Identification of murine H-2Db histocompatibility antigens in cells transfected with cloned H-2 genes

Clones of mouse L-cells transformed with 21 cosmids containing 15 major histocompatibility complex class I genes of C57BL10 (H-2b) sperm cell DNA were analyzed for the expression of their transfected H-2 and Qa/Tla genes. Three cosmids contained a single gene, mapping to the H-2D region. This gene encodes the H-2Db alloantigen: mouse L-cells transfected with cosmids containing this gene reacted with monoclonal antibodies and alloantisera specific for the H-2Db antigen and expressed a 46-kd H-2 heavy chain associated with beta 2-microglobulin in their cell membranes. Furthermore, these transfected cells were stimulators of, and targets for, anti-H-2Db cytotoxic T-lymphocytes. Eighteen cosmids contained 14 different genes mapping to the Qa and Tla regions. L-cells transfected with these genes did not express class I genes reacting with alloantisera or monoclonal antibodies against Qa2, Qa4 or TL differentiation antigens. In particular, the Qa2,3 gene of C57BL10 was not identified.     

Structural studies on H-2Db and H-2Kd molecules indicate that murine major histocompatibility b and d haplotype alloantigens share structural features

Structural properties of the H-2D^b and H-2K^d murine major histocompatibility complex (MHC) antigens were examined by radiochemical methods. Radiolabelled preparations of the H-2D^b and H-2K^d antigens were obtained by indirect immune precipitation of NP-40 lysates of the lymphoid tumor cell lines EL-4 (H-2^b) and C14 (H-2^d), respectively. After preparation of the 37,000 molecular weight papain fragment the antigens were cleaved with CNBr. The H-2K^d antigen yielded four major CNBr fragments whereas the H-2D^b molecule provided six. These CNBr fragments were subjected to partial NH₂-terminal amino acid sequence analysis and aligned by homology to the H-2K^b glycoprotein. Comparison of the structural properties of the H-2K^d and H-2D^b molecules with previously published data on the other known major transplantation antigens of the b and d haplotypes (H-2K^b, H-2D^d and H-2L^d) reveal a marked structural similarity. First, the data show that certain methionine residues have been highly conserved and that cleavage by CNBr at these positions provides an initial strategy for the study of these molecules. Secondly, disulfide-linked peptides obtained after CNBr cleavage could be aligned and the data suggest the presence of disulfide bridges in homologous positions. Third, after CNBr cleavage both the H-2K^d and H-2D^b molecules yielded two glycopeptides which were homologous to glycopeptides from the H-2K^b molecule. Fourth, overall homology for a limited number of comparable positions is about 81% between the H-2K^b and H-2K^d gene products and 88% between the H-2K^b and H-2D^b gene products.     

Presentation of viral antigens restricted by H-2Kb,Db or Kd in proteasome subunit LMP2-and LMP7-deficient cells

In the class II region of the major histocompatibility complex (MHC), four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP 1 and TAP 2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP 2 and LMP 7) code for subunits of the proteasome. While TAP 1 and TAP 2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP 2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP 1/2 and LMP 2/7 genes, it was recently shown that expression of TAP 1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP 2/7, we transfected T2 cells with TAP 1, TAP 2 and either of the H-2K^b, D^b or K^d genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP 1/2 gene products, presented all tested viral antigens restricted through either the H-2K^b, D^b or K^d class I molecules. We conclude that the proteasome subunits LMP 2/7 as well as other gene products in the MHC class II region, except from TAP 1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP 2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.     

A tentative new model for the organization of the mouse H-2 histocompatibility system: two segregant series of antigens

Based on serological and genetic evidence from the HL-A system, a tentative new H-2 model of two segregant series of antigens is suggested. It is proposed that each H-2 allele from the two series determines only one antigen and that the other H-2 specificities associated with each allele are caused by crossreacting antibodies. The new model amplifies the homology between the H-2 and HL-A systems.     

H-2K and H-2D antigens are independently regulated in mouse embryo fibroblasts

The concentration of MHC class I (H-2K and H-2D) antigens on stimulating cells and target cells may influence the induction of responses in cytotoxic T-cell precursors, and the efficiency of cytotoxicity mediated by activated T cells, respectively. Using FACS analysis, we found that exposure of BALB/c mouse embryo fibroblasts (MEF) under different culture conditions to the supernatant from concanavalin A(Con A)-stimulated splenocyte cultures (CS) could result either in H-2K antigens increasing significantly more than H-2D antigens or in H-2D antigens increasing significantly more than H-2K antigens. Furthermore, we observed that the concentrations of H-2K antigens on MEF that were not exposed to CS in vitro could vary independently of H-2D antigen concentration during a period of several days in vitro. Thus we propose that the cell surface concentration of H-2K and H-2D antigens may be independently regulated, and we discuss how such regulation may be biologically advantageous during protective T-cell responses.     

