrying a new specificity in addition to two Ia subsets carrying the established DR specificities. The new specificity was detected by putative anti-DRw4 and anti-DRw7 antisera and was closely associated with DRw4 and DRw7 at population level. It was thus designated provisionally as BR4X7. These results suggest that the BR4X7 specificity is coded for by a separate Ia locus closely linked to HLA-DR locus. The determinant(s) responsible for BR4X7 was located on the small subunit of Ia molecules.

Transplantation in Miniature Swine

Abstract The complexity of the I region analog associated with the MHC of miniature swine has been probed by sequential antibody precipitation studies of Ia antigens. Treatment of solubilized lymphocyte preparations from MSLA homozygotes of the DD haplotype with excess AA anti-DD alloantiserum led to precipitation of only a portion of the total Ia antigens, as determined by secondary precipitation of the remaining material with CC anti-DD serum. The presumed I region of miniature swine must therefore code for more than one Ia antigen-bearing polypeptide chain. In addition, certain mouse alloantisera that had previously been shown to react with rat Ia antigens were tested for reactivity with swine Ia antigens. Anti-Iak mouse alloantisera precipitated Ia molecules from every swine extract tested, regardless of MHC type, precluding genetic mapping studies. However, sequential precipitation studies demonstrated that the cross-reactive mouse alloantisera reacted only with a subclass of swine Ia antigens, again suggesting genetic complexity of the pig Ia locus.

Ir Gene Control of Cytolytic T Lymphocytes Induced by TNP Modified Syngeneic Cells in Normal and Chimeric Mice

Abstract Cytolytic T lymphocytes (CTL) generated to trinitrophenyl (TNP) modified syngeneic cells from mice bearing the H-2b,d,r,s haplotypes exhibit cytolytic activity toward TNP modified syngeneic target cells, however they also demonstrate significant levels of cross reactive lysis of TNP modified allogeneic targets. In contrast, CTL from strains bearing the H-2k haplotype are H-2 restricted, only lysing TNP modified syngeneic targets. This inability to cross-reactively lyse TNP modified allogeneic targets is a defect of the responder cell and not the stimulator. Specifically, (B6 × C3H)F1 spleen cells demonstrate cross-reactive lysis when stimulated by either TNP modified C57BL/6 or TNP modified C3H stimulator cells. Utilizing H-2 recombinant strains we have mapped the genes controlling this H-2 retriction to the K and/or IA loci. Thus, this appears to be an Ir gene-like phenomenon. Bone marrow cells from C3H mice were allowed to mature in a lethally irradiated (B6 × C3H)F1 host to determine whether the host environment influenced CTL specificity.

Immunochemical evidence for three Ia loci in the I-region of the H-2 complex.

Sequential immunoprecipitation analyses of Ia antigens were performed with 125I-labeled B10, B10.A(3R), and B10.A(5R) spleen cell lysates. The results suggest the presence of one I-region associated (Ia) locus (Ia-1) in the I-A subregion. In addition, they indicate that two Ia loci map between the I-J and S regions. One locus, Ia-5, maps in the I-E subregion. The second locus, most likely Ia-3, maps in either the I-E or I-C subregion. Taken together, the data demonstrate that at least three Ia loci in the I-region of the H-2 complex code for molecules detectable by immunoprecipitation.

Histocompatibility antigens controlled by the I region of the murine H-2 complex. I. Mapping of H-2A and H-2C loci.

Skin grafts were reciprocally exchanged in pairs of congenic lines identical in all genes except those located in the central portion of the H-2 complex. Seven such lines were tested: 6R, B10.AQR, A.TL, A.TH, 7R, 9R, and B10.HTT. In all donor-recipient combinations at least some grafts were rejected. In combinations differing at the IA subregion (and other central H-2 regions or subregions), all first-set grafts were rejected within 3 wk after transplantation, and all second-set grafts were rejected within 10 days. In combinations differing at the IC subregion (and other central regions, but not at the IA subregion) between 60 and 100% of first-set grafts were rejected, but some grafts survived for over 100 days. Most of the second-set grafts were rejected within 1 mo after grafting. This behavior of skin grafts indicated the presence of two histocompatibility loci in the I region, a strong one and a weak one. This conclusion was confirmed by genetic mapping which placed the strong locus in the IA subregion and the weak locus in the IC subregion. We designate the former locus H-2A and the latter H-2C. The same strain combinations used for the skin grafting were also used for determination of the capacity of I-region antigens to function as targets in the in vitro cell-mediated lymphocytotoxicity (CML) assay. Spleen cells from mice presensitized in vivo by skin grafting were restimulated in vitro and tested against 51Cr-labeled concanavalin A or lipopolysaccharide blasts. The testing revealed the presence in the I region of two loci coding for CML-target antigens. The two loci comapped with the H-2A and H-2C loci and were most likely identical to them. As in the skin grafting test, in the CML test, the H-2A antigens evoked stronger response than the H-2C antigens. Rejection of skin grafts across the H-2A and H-2C loci was accompanied by the production of Ia antibodies. Direct cytotoxic and absorption tests with Ia antibodies directed against antigens coded for by the IC subregion revealed the presence of IaC antigens on epidermal cells. We suggest that the products of Ia loci might function as transplantation antigens.

Genetic nomenclature for new lymphocyte antigens controlled by theI region of theH-2 complex

Several laboratories have recently reported studies of new alloantigenic specificities preferentially expressed on lymphocytes and apparently controlled by genes in the Ir region [hereafter designated the I region (Klein et al. 1974)] of the H-2 gene complex (Hauptfeld et al. 1973, David et al. 1973, Sachs and Cone 1973, G~Jtze et al. 1973, Hammerling et al. 1973). These antigens have been designated by various laboratories as Ir-l.1 (Hauptfeld et al. 1973), Lna (David et al. 1973b) and ~ (Sachs and Cone 1973). To avoid confusion in the literature and to establish a standard nomenclature for these new antigens, representatives of the laboratories concerned met during the H-2 workshop in Bar Harbor, Maine, October 8-10, 1973, and agreed upon the following notation: 1) The system of antigens and the controlling genetic element(s) will be provisionally designated Ia (I-region-associated antigens). This symbol was chosen as a general, descriptive term having no specific connotations with regard to possible relationships of these antigens with It-1 or Lad genes or the tissue or cellular distribution of these antigens. When further information on these questions becomes available, the notation can be modified as necessary. 2) If it is found that these antigens are controlled by more than one genetic locus in the I region, the separate loci will be designated by numbers, Ia-1, Ia-2, Ia-3, etc. 3) If it is found that there are multipe Ia loci, some of which control antigens specific for B lymphocytes (bursa-equivalent lymphocytes) and some of which control antigens specific for T lymphocytes (thymusderived lymphocytes), those loci may be distinguished by the symbols Iab and Iat respectively. Multiple loci of each type would be denoted Iab-1, Iab-2, etc. and Iat-1, Iat-2, etc. 4) Alleles at the Ia loci should be designated by a superscript which