Polymorphism of Hh-1, the mouse hemopoietic histocompatibility locus

The major goal of these studies is to more fully assess the polymorphism of the hemopoietic histocompatibility (Hh) genetic system. H-2 homozygosity is required for optimal immunogenicity of bone marrow cell (BMC) grafts, and hybrid resistance to grafts of parental strain BMC by irradiated H-2 heterozygous F₁ hybrid mice suggests that Hh-1 antigens are inherited recessively. The Hh-1 antigens are also expressed on other normal hematopoietic cells and lymphoid tumors, and natural killer cells are the effectors which mediate the elimination of BMC grafts in an Hh-specific manner. Previous studies have demonstrated three different antigens mapping to the Hh-1 locus near H-2D. We test the expression of Hh-1 on BMC of all nonrecombinant H-2 haplotypes of independent origin and H-2 ^j , a presumed natural recombinant. Hh-1 typing is based on the pattern of growth and rejection in a panel of hosts. F₁ hybrids with H-2 ^b , H-2 ^d , and H-2 ^k are produced and used as donors and hosts to confirm the phenotype. Grafts of b-, d-, and j-haplotype marrow serve as prototypical examples of determinants that are provisionally designated as 1, 2, and 3, respectively. We describe a new determinant, 4, in the k haplotype. It is non-codominantly expressed, maps to H-2D, and is also expressed on H-2^b BMC. NZW, H-2^Z grafts exhibit a phenotype similar to k, but express a unique determinant 5 which can be distinguished from determinant 4. This additional determinant is also expressed by the b haplotype. The d, f, and p haplotypes all express determinant 2, and grafts of j-haplotype marrow are found to express determinants 2 and 5 in addition to determinant 3. The q and r haplotypes are null for all known determinants. Finally, we describe a phenotype which is a new combination of previously described determinants: s-haplotype grafts express determinants 1, 2, and 4. The polymorphism of Hh-1 detected thus far consists of seven alleles which are combinations of five distinct determinants.     

Bone marrow cell transplants involving intra-H-2 recombinant inbred mouse strains. Evidence that hemopoietic histocompatibility-1 (Hh-1) genes are distinct from H-2D or H-2L

Hemopoietic histocompatibility (Hh) Ag are noncodominantly expressed on bone marrow stem cells and other normal and neoplastic cells of hemopoietic origin. H-2/Hh-1 allogeneic or parental-strain bone marrow grafts are eliminated in a determinant specific manner by NK cells. In inbred mouse strains, seven Hh-1 alleles representing combinations of five different Hh-1 antigenic determinants are described. Each Hh-1 allele maps in the vicinity of H-2D, and the genes that map to Hh-1 are transacting regulatory genes. The expression of a particular determinant depends on the absence of the regulatory gene and the presence of the appropriate structural gene. The primary focus of this study is to ascertain whether the Hh-1 phenotype and the serologic H-2DL typing are always correlated or whether recombinant can separate the two. To achieve this, we used a panel of irradiated hosts that are able to recognize the different Hh-1 determinants on the bone marrow cells of congenic intra-H-2 recombinant donors. We report: 1) the majority of strains show a correlation between Hh-1 and H-2DL: 2) B10.RQDB and B10.WB strains dissociate Hh-1 from Lb: 3) nine H-2S/D interval recombinant strains exhibit no correlation between the H-2DL type and Hh-1 phenotype; and 4) in two strains from this group, B10.D2 (R106) and B10.RSF5, H-2S/D crossovers occurred within Hh-1r, (Hh-1 regulatory). We conclude that Hh-1r is a distinct regulatory locus mapping telomeric of H-2S and centromeric of, although probably closer to, H-2D.     

Homopoietic histocompatibility (Hh-1) phenotype and the regulation of its expression

Hybrid resistance (HR) is primarily controlled by the genes of the Hemopoietic histocompatibility-1 (Hh-1) locus within the H-2 complex. HR is a consequence of the Hh-1-controlled target determinants in homozygous parental strain mice and their absence in heterozygous F₁ hybrid mice. To examine the mechanism that controls the Hh-1 phenotype, three independent clones of somatic cell hybrids between parental lines EL-4 (C57BL/6 origin, H-2 ^b ) and R1 (C58 origin, H-2 ^k ) were studied. The line EL-4 is Hh-1^b-positive and is subject to HR by H-2 ^b heterozygous F₁ mice, but R1 lacks the Hh-1 ^b allele and is not susceptible to HR. Of the three hybrid clones, F263.2 is Hh-1^b-positive, whereas the other two, F262.2 and F264.2, are Hh-1-negative, as judged by these cells' capacity to compete in vivo with the grafted parental C57BL/6 bone marrow cells in the resistant (C57BL/6 × C3H)F₁ mice. All three clones express the H-2^b and H-2^k class I antigens equally well, are susceptible to activated NK cells to the same extent, and all carry four copies of chromosome 17. However, Southern analysis reveals that clone F263.2 contains three copies of H-2 ^b chromosome and one H-2 ^k , whereas the other two clones carry two copies each of the parental chromosome 17. The results suggest that the relative copy number of specific alleles is the crucial determinanr of the Hh-1 phenotype, and render unlikely both the gene dosage hypothesis and the trans-acting dominant suppression hypothesis to account for the noncodominant expression of the Hh-1 phenotype.     

