Crystallization of murine major histocompatibility complex class I H-2Kb with single peptides.

X-ray quality crystals of a soluble murine class I H-2Kb molecule complexed with three different peptide antigens were grown in several forms by streak seeding and macroseeding methods. Co-crystals with VSV-8 (RGYVYGQL), OVA-8 (SIINFEKL) and SEV-9 (FAPGNYPAL) peptides were grown either from NaH2PO4/HPO4 or from polyethylene glycol 4000 within the pH range 5.0 to 7.5, with the use of 4-methyl-2-pentane diol (MPD) as an additive. The VSV-8 crystals grew in space groups P1, with cell dimensions a = 63.1 A, b = 69.1 A, c = 72.0 A, alpha = 89.9 degrees, beta = 77.1 degrees, gamma = 123.3 degrees and P2(1)2(1)2, with a = 138.1 A, b = 88.6 A, c = 45.7 A, and diffract to 2.9 and 2.3 A, respectively. Crystals of the SEV-9 complex grew from similar crystallization conditions to those of the orthorhombic VSV-8 complex with similar cell parameters and diffract to at least 2.5 A resolution. Crystals of the OVA-8 complex were obtained from either phosphate (space group C2, a = 118.7 A, b = 61.6 A, c = 85.3 A, beta = 108.4 degrees) or polyethylene glycol (space group P1, a = 64.5 A, b = 71.0 A, c = 66.3 A, alpha = 89.7 degrees, beta = 95.7 degrees, gamma = 123.3 degrees) and diffract to 3 A resolution. The crystallization procedures used here significantly increased the rate and production of X-ray quality crystals.     

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.     

Restriction fragment length polymorphism of the murine C4 and Slp genes: two C4 groups.

We have examined the related H-2 genes coding for the fourth component of complement (C4) and the sex-limited protein (Slp) from 30 inbred mouse strains by Southern blot analysis. With four restriction enzymes, 11 RFLP patterns distributed among 26 different H-2 haplotypes have been identified. Strains of the same serologic H-2 haplotype were found to have identical RFLP patterns. It was confirmed that the number of C4-related genes in most haplotypes is two, Slp and C4; but H-2SWI6 (SWI6) and SWI9, which have the same RFLP pattern, have four and Sw7 has five. Although C4 and Slp have many similarities, they also were found to contain distinctive features: relative to Slp, each C4 allele examined has two insertions totaling 1.1 kb located in introns 14 and 15; and each Slp allele examined, excluding hybrids, has a provirus insertion upstream. No other large deletions or insertions were detected. The RFLP patterns are also due to 10 polymorphic restriction sites, which have been placed on standard maps; two are associated with Slp and eight are associated with C4.Sk strains, the only strains that express low serum levels of C4, have the same RFLP phenotype as Sw14, Sw18, and Swx; Sk may have arisen from a recent common ancestor of these strains. Homologous recombination has been important in the formation of existing C4 alleles. However, based on complete linkage disequilibrium between three RFLP internal to C4, the haplotypes have been divided into two groups that may have functional significance.     

Immunoprecipitation of insulin receptors by antibodies against Class 1 antigens of the murine H-2 major histocompatibility complex

In rat lacrimal gland, cholinergic, α- or β-adrenergic or methylxanthine stimulations of protein secretion are extracellular calcium dependent. 10 μM trifluoperazine (TFP) inhibited only cholinergic and α-adrenergic stimulations. Half maximal effect was observed at 30 μM, with all inducers except norepinephrine (3 μM). 10 or 30 μM TFP also suppressed the decrease of L-[³H]leucine incorporation into protein due to carbamylcholine. 100 μM TFP inhibited protein secretion and L-[³H]leucine incorporation. 500 μM TFP promoted cell lysis. It is suggested that: (a) at 100 μM TFP, inhibition is not specific for protein secretion; (b) at 30 μM TFP, inhibition could be related to a role of calmodulin in the secretory regulation process.     

