Histocompatibility-2 system in wild mice. VI. Histogenetic analysis of sixteen B10.W congenic lines.

Sixteen B10.W congenic lines carrying wild derived H-2 haplotypes on C57BL/10Sn or B10 background were typed by the allogeneic cell-mediated lymphocytotoxicity (CML) assay; in addition, selected lines were also typed by the TNP-CML assay and by skin grafting. The analysis revealed similarity or identity of two strain pairs: SNA57 (H-2w21) ssems to carry a similar haplotype as B10.SM (H-2v), and STA10 and STA12 seem to share the same H-2K and H-2D alleles. All other B10.W strains were different from each other and from B10 congenic lines carrying inbred-derived H-2 haplotypes. These results agree with the results of the serologic typing with two exceptions: the KPA42, KPA132, and SNA57 lines, which were serologically indistinguishable from each other and from B10.SM, were distinguished by histogenetic typing. The presence among wild mice of a haplotype (H-2u21) that appears to be very similar to a haplotype (H-2v) carried by an inbred strain (B10.SM) has some interesting implications for considerations of H-2 gene mutability. The finding that haplotypes derived from different localities are different provides further evidence that the H-2 polymorphism is extensive, indeed.     

Serology and polymorphism ofH-2L- locus encoded antigens

Thirty B10.W congenic lines were analysed serologically, both by direct cytotoxicity and by absorption, for the presence of H-2L antigens. Three new H-2L antigens, 73, 74, and 75, were discovered. The B10.W lines and the inbred strains can be classified into at least six H-2L phenogroups on the basis of their reactivity withH- 2^dm2 anti-H- 2^d serum: BALB/c, B10.BUA1, B10.GAA37, B10.BUA16. B10.KPB128, and the negative group. Twenty-oneD-end recom-binants were analysed for the possible separation ofH-2D andH-2L loci. The failure to find such a separation indicates that theH-2D andH-2L loci are tightly linked. Serological analysis also indicated that theH- 2^dm1 has lost most of its H-2L antigens but retained at least one specificity which can be detected byH- 2^dm2 anti-H- 2^d serum.     

H-41, a new minor histocompatibility locus. I. Histogenetic analysis

The B10.STA12 mouse congenic line inherited from the wild mouse parent not only the H-2w13 haplotype but also an allele at a minor H locus, which we designate H-41. This allele (H-41a) differentiates the B10.STA12 line from B10.STA10 and B10.LIB55, which carry identical H-2w13 haplotypes but a different H-41 allele (the H-41b, also present in the background strain C57BL/10Sn). The B10.STA12 and B10.STA10 lines reject each other's skin grafts and generate cytolytic T lymphocytes (CTL) after in vivo immunization and in vitro restimulation with cells of the partner strain. The B10.STA12 anti-B10.STA10 CTL react with B10.STA10, B10.LIB55, and B10.STA39 target cells and with cells of F1 hybrids between the responder strain B10.STA12 and strains C57BL/6, C57BL/10, C57L, BALB/c, A, AKR, WB, DBA/1, and DBA/2 but fail to react with (C3H x B10.STA12) F1 and (CBA x B10.STA12) F1 cells. The B10.STA10 anti-B10.STA12 CTL react with B10.STA12, B10.P, and C3H.NB cells but fail to react to (B6 x B10.STA10) F1 target cells. The CTL reactivity in both combinations is Dp restricted. The B10.STA10 anti-B10.STA12 CTL exhibit, in addition, a cross-reactivity with B10.SAA48 cells that may be directed at one of the alloantigens controlled by the H-2 haplotype of this strain.     