The cytotoxic T-cell response to H-1 minor histocompatibility antigen differences

H-1-specific cytotoxic T cells were generated in in vitro secondary cultures. Effectors were assayed on H-2 compatible, peritoneal exudate cell targets in a ⁵¹Cr release assay. Target-cell lysis appeared to be specific for the H-1 type of the stimulator cells. Effector cells were T cells since they expressed Thy 1.2 alloantigen and required H-2 compatibility between donors of the stimulator cells, responder cells, and target cells for efficient lysis. Peritoneal exudate cells were found to be efficient specific competitors in the cytotoxicity assay. There appeared to be no strict correlation between in vitro cytotoxic T-cell activity and mean skin graft rejection times for a number of minor H and H-2D differences.     

The purification of murine histocompatibility antigens (H-2b) from RBL-5 tumor cells using detergents.

A detergent-solubilized form of H-2b (dH-2b) has been purified 1500-fold from RBL-5 tumor cells. The purification was accomplished by deoxycholate solubilization of purified plasma membranes, gel filtration, Lens culinaris lectin affinity chromatography, and affinity chromatography on a sheep anti-dH-2b immunoadsorbent. Both alloantigen and beta 2-microglobulin were monitored by radioimmunoassay during purification. The final product was judged to be greater than 90% pure by the following criteria: 1) sodium dodecyl sulfate-polyacrylamide electrophoresis which showed the expected 2-component structure of histocompatibility antigens, i.e. a heavy chain and beta 2-microglobulin; 2) amino acid composition which was comparable to the known compositions of other H-2 and HLA molecules; 3) NH2-terminal sequencing which gave a unique sequence for the heavy chain, and the reported sequence for beta 2-microglobulin; and 4) immunoprecipitation of the bulk of the preparation by appropriate alloantisera.     

Expression of hemopoietic histocompatibility antigens on H-2-loss variants of F1 hybrid lymphoma cells: evidence consistent with trans gene regulation

H-2 heterozygous marrow stem cells, lymphoid progenitor cells, and leukemia/lymphoma cells do not express hemopoietic or hybrid histocompatibility (Hh) antigens, which are important transplantation antigens recognized during the rejection of normal or neoplastic hemopoietic cells. The Hh-1b determinant of the H-2b haplotype maps to the D region of H-2. We have tested the hypothesis that gene(s) at or near H-2D of the H-2d haplotype down-regulate the expression of Hh-1b in the trans configuration. We used Abelson leukemia virus-transformed pre-B lymphoma cells (ACCb) of BALB/c X BALB.B (H-2d X H-2b) origin, as well as variant lines of ACCb, which were selected for resistance to monoclonal anti-H-2 antibodies plus complement. B6D2F1 (H-2b X H-2d), C3B6F1 (H-2k X H-2b), or B6 (H-2b) mice were infused with inocula of 5 X 10(6) B6 bone marrow cells (BMC). Proliferation of donor-derived marrow cells was judged in terms of DNA synthesis by measuring the splenic incorporation of 5-iodo(125I)-2'-deoxyuridine (IUdR) 5 days after cell transfer. B6 BMC grew much better in B6 than in F1 hybrid host mice, an expression of "hybrid resistance". As observed previously, the injection of EL-4 (H-2b, Hh-1b) tumor cells prior to infusion of B6 (H-2b, Hh-1b) BMC enhanced the growth of B6 BMC in F1 hybrid mice. Therefore, this in vivo "cold target cell competition" type of assay can be used to detect the expression of Hh-1b antigens. Unlike EL-4 (H-2b) cells, hybrid resistance was not affected by prior infusion of (H-2b X H-2d) heterozygous ACCb cells. In contrast, three ACCb variant cell lines, H-2d-, Ld-Dd-, and Dd-, enhanced the growth of B6 BMC in F1 hosts. The ACCb H-2b- cell line did not affect hybrid resistance to B6 BMC. The loss of gene expression on the H-2d chromosome at or very near the H-2Dd locus is correlated with the appearance Hh-1b, as determined by the in vivo cold target competition assay. These results support the hypothesis that heterozygous cells possess trans-acting, dominant, down-regulatory genes mapping near H-2D that control the Hh-1 phenotype of lymphoid tumor cells.     