Synthesis and immunochemical study of the antigenic determinant of the H-2Db molecule of the murine major histocompatibility complex

Primary structure of murine class I histocompatibility antigens has been analysed to select possible antigenic determinant. Hexapeptide Leu-Gln-Gln-Leu-Ser-Gly, homologous to the region 95-100 of the H-2Db antigen heavy chain, was synthesised by stepwise elongation of peptide chain beginning from the COOH-terminal Gly. Rabbit anti-hexapeptide antibodies were obtained and shown to interact specifically with purified H-2Db antigen as well as with the native antigen on cell surface. These antibodies bind to lymphocytes of H-2b haplotype (C57BL/6 mice) but not H-2d (BALB/c) or H-2k (CBA). These data suggest that the region 95-100 is responsible for serologic differences between the alleles of H-2 antigens, i.e. it may be a xenotypic as well as an allotypic antigenic determinant. The latter was confirmed by study of interaction of the hexapeptide with allogeneic monoclonal antibodies specific to H-2Db antigen.     

New genes in the class II region of the human major histocompatibility complex

A detailed map of the class II region of the human major histocompatibility complex has been constructed by pulsed-field gel electrophoresis. This map revealed clusters of sites for enzymes that cut preferentially in unmethylated CpG-rich DNA often found at the 5' ends of genes. Three of these clusters have been cloned by cosmid walking and chromosome jumping. Analysis of the clones encompassing these regions through the use of zoo blots, Northern blots, and cDNA libraries resulted in the discovery of four novel genes. The D6S111E and D6S112E genes are centromeric to the HLA-DPB2 gene, while D6S113E and D6S114E are between HLA-DNA and HLA-DOB. Preliminary characterization of the new genes indicates that they are unrelated to the class II genes themselves, although D6S114E expression, like class II expression, is inducible with interferon. In addition, the HLA-DNA gene has been accurately positioned and oriented for the first time.     

Mapping of C2, Bf, and C4 genes to the swine major histocompatibility complex (swine leukocyte antigen)

Three miniature swine lines, inbred for swine leukocyte antigen (SLA) haplotypes, a, c, and d, and a recombinant line, haplotype g, were analyzed for possible restriction fragment length polymorphisms (RFLP) by Southern blot hybridization with human C2, factor B (Bf), and C4 specific probes. The search for RFLP by using a human C2 probe failed to reveal any variants. However, a Taq I polymorphism was identified with the human Bf probe and Bam HI and Pvu II polymorphisms were identified with the human C4 probe. Overlapping restriction fragments were found with the C2 and Bf probes, which strongly suggests close linkage of C2 and Bf genes in swine. Segregation analyses of the Bf and C4 polymorphisms indicated that the polymorphic fragments followed a Mendelian pattern of inheritance. The recombinant haplotype g, which expresses class I genes of haplotype c and class II genes of haplotype d, was shown to produce an identical RFLP pattern, by using the Bf and C4 probes, as haplotype d, but different from that of haplotype c. This indicates that there is a close association of [C4-Bf-C2] and class II genes in miniature swine. Although these data do not show conclusively the location of the [C4-Bf-C2] genes, it is hypothesized that swine [C4-Bf-C2] genes are located between the class II and class I genes, as has been demonstrated in mouse and man.     

Composite origin of major histocompatibility complex genes

Major histocompatibility complex WHO genes have now been cloned from representatives of all vertebrate classes except Agnatha. The recent accumulation of sequence data has given great insight into the course of evolution of these genes. Although the primary structure of the MHC genes varies greatly from class to class and also within the individual classes, the general features of the tertiary and quaternary structure have been conserved remarkably well during more than 400 million years.of evolution. The ancestral MHC genes may have been assembled from at least three structural elements derived from different gene families. Class II MHC genes appear to have been assembled first, and then to have given rise to class I genes.     

A histocompatibility region (H-2IC) in theIC region of theH-2 complex of the mouse

Skin graft rejection in congenic pairs of mice differing only at theH-2 complex appears to be influenced by at least 3 genes (H-2K, H-2D, H-2I); we now describe a fourth,H- 2IC: Grafts transplanted across anIC difference are sometimes rejected. TheI-C regions of three differentH-2 haplotypes (d,k,s) were studied in different combinations, and variable patterns emerged: (a)IC ^d : B10.S(7R) show delayed or no rejection of first B10.S(9R) grafts, but grafts to immunized recipients were usually rejected in 20 days; (b)IC ^k : in two combinations (A.AL A and B10.HTT B10.S[9R]) first grafts were rejected by day 30, although grafts to immunized mice showed a different pattern. In the third combination (B10.HTTB10.S[7R]) first grafts were retained but immunized mice rejected their grafts, (c)IC ^s : B10.S(9R) regularly reject B10.S(7R) first grafts, but immunized mice retain their grafts. In two other combinations first grafts were retained but grafts to immunized recipients were rejected; while in a third combination rejection did not occur at all. The background of the recipient appeared to be important in determining the variable pattern of rejection, and there is evidence for a similarity of the H-genes inIC ^s andIC ^k , and inIC ^k andIC ^p . Graft rejection occurred independently of known differences in Ia specificities, indicating thatH-2IC and the genes determining Ia specificities are probably different, although when grafts were performed in the presence of known la differences, graft rejection usually occurred.     