The histocompatibility-2 system in wild mice

The I-region gene products of 29 wild-derivedH-2 haplotypes on a B10 background (B10.W congenic lines) were typed with alloantisera which detect 17 inbred I-region antigens. Five new I-region antigens were defined by expanding the inbred line panel ofH-2 haplotypes to includeH-2 ^u , H-2^v, andH-2 ^j . Based on serological analyses of the inbred and B10.W lines, the polymorphism of theIA gene (or genes) is estimated to be at a minimum of 15 alleles and theIE gene (or genes) at a minimum of 4 alleles. These results indicate that theIA subregion is more polymorphic than theIE subregion. By combining the I-region typing data with theH-2K andH-2D region typing data reported previously, a total of 11 new natural recombinants of inbredH-2 alleles were detected among the B10.W lines.     

Sixteen newH-2 haplotypes derived from wild mice

Wild mice captured in Texas, Scotland, Federal Republic of Germany, Denmark, Spain, Greece, Israel, Egypt, and Chile were mated to inbred strains and through successive backcross matings and H-2 typing lines homozygous for wild-derived H-2, haplotypes were established. The lines, which are neither congenic nor inbred, were then typed with antibodies defining known H-2 alleles at class I and class II loci. In addition, antisera were produced by the immunization of inbred strains with tissues of the new lines. Sixteen of the lines were characterized in this manner. The characterization resulted in the identification of 16 new H-2 haplotypes, 11 new K alleles, 10 new D alleles, and 21 new class I antigenic determinants, most of them of the private type. Most of the haplotypes represent natural recombinants sharing segments of the H-2 complex with previously identified haplotypes. A number of haplotypes are recombinants between the K and the A loci, which in genetic studies have proved difficult to separate. The lines, however, also provide evidence for preservation of blocks of genes in the H-2 complex, particularly in the class II region. Some of class I alleles previously found in wild mice from Michigan have now been found again in these mice. Several class II alleles of these lines appear to be the same as those found in inbred strains. Identical or nearly identical class I and class II alleles thus commonly occur in different populations. These findings strengthen the argument that in populations, H-2 alleles are relatively stable.     

Genetic control of T-cell proliferative responses to poly(glu40ala60) and poly(glu51lys34tyr15): subregion-specific inhibition of the responses with monoclonal Ia antibodies

The relationship between Ir genes and Ia antigens was studied in the T-cell proliferative responses to two synthetic polypeptides poly(glu40ala60) (GA) and poly(glu51lys34tyr15) (GLT15). The response to GA was found to be controlled by an Ir gene in the I-A subregion, whereas the anti-GLT15 response was shown to be under dual control, one Ir gene mapping probably in the I-A subregion, and the other in the I-E subregion. We obtained two different lines of evidence suggesting identity of Ir and Ia genes. First, the presence of certain serologically identified allelic forms of the I-A-encoded A molecule correlated with the responder status to GA both in inbred strains and in B10.W lines, the latter carrying wild-derived H-2 haplotypes. Thus the Ir and Ia phenotypes were not separable in strains of independent origin. Second, the anti-GA response was completely inhibited by monoclonal antibodies against determinants on the A molecule (Ia.8, 15, and 19), but not by a monoclonal antibody against a determinant on the E molecule (Ia.7). In contrast, the anti-GLT15 response was only inhibited by a monoclonal antibody against the E molecule, but not by antibodies against the A molecule. Our data support the hypothesis that Ia antigens, as restriction elements for T-cell recognition, may in fact be the phenotypic manifestation of Ir genes.     

Comparison of one-dimensional IEF patterns for serologically detectable HLA-A and B allotypes