Differential induction of H-2K vs H-2D class I major histocompatibility complex antigen expression by murine recombinant interferon-gamma

The results presented here indicate that recombinant murine interferon-gamma can cause a dramatic differential induction of two distinct class I MHC molecules. Thus, IFN-gamma treatment of the murine leukemia virus (MuLV)-induced AKR SL3 tumor, a cell line that normally expresses moderate levels of class I MHC antigens, resulted in a large increase in H-2Dk expression, but no change or a slight decrease in H-2Kk expression as measured by cytofluorography. Explanations of the selective enhancement of Dk expression based on increased Fc receptor display or differential kinetics of induction were ruled out. The phenomenon was observed over a wide range of doses of IFN-gamma and with two different monoclonal antibodies to Kk, the latter finding making it unlikely that an altered form of the Kk molecule was induced. The same differential induction of the Dk antigen was observed for the LBRM.5A4 tumor cell line. Because LBRM.5A4 is also MuLV+ but of congenic B10.BR (H-2k) origin, these results were consistent with the possibility that such differential induction was associated with the H-2k haplotype and/or MuLV. The implications of these results, as a possible mechanism of tumor cell escape from an immune surveillance system monitored by class I MHC-restricted T cells and as a useful model system to dissect the mechanism of IFN-gamma induction of class I MHC antigens, are discussed.     

Stability of surface H-2K(b), H-2D(b), and peptide-receptive H-2K(b) on splenocytes.

We have used flow cytometry to study the stability and peptide-binding capability of MHC class I (MHC-I) on the surface of normal C57BL/6 mouse T lymphoblasts. The MHC-I molecules on each cell are nearly evenly divided into two populations with mean half-life values of approximately 1 and 20 h. Our observations suggest that members of the later contain peptide bound with medium to high affinity. Cell surface MHC-I molecules capable of binding exogenous peptide (thus, "peptide-receptive") belong almost entirely to the less stable population. Before exogenous peptide can bind, MHC-I must undergo a change, probably loss of a very low affinity peptide. For MHC-I-K(b), we found that the maximum rate for binding of exogenous peptide corresponds to a t(1/2) value of 12 min. To maintain the 50:50 steady-state distribution of long- vs short-lived MHC-I molecules on the cell surface, approximately 20 short-lived molecules must be exported to the cell surface for each long-lived molecule.     

Anti-major histocompatibility complex immunity detected prior to intentional alloimmunization. I. Naturally occurring H-2-specific antibodies in C57BL/KaLwRij (H-2b) mice

Naturally occurring, H-2-specific, lymphocytotoxic antibodies were detected in 3-10% of young adult and in 10-40% of aged C57BL/KaLwRij (H-2b) mice. The antibodies were of the IgM class and occurred in low titers, but occasionally a high titer was found. The antibodies detected public lymphocyte-membrane antigens controlled by genes identical with, or closely linked to class-I H-2K and H-2D genes. Antibodies against 7 different allogeneic H-2 haplotypes were detected but sera of individual mice exerted different reaction patterns and some specificities occurred more frequently than others. Although the occurrence of the antibodies was age dependent, thymus involution, gammapathies, autoimmunity, the presence of other natural lymphocyte-specific antibodies, and polyclonal or nonspecific stimulation could not be related to the occurrence of natural H-2-specific antibodies. Several possible explanations of natural H-2-specific antibodies exist. We propose that determinants of complex altered self-MHC (MHC + X) antigen(s) triggered the production of H-2-restricted antibodies that recognize H-2-public determinants on normal allogeneic cells.     

F1 hybrid anti-parental H-2k cell-mediated lympholysis. I. Stimulator and target determinants controlled by the H-2K region.

H-2k-heterozygous F1 hybrid mouse spleen cells cultured with irradiated H-2k-homozygous stimulator cells generated specific anti-parent cytolytic effectors. The parental antigenic determinants recognized by responder cells during induction (afferent arm) and by effector cells during cytolysis (efferent arm) were coded for, or regulated by, the H-2K-Hh3 region of the MHC, according to recombinant analysis. There were no detectable influences by other linked or unlinked genes on the phenotypic expression of parental antigens; however, the anti-parent responsiveness was modulated by background genes of responder cells. These experiments establish that the K end of H-2 controls determinants of F1 anti-parental H-2k CML, like the D end controls those of F1 anti-parental H-2b CML, thus confirming the basic symmetry of the H-2 complex. The relationship of this primary in vitro cell-mediated response with natural in vivo resistance to parental and allogeneic bone marrow grafts is discussed.     