Differential effect of adenovirus 2 E3/19K glycoprotein on the expression of H-2Kb and H-2Db class I antigens and H-2Kb- and H-2Db-restricted SV40-specific CTL-mediated lysis

The E3/19-kDa glycoprotein (E3/19K) coded by adenovirus type 2 (Ad2) is known to inhibit the cell-surface expression of major histocompatibility complex (MHC) class I antigens by binding to the MHC antigens intracellularly, and thus reduces recognition of antigens by MHC-restricted cytotoxic T lymphocytes (CTL). We have studied the effect of the E3/19K expression in SV40-infected monkey cells, TC-7/H-2Kb and TC-7/H-2Db expressing transfected H-2Kb and H-2Db antigens, respectively, on the cell-surface H-2 class I antigens and on lysis of the cells by SV40 large tumor (T)-antigen-specific H-2Kb- and H-2Db-restricted CTL clones. H-2Db antigen expression on TC-7/H-2Db cells was drastically reduced by infection with Ad2 but not with an E3/19K-negative SV40-Ad2 hybrid virus, Ad2+ND1, as early as 12 hr postinfection. However, H-2Kb antigen expression on Ad2-infected TC7/H-2Kb cells remained unaltered, even at 24 hr postinfection. Specific lysis of SV40-infected TC-7/H-2Db cells by H-2Db-restricted SV40 T-antigen-specific CTL clones, Y-1 and Y-3, was strongly reduced by coinfection of the target cells with Ad2 but not with Ad2+ND1. Lysis of SV40-infected TC-7/H-2Kb cells by a H-2Kb-restricted SV40 T-antigen-specific CTL clone Y-4 was also reduced significantly by Ad2 infection, but not Ad2+ND1. These results indicate that the E3/19K protein affects cell-surface expression of H-2Db antigen but not H-2Kb antigen.     

Antigen-dependent,H-2-restricted cytolytic and noncytolytic T cell lines with specificity for minor histocompatibility antigens

Several cloned T cell lines were isolated from primed mixed lymphocyte cultures immunized against minor histocompatibility antigens. These lines were selected with irradiated stimulator cells as antigen and require restimulation at intervals to keep growing. They are responsive, as measured by proliferation, to interleukin 2 (T cell growth factor) but cannot be grown in it continuously. These T cell lines have either the H-2^d or H-2^k haplotype. They all show exquisite H-2 restriction and minor histocompatibility antigen specificity. We did not observe any alloreactivity on 8 different H-2 haplotypes. For the H-2^k T cell lines, the restriction element could be mapped to either the K or D end of the H-2 complex. No I-A-restricted cell line was found. It is of interest that all these T cell lines need the presence of T cells in the irradiated stimulator cell population. This suggests a more complex interaction between irradiated stimulators and responder T cells than just H-2K- or D-restricted antigen interaction. This recognition, though necessary, does not seem sufficient to induce the T cell clones to proliferate.     

Tumorigenicity of mouse T lymphoma cells is controlled by the level of major histocompatibility complex class I H-2Kk antigens

We have previously found that an increased tumorigenicity and spontaneous metastatic potential of BW5147-derived T lymphoma cells was associated with a decrease in major histocompatibility complex (MHC) class I H-2K^k antigen expression. This suggested that H-2K^k antigens may control the tumorigenic potential of BW T lymphoma cells. Our current experiments aimed to prove this association by specifically altering H-2K^k expression by gene transfection. Transfected cells expressing a high level of H-2K^k antigens were significantly less tumorigenic and metastatic after subcutaneous inoculation. However, there was selectionin vivo for cells expressing a reduced level of H-2K^k antigens, which concomitantly led to an increased tumorigenicity. These data further confirmed the strong association between H-2K^k expression and tumorigenicity. We subsequently tested whether the immune system is implicated in this phenomenon by inoculating the H-2K^k transfectants into irradiated, immunocompromised recipients. Our results indicate that the reduced tumorigenicity of the BW H-2K^k transfectants is due to an immune rejection mechanism, mediated by CD8⁺ immune effector cells, as revealed byin vivo depletion experiments with anti-CD8 antibodies. Hence, we hereby demonstrated that H-2K^k antigens increased the immunogenicity of BW cells, via a CD8-dependent mechanism, which consequently reduced their tumorigenicity.     

H-2 antigens on nuclear membranes

Nuclei and nuclear membranes were examined for the presence of H-2 histocompatibility antigens in the mouse. These were detected on thymus as well as on liver preparations by means of ⁵¹Cr-cytotoxocity inhibition and complement fixation tests, absorption experiments and by the immunogenic properties of these preparations.     