A new mouse monoclonal antibody (MoAb) 4E, which detects an epitope shared by HLA-B locus antigens, together with the MoAb W6/32, detecting a common HLA, B, C, determinant, and the MoAb 4B, detecting HLA-A2 and A28, were used to isolate HLA-A and -B antigens in sequential immunoprecipitation. The HLA antigens obtained from metabolically labeled cell extracts of B-lymphoblastoid cell lines or from phytohemagglutinin (PHA) activated peripheral blood lymphocytes were compared by one-dimensional isoelectric focusing (1D-IEF). The IEF banding patterns obtained with native HLA antigens segregated in a family with HLA. Neuraminidase treatment of isolated antigens reduced the number of bands to one or two, simplifying the analysis of characteristic patterns. Thus, we have cataloged IEF banding patterns for the majority of the serologically recognized HLA-A and -B allotypes obtained from 57 unrelated American Caucasians. While no HLA-A locus or HLA-B locus specific banding patterns were observed, the HLA-A antigens had, in general, slightly higher pl values than the HLA-B antigens. HLA-C antigens could not be detected in this assay system. The polymorphism detected by IEF banding patterns was as extensive as the serologically detected polymorphism identified by classical HLA serology. Moreover, variants for some HLA allotypes could be detected by this method. In addition to previously recognized A2 variants, new variants were identified for HLA-A1, A26, and Bw44. Each A1 and Bw44 variant was associated with particular haplotypes. The HLA-A2 antigens occurred on 43 HLA haplotypes in the unrelated Caucasian population. Only one of each A2 variants was identified in this population.     

Genetic control of acquired resistance to visceral leishmaniasis in mice

A series of H-2 and non-H-2 congenic resistant (CR) strains on a C57BL/10Sn background were infected with 10(7) amastigotes of Leishmania donovani. Non-H-2 congenic strains B10.LP-H-3b and B10.CE(30NX) and (B10.LP-H-3b x B10)F1 hybrids showed a very rapid decrease in liver-parasite burdens beyond day 21. Parasite counts for these strains at day 35 were significantly lower than for all other strains tested. The rapid decrease in parasite numbers, massive lymphocellular infiltration into the liver and strong delayed hypersensitivity reactions to parasite antigens in strains congenic for a portion of chromosome 2 indicated that acquired immunity to L. donovani was controlled by a dominant gene at or near the Ir-2 locus. In addition, B10.129(10M) mice, which differ from C57BL/10Sn at the H-11 locus, showed highly significant increases in parasite numbers at day 35. Other observations supporting the absence of acquired immunity in B10.129(10M) included negative delayed hypersensitivity tests to parasite antigens and the absence of lymphocellular infiltrate into the liver. Although the differences were not as pronounced, H-2 CR strains with H-2b, H-2a, and H-2k haplotypes also showed significantly greater decreases in parasite numbers by day 35 as compared to other H-2 CR strains.     

Genetic structure and origin of t haplotypes of mice, analyzed with H-2 cDNA probes

We investigated the genetic organization and evolutionary origin of t chromosomes of mice by examining the restriction fragment patterns of DNA from t haplotypes and normal chromosomes with cDNA probes to H-2 class I genes. On genomic DNA blots, the restriction fragments containing H-2-related sequences were highly variable among different inbred strains of mice, whereas they were very similar among different t haplotypes even when the t haplotypes carried serologically different H-2 haplotypes. These observations suggest that all t haplotypes have a common origin and are not products of independent mutational events. We also mapped the position of several restriction fragments characteristic of t DNA by using a battery of recombinant t haplotypes, defined with respect to their t-lethal factors and H-2 haplotypes. We thus show that restriction fragments containing H-2-related sequences map to the left of the H-2 class I genes in t chromosomes, a region in which the tw32 b-lethal factor also maps. The cloning of these fragments can be expected to provide an entry for the structural analysis of t DNA.     

Two new recombinant H-2 haplotypes, one of which juxtaposes Kb and Ik alleles.