The response of lymphocytes to H-2D and H-2K region antigens: I. Differential effect of cortisone acetate

Cell surface antigens controlled by separate portions of the H-2 region differentially stimulate lymphocytes. Cells responding to antigens controlled by loci in or near the H-2D region transform later (5 days) than cells responding to antigens controlled by loci in or near the H-2K region (3 days). Treatment of lymphocyte donors with cortisone acetate shows that lymphocytes responding to some H-2K-associated antigens are cortisone resistant and that lymphocytes responding to H-2D-associated antigens are cortisone sensitive. Parallels are drawn between these characteristics and the lymphocytes responsible for cellular and humoral immunity.     

Anti-major histocompatibility complex immunity detected prior to intentional alloimmunization. II. Monoclonal H-2-specific antibodies obtained from an unimmunized C57BL/KaLwRij (H-2b) mouse

A monoclonal 'natural' anti-H-2 IgM antibody produced by a hybridoma cell line OL-3.17 (H-2 m. 209) is described. The OL-3.17 monoclonal antibody was obtained by hybridization of spleen B cells from an unimmunized C57BL/Ka (H-2b) mouse in the serum of which simultaneously an IgM kappa paraprotein of high concentration and a natural H-2-specific antibody of high titer was detected. The monoclonal antibody OL-3.17 reacted strongly with H-2d and H-2s and weakly with H-2k,q,r lymphocytes, thereby detecting a hitherto unknown H-2 public determinant. The target molecules for OL-3.17 cocapped with class-I H-2 antigens, but immunoprecipitation of H-2 antigens was not achieved. This is the first monoclonal H-2-specific antibody obtained from a mouse without intentional immunization and, with high probability, was derived from a B-cell clone which produced natural H-2-specific antibodies detectable in the serum of the original mouse.     

Differential sensitivity of GLUT1- and GLUT2-expressing beta cells to streptozotocin.

Streptozotocin injection in animals destroys pancreatic beta cells, leading to insulinopenic diabetes. Here, we evaluated the toxic effect of streptozotocin (STZ) in GLUT2(-/-) mice reexpressing either GLUT1 or GLUT2 in their beta cells under the rat insulin promoter (RIPG1 x G2(-/-) and RIPG2 x G2(-/-) mice, respectively). We demonstrated that injection of STZ into RIPG2 x G2(-/-) mice induced hyperglycemia (>20 mM) and an approximately 80% reduction in pancreatic insulin content. In vitro, the viability of RIPG2 x G2(-/-) islets was also strongly reduced. In contrast, STZ did not induce hyperglycemia in RIPG1 x G2(-/-) mice and did not reduce pancreatic insulin content. The viability of in vitro cultured RIPG1 x G2(-/-) islets was also unaffected by STZ. As islets from each type of transgenic mice were functionally indistinguishable, these data strongly support the notion that STZ toxicity toward beta cells depends on the expression of GLUT2.     

Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol

Recent studies indicate that membrane cholesterol can associate with G protein-coupled receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 cholecystokinin receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 cholecystokinin receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wild-type and CCK2R/CCK1R chimeric receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs.     

Differential allo-response of murine thymocytes to H- 2 K region different recombinants and to H-2Kb mutants

Thymocytes used as responding cells in a mixed leukocyte culture with x-irradiated splenic stimulating cells generate highly significant proliferative and cytotoxic responses when responding and stimulating cells differ by the entire H-2 complex. On the other hand, when the genetic difference between responding and stimulating cells is only a K region, very little, if any, proliferative response is detectable and no cytotoxic response is found. In contrast, when responding and stimulating cell donors differ by a spontaneous mutation in the K region of the H-2 complex, as found in B6.C-H-2ba, B6-H-2bd and B6.C-H-2bf, highly significant proliferative and cytotoxic responses can be obtained. These results, thus, argue that the H-2 mutants cannot, with regard to their relationship to the parental strain, be readily equated with a K region difference as defined in the recombinant inbred strains.     

H-33-A histocompatibility locus to the left of theH-2 complex

Another minor histocompatibility locus, calledH-33, was found on chromosome 17. This locus was revealed by skin graft experiments between BALB/c and a new congenic strain, BALB.TTF.     

Discrimination between mammalian RNases H-1 and H-2.

The two principal RNases H in mammalian cells, H-1 and H-2, differ in their responses to sale, divalent metal, and sulfhydryl inhibition. Specific reaction conditions that provide unambiguous discrimination between RNases H-1 and H-2 with only two assays are described. The assays were used for identification in a new purification procedure for RNases H-1 and H-2.