Intradomain H-2Kd/Dd recombinants define the same regions as crucial for recognition by alloreactive or major histocompatibility complex-restricted cytolytic T cells

To identify residues on class I major histocompatibility complex (MHC) antigens that are important in T cell recognition, we analyzed a series of 11 intradomain recombinant mouse MHC (H-2) molecules in which N-terminal H-2Kd segments of varying lengths are followed by H-2Dd segments. Lysis of L cell transfectant target cells by a series of alloreactive cytolytic T cell (CTL) clones specific for Kd or for Dd revealed several regions that contain residues critical for specific recognition. These residues map within the presumed antigen-binding site of the MHC molecule. Of particular interest was the finding that the two regions identified as important for Kd allorecognition match those that influence the recognition of synthetic peptide antigens by Kd-restricted CTL.     

The effect of histocompatibility antigens on lymphocyte migration in the mouse

Migration patterns of ⁵¹Cr-labelled murine lymph node cells were studied in syngeneic, allogeneic, semi-allogeneic and congenic strain combinations. In certain allogeneic and semi-allogeneic combinations with strong H-2 histoincompatibility, reduced migration of a donor population representing recirculating lymphocytes into recipient lymph nodes was observed. This phenomen could be reproduced in congenic strains differing only at the H-2 locus, and was not seen in congenic strains differing at the Ly-A, Ly-B, RZ, M, or θ loci.

The kinetics of reduced lymphocyte homing to allogeneic lymph nodes and the role of host and donor recognition were investigated. These studies are discussed in relation to possible mechanisms of action, and relevance to factors regulating lymphocyte recirculation.

     

Serological analysis of H-2 antigens expression on the cell surface of selected mouse tumors

Studies on histocompatibility antigens expression on different selected mouse tumor cells revealed very variable pattern. No qualitative alterations in H-2 antigens expression on the cells of the following tumors: RL male 1 and MP26a of BALB/c (H-2d) mice, ASL-1 and RADA-1 of A (H-2a) mice, EL-4 and E male G2 of C57B1/6 (H-2b) mice, K36 and AKSL-4 of AKR (H-2k) mice could be detected. However, the quantitative differences in H-2 antigens expression on some of these tumors were observed.     

Identification of murine leukemia viral antigens detected on mouse cells by H-2 alloantisera.

H-2 alloantisera have been previously reported to contain antibodies against murine leukemia viral antigens, but the nature of the viral antigens on mouse cells which interact with these antibodies has not been established. We have found that H-2 alloantisera recognize components of molecular weight 70 000-80 000 mouse lymphocytes and leukemia cells. These components were also detected by a goat antiserum against the murine leukemia virus (MuLV) glycoprotein (gp 70) and are therefore closely related to or identical with that viral protein. Although most H-2 alloantisera detected gp 70-like molecules on lymphocytes and leukemia cells from a great variety of mouse strains, only one H-2 alloantiserum was found to interact with a gp 70 component on cells from C57BL/10 and C57BL/6 mice. Animals such as C57BL/10 mice that lacked the component reacting with most H-2 alloantisera showed increased serum levels of anti-MuLV antibodies after injection of B10.A spleen cells having a gp 70 component detectable by other H-2 alloantisera. In contrast, strains with cells reactive to antiviral antibodies in the H-2 alloantisera had low responses to MuLV antigens after a similar immunization procedure. Serum levels of anti-MuLV antibodies in both groups of mice, however, were increased after injection of Freund's adjuvant. These observations suggest that anti-MuLV antibodies in mouse alloantisera may arise from a response to viral antigens on the immunizing cells and general stimulation of the immune system.     

Variable binding affinities of listeriolysin O peptides for the H-2Kd class I molecule

Previously we used the peptide-binding motif for the murine class I major histocompatibility complex molecule H-2K^d to identify a nonamer peptide of the Listeria monocytogenes listeriolysin (LLO) protein that was recognized by cytotoxic T lymphocytes (CTL) in association with H-2K^d. Eleven nonamer peptides contained in the LLO sequence were synthesized and one, LLO 91-99, proved to be a CTL target. Using peptide binding competition assays with H-2K^d-restricted CTL, we show that 3 out of the 11 LLO peptides, including the CTL epitope, have a high binding affinity for H-2K^d; 2 of 11 peptides have approximately 10-fold lower affinity, while the remaining 6 peptides have no or very low affinity for H-2K^d. Single residue changes were made in the LLO 91-99 peptide and two other LLO peptides to identify non-anchor amino acids that might interfere with peptide binding. In addition, we used the LLO peptides which bound well to H-2K^d to attempt to restimulate a secondary CTL response from L. monocytogenes-primed spleen cells. Only LLO 91-99 was able to induce such a response. Thus only a fraction of nonamer peptides which fit the original binding motif have a high affinity for the H-2K^d class I molecule, and only a fraction of these serve as CTL epitopes.     