Two new recombinant H-2 haplotypes have been detected and established as congenic resistant lines on the C57BL/10 background. On the basis of serologic testing and immunoprecipitation analyses, the sublocus composition of the first recombinant haplotype, H-2bq1 (B10.MBR) has been shown to be KbIkDq, and that of the second recombinant, H-2sq3 (B10.SQR) to be KsIsSsDq. The occurrence of the Kb I-Ak juxtaposition after a recombination between H-2b and H-2m contrasts with the almost uniform failure to observe H-2b/H-2k recombinants in previous studies. This finding and the occurrence of a second recombination event involving the same chromosome soon after the first in our studies may imply that recombination within H-2 is not generally a random event. The B10.MBR line has proved useful in the production of specific anti-H-2Kb and anti-I-Ab antisera previously quite difficult to obtain contamination by anti-Ia or anti-H-2K antibodies, respectively.     

The histocompatibility-2 system in wild mice. XI. Ss and Slp properties of wild-derived H-2 haplotypes

In this study, 14 B10.W lines were examined for genetic traits associated with the Ss protein and Slp alloantigen. Three B10.W lines were found to possess low levels of serum Slp alloantigen. Of these three Slp-positive lines, expression of the alloantigen was sex-limited in two lines (B10.LIB55 and B10.STA12) and constitutive, i.e., found in both sexes, in the remaining line (B10.KPB128). Lines which were found to be Slp-negative were also tested for IA-controlled immune response to Slp alloimmunization. The finding that one of these Slp-negative lines produced no detectable anti-Slp indicates that nonresponder alleles exist in wild populations. Further, the discovery of an unexpected immune response to Slp in F1 hybrids involving lines B10.LIB55 and B10.STA12 suggests the existence of a variant form of Slp alloantigen in these lines.     

Myelin proteolipid protein-induced experimental allergic encephalomyelitis. Variations of disease expression in different strains of mice

Strains of mice with diverse genetic backgrounds were tested for susceptibility to experimental allergic encephalomyelitis (EAE) induced by myelin proteolipid protein. EAE was elicited in all strains of mice tested, but the clinical and histologic features varied. SJL (H-2s) mice had a high incidence of both clinical and histologic disease characterized by early onset of clinical signs. Inguinal lymph node T cells from diseased animals responded specifically [( 3H]thymidine incorporation) to proteolipid protein and not to myelin basic protein. In contrast, BALB/c (H-2d), DBA/1 (H-2q), C57BL/6 (H-2b), AKR (H-2k), CBA (H-2k), C3H (H-2k), B10.BR (H-2k), and C57BR (H-2k) mice showed a later onset of clinical signs and typically a lower disease incidence. However, the most marked variations in disease incidence occurred among BALB/c (H-2d) substrains in which the incidence of EAE ranged from eight of nine (BALB/cPt) to complete resistance (BALB/cWt and BALB/cORNL). Because these BALB/c substrains were initially derived from the same inbred genetic source and are serologically identical at H-2, these results suggest that expression of proteolipid protein-induced EAE in the mouse involves additional loci outside the MHC.     

Genetic control of T-cell proliferative responses to poly(glu40ala60) and poly(glu51lys34tyr15): Subregion-specific inhibition of the responses with monoclonal Ia antibodies

The relationship betweenIr genes and Ia antigens was studied in the T-cell proliferative responses to two synthetic polypeptides poly(glu⁴⁰ala⁶⁰) (GA) and poly(glu⁵¹lys³⁴tyr¹⁵) (GLT¹⁵). The response to GA was found to be controlled by anIr gene in theI-A subregion, whereas the anti-GLT¹⁵ response was shown to be under dual control, oneIr gene mapping probably in theI-A subregion, and the other in theI-E subregion. We obtained two different lines of evidence suggesting identity ofIr and Ia genes. First, the presence of certain serologically identified allelic forms of the I-A-encoded A molecule correlated with the responder status to GA both in inbred strains and in B10.W lines, the latter carrying wild-derivedH-2 haplotypes. Thus the Ir and Ia phenotypes were not separable in strains of independent origin. Second, the anti-GA response was completely inhibited by monoclonal antibodies against determinants on the A molecule (Ia.8, 15, and 19), but not by a monoclonal antibody against a determinant on the E molecule (Ia.7). In contrast, the anti-GLT¹⁵ response was only inhibited by a monoclonal antibody against the E molecule, but not by antibodies against the A molecule. Our data support the hypothesis that Ia antigens, as restriction elements for T-cell recognition, may in fact be the phenotypic manifestation ofIr genes.     