Class II major histocompatibility complex genes of the sheep

The class II genes of the sheep major histocompatibility complex (MHC) have been cloned from two unrelated heterozygous sheep into cosmid vectors. By restriction mapping and hybridization with a number of class II probes of human and mouse origin, the cloned genetic material has been assigned to seven distinct alpha genes, 10 distinct beta genes and 14 beta-related sequences. It was difficult to identify homologues of specific HLA class II genes because of a tendency for the ovine genes to cross-hybridize between HLA probes representing different loci. Such cross-hybridization was especially marked among the beta genes. While DQ and DR homologues have been tentatively identified by several criteria, no genes corresponding to DP have been identified. Cosmids containing class II alpha and beta genes have been transfected into mouse LTK- cells, and surface expression of a sheep class II molecule has been obtained.     

The complement components of the major histocompatibility locus

Polymorphism of complement components, recognized by differences in either their antigenic specificity or their electrophoretic mobility, together with studies of inherited deficiencies, has enabled many of their structural genes to be mapped. In humans, three genes (for C2, C4, and factor B) have been placed between HLA-D and HLA-B on chromosome 6 and in mice, C4 between H2-I and H2-D, chromosome 17. Structural studies show that these components have exceptional features. C2 and factor B which contain the proteolytic active site of the C3 and C5 convertases are of the classical and alternative pathway respectively and are similar in structure and function. Both are novel types of serine proteases. C4 (as C3) contains an intrachain thioester bond essential for hemolytic activity. Molecular genetic investigations are determining the relative positions of these genes, and their precise structure, and should clarify their relation to the inherited diseases which are associated with defects in this section of the human genome.     

Genetic sophistication of human complement components C4A and C4B and RP-C4-CYP21-TNX (RCCX) modules in the major histocompatibility complex

Human populations are endowed with a sophisticated genetic diversity of complement C4 and its flanking genes RP, CYP21, and TNX in the RCCX modules of the major histocompatibility complex class III region. We applied definitive techniques to elucidate (a) the complement C4 polymorphisms in gene sizes, gene numbers, and protein isotypes and (b) their gene orders. Several intriguing features are unraveled, including (1) a trimodular RCCX haplotype with three long C4 genes expressing C4A protein only, (2) two trimodular haplotypes with two long (L) and one short (S) C4 genes organized in LSL configurations, (3) a quadrimodular haplotype with four C4 genes organized in a SLSL configuration, and (4) another quadrimodular structure, with four long C4 genes (LLLL), that has the human leukocyte antigen haplotype that is identical to ancestral haplotype 7.2 in the Japanese population. Long-range PCR and PshAI-RFLP analyses conclusively revealed that the short genes from the LSL and SLSL haplotypes are C4A. In four informative families, an astonishingly complex pattern of genetic diversity for RCCX haplotypes with one, two, three and four C4 genes is demonstrated; each C4 gene may be long or short, encoding a C4A or C4B protein. Such diversity may be related to different intrinsic strengths among humans to defend against infections and susceptibilities to autoimmune diseases.     

The allogeneic effect: M-locus differences substitute for differences in the H-2 major histocompatibility complex.

The primary immune response against sheep red blood cells in T cell-deficient spleen cell cultures from nude mice was tested in the absence and presence of allogeneic spleen cells. The allogeneic spleen cells differed either in regard to the major histocompatibility complex (H-2) or only with respect to the M-locus. Surprisingly the M-locus different spleen cells were almost as efficient in enhancing the anti-sheep red blood cell response in nude cultures as were the cells differing on the complete H-2 complex. Evidence is presented that AKR anti-theta serum sensitive T cells are responsible for the M-locus-dependent effect edscribed. This effect is shown to be mediated by a factor released from actived T cells stimulated in M-locus different mixed lymphocyte cultures. Since almost identical parameters have been observed in both the M-locus-dependent situation as in the "classical" allogeneic situation we concluded that an allogeneic effect can be induced by T cells responding to a complete set of the major histocompatibility complex (H-2) as well as to lymphocyte-activating determinants (M-locus) alone.