Abrogation of negative selection by GVHR induced by minor histocompatibility antigens or H-2D antigen alone

Allogeneic bone marrow chimeras were prepared by donor and recipient combinations that differed in minor histocompatibility loci or H-2D locus alone. When 1 x 10(5) splenic T cells were inoculated in addition to T cell-depleted bone marrow cells (1 x 10(7)), clinically detectable GVHR was induced. In these GVHR chimeras, substantial numbers of T cells reactive to either donor or recipient antigens were both phenotypically and functionally detected. The mechanisms underlying the abrogation of intrathymic negative selection are discussed.     

Evidence for a histocompatibility locus probably linked to but distinct from Mls and H-25

BALB/c (Mlsb) and BALB.D2-Mlsa strains of mice, both H-2d, are congenic and differ for the Mls locus (and linked genes) located on chromosome 1. The BALB.D2-Mlsa strain was obtained by introducing the Mlsa allele of DBA/2 mice into BALB/c mice. In previous studies we showed that BALB.D2-Mlsa recipients reject, relatively rapidly, all skin grafts from BALB/c donors. We and other groups have questioned whether the rejections observed were indeed due to the incompatibility for Mlsb products or for products of a histocompatibility (non-H-2) locus linked to, but distinct from, Mlsb. To answer this question, several hybrids carrying either Mlsa or Mlsb in various genetic contexts were grafted with skin from Mls-compatible BALB/c or BALB.D2-Mlsa donors; in the genetic combinations selected, any rejection which might occur would reflect the effects of a non-Mls incompatibility between BALB/c and BALB.D2-Mlsa strains. In certain of the donor-recipient combinations studied, the skin grafts were tolerated for greater than 200 days, but a relatively rapid rejection of BALB/c skin grafts was observed in (B10.D2 x BALB.D2-Mlsa)F1 and (B10.BR x BALB. D2-Mlsa)F1 hybrid recipients. These results indicated that in addition to Mls, the BALB/c and BALB.D2-Mlsa strains differ for at least one other non-H-2 histocompatibility locus. The possible involvement of H-25 was then investigated. Indeed, disparity for H-25, which maps on chromosome 1 close to Mls, can induce relatively rapid skin graft rejection. The H-25 allele of the DBA/2 strain has not been defined: we considered, therefore, that BALB/c and DBA/2 could be disparate at the H-25 locus, and that H-25 (transmitted by DBA/2 to the BALB.D2-Mlsa strain, together with the Mlsa allele) could be responsible for the skin graft rejection we observed. Our results showed, however, that DBA/2, BALB/c and BALB.D2-Mlsa strains of mice all share the H-25c allele; they therefore ruled out a role for H-25 incompatibility in the skin graft rejections we observed, and indicated that these rejections are due to the effects of a yet undefined histocompatibility locus (locus 'x'), probably linked to, but distinct from, the Mls locus. Further experiments showed that the histocompatibility effect of locus 'x' cumulates with that exhibited by Mlsb (or by a putative histocompatibility locus linked to Mlsb).     

H-Y and antigens of the major histocompatibility complex.: H-Y and H-2D antigens are associated on unfixed lymphocytes and thymocytes but not on testicular cells

The results of an antibody blocking technique indicate that H-Y and H-2D antigens are close to one another on the surfaces of unfixed thymocytes and lymphocytes, but distant on testicular cells. The findings are the same using cells from mice of several inbred strains (C57BL/10, BALB/c, B6.H-2^k). The data indicate moreover that H-2K antigens and H-Y are distant on all cell types tested. The gonad specific H-Y receptor-presumed necessary for H-Y directed testicular differentiation is associated with neither H-2D nor H-2K antigens.     

Separation of cells with different histocompatibility (H-2) antigens by passage through cellular immunoadsorbent columns.

The anti-Ig column technique of Wigzell et al. (Scand. J. Immunol. 1972. 1:75) was adapted to the separation of mouse cells with different H-2 antigens. Fractionation was achieved by a two-step procedure: first the cells were treated with anti-H-2 sera, and thereafter passed over anti-mouse Ig columns. Antibody-coated cells were selectively retained in the anti-Ig column. The method could be applied to the separation of normal mouse lymphocytes or lymphoma cells with different H-2 antigen complexes and for the selective isolation of H-2 loss variants from a mouse carcinoma/fibroblast hybrid.