Quantitative variations in the expression of the mouse serum antigen Ss and its sex-limited allotype Slp

A radial immunodiffusion assay for quantitation of the Ss and Slp serum antigens is described. Significant differences between the mean serum concentrations of Ss and Slp were found among various inbred strains. Some of these differences have been shown to be associated with the H-2 haplotype. The quantitative difference between Slp levels associated with the H-2 ^a and H-2 ^S haplotypes has been used as a marker for the S region in the analysis of certain H-2 recombinant strains [A.TH, B10.S(7R), B10.S(9R), and B10.BSVS]. Male mice of two strains with the H-2 ^b haplotype have been shown to have significantly lower levels of Ss compared to males of the other strains tested. Male mice of every strain examined were found to have significantly higher levels of Ss in their serum than females of the same strain. The molecular relationship and developmental patterns of the Ss and Slp antigens have also been investigated using the radial immunodiffusion assay.     

Characterization of newly isolated monoclonal antibodies against MHC of a Japanese wild mouse

We have already developed nine B10.MOL congenic strains carrying H-2 haplotypes derived from Japanese wild mice, Mus musculus molossinus, with the C57BL/10 genetic background. To obtain monoclonal antibodies against the H-2 antigen of the Japanese wild mouse, we carried out cell fusion using spleen cells from the animal immunized with one of the B10.MOL strains, B10.MOL-SGR (H-2 ^wm7). As a result, 19 hybridomas producing monoclonal antibodies were produced. Analysis with the intro-H-2 recombinants derived from B10.MOL-SGR indicated that 8 of them reacted with the class I and II with the class II molecule. The class I antibodies were tested for their cross -reactivities on wild mice and on the panels of standard inbred and B10.MOL strains. Most of the antibodies reacted with both the Japanese wild mice and the other subspecies, including standard inbred, while two antibodies highly specific for the donor H-2K region reacted with only three wild-derived mice, two M. m. molossinus from Anj o and Shizuoka, Japan, and one M. m. domesticus from Pigeon, Canada. In addition, all of the other four antibodies reactive with the K antigen of B10.MOL-SGR also reacted with the same three wild mice. The wild mice belonging to different subspecies might share very similar H-2K antigenic determinants in spite of their genetic and geographical remoteness.     

Mapping immunodominant antigens and H-2-linked antibody responses in mice urogenitally infected with Chlamydia muridarum

To identify immunodominant antigens and MHC-restricted antibody responses, seven different strains of mice were intravaginally infected with Chlamydia muridarum and compared for antibody responses to 257 C. muridarum proteins. The 7 strains of mice recognized a total of 109 proteins as antigens, of which, 5 antigens (TC0660, TC0727, TC0828, TC0726 & TC0268) were each recognized by 60% or more mice from each mouse strain and thus designated as immunodominant antigens. Furthermore, antibody responses to 19 other antigens displayed strong associations with mouse H-2 haplotypes, including 6 antigens (TC0480, TC0912, TC0229, TCA04, TC0289 & TC0892) whose antibody responses were linked to H-2(b), 8 (TC0035, TC0387, TC0052, TC0781, TC0373, TC0117, TC0066 & TC0396) to H-2(d) and 5 (TC0512, TC0177, TC0589, TC0794 & TC0596) to H-2(k) haplotypes respectively. Interestingly, H-2(b) was negatively associated with antibody responses to most of the antigens that were positively linked to H-2(d) or H-2(k) haplotypes. These results by mapping Chlamydia trachomatis antigens commonly recognized by mice with different strain background and H-2 genes and revealing antigen association with H-2 haplotypes have provided important information for developing chlamydial subunit vaccines and understanding chlamydial pathogenesis.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. IV. Partial tissue distribution and mapping of immunodominant antigens

The immunization of C57BL/6 responder mice with spleen cells from H-2-matched BALB.B donors, which differ by multiple non-H-2 histocompatibility (H) antigens, results in the generation of cytotoxic T lymphocytes (CTL) that are specific for only a limited number of immunodominant antigens. Previous analysis of the genes encoding these dominant antigens has not mapped these genes to any of the non-H-2 H loci defined by congenic strains. It would have been expected that the histogenetic techniques employed for congenic strain selection would have preferentially identified the "strongest" H antigens. Therefore, we have investigated the possibility that immunodominant antigens do not belong to the class of non-H-2 H antigens encoded by genes mapping to H loci defined and mapped by congenic strains. The first experiments were aimed at identifying antigens that were expressed by independently derived inbred strains and were cross-reactive with the immunodominant cytotoxic T cell target (CTT-1) antigen of BALB.B. Strong cross-reaction with the C3H.SW (H-2b) strain was observed; the C3H gene encoding this antigen was mapped with BXH recombinant inbred strains. Contrary to the mapping of the CTT-1 gene to chromosome 1 in BALB.B, the C3H gene was shown to map to either chromosome 4 or chromosome 7. This result indicates that identical, or at least extensively cross-reactive, non-H-2 antigens may be encoded by genes mapping to independently segregating loci in different inbred strains. The tissue distribution of immunodominant antigens was approached by determining the reactivity of CTL specific for these antigens with either lymphoid-derived or fibroblast-derived targets. These CTL effectively lysed lymphoblast and lymphoid tumor targets but did not lyse an SV40-transformed fibroblast line that was shown to be efficiently lysed by CTL specific for non-H-2 H antigens defined by congenic strains. Therefore, it was concluded that immunodominant antigens detected by B6 anti-BALB.B CTL have a restricted tissue distribution in comparison to non-H-2 H antigens defined by congenic strains. The implications of these results for our understanding of the origin and heterogeneity of non-H-2 cell-surface antigen recognized by effector T cells are discussed.     

Comparative anatomy of the primate major histocompatibility complex DR subregion: evidence for combinations of DRB genes conserved across species.

The class II region of the human major histocompatibility complex (HLA) is made up of three major subregions designated DR, DQ, and DP. With the aim of gaining an insight into the evolution and stability of DR haplotypes, a total of 63 cosmid clones were isolated from the DR subregion (Gogo-DR) of a western lowland gorilla. All but one of these cosmid clones were found to fall into two clusters. The larger cluster, A, was defined by 41 overlapping cosmid clones and contained a DRB gene segment made up of exons 4 through 6 and four DRB genes, designated Gogo-DRB6, Gogo-DRB5*01, Gogo-DRB8, and Gogo-DRB3*01. The total length of this cluster was approximately 180 kb. The second cluster, B, encompassed a contiguous DNA stretch of approximately 145 kb and was composed of 21 overlapping cosmid clones. Cluster B contained three DRB genes, designated Gogo-DRB1*08, Gogo-DRB2, and Gogo-DRB3*02. One cosmid clone (WP1-9) containing a DRB pseudogene could not be linked to either cluster A or B. Neither the organization of cluster A nor that of cluster B was identical to that of known HLA-DR haplotypes. However, two gorilla DRB genes, Gogo-DRB6 and Gogo-DRB5*01, the human counterparts of which are linked in the HLA-DR2 haplotype, were found to be located next to each other in cluster A. The arrangement of the Gogo-DRB genes in cluster B, which is presumed to be the gorilla DR8 haplotype, was similar to that of HLA-DR3/DR5/DR6 haplotypes and to that of the presumed ancestral HLA-DR8 haplotype.(ABSTRACT TRUNCATED AT 250 WORDS)