Using the emerging Collaborative Cross to probe the immune system.

The Collaborative Cross (CC) is an emerging panel of recombinant inbred (RI) mouse strains. Each strain is genetically distinct but all descended from the same eight inbred founders. In 66 strains from incipient lines of the CC (pre-CC), as well as the 8 CC founders and some of their F1 offspring, we examined subsets of lymphocytes and antigen-presenting cells. We found significant variation among the founders, with even greater diversity in the pre-CC. Genome-wide association using inferred haplotypes detected highly significant loci controlling B-to-T cell ratio, CD8 T-cell numbers, CD11c and CD23 expression. Comparison of overall strain effects in the CC founders with strain effects at QTL in the pre-CC revealed sharp contrasts in the genetic architecture of two traits with significant loci: variation in CD23 can be explained largely by additive genetics at one locus, whereas variation in B-to-T ratio has a more complex etiology. For CD23, we found a strong QTL whose confidence interval contained the CD23 structural gene Fcer2a. Our data on the pre-CC demonstrate the utility of the CC for studying immunophenotypes and the value of integrating founder, CC and F1 data. The extreme immunophenotypes observed could have pleiotropic effects in other CC experiments.

Allelic diversity at the DLA-88 locus in Golden Retriever and Boxer breeds is limited.

In the dog, previous analyses of major histocompatibility complex class I genes suggest a single polymorphic locus, dog leukocyte antigen (DLA)-88. While 51 alleles have been reported, estimates of prevalence have not been made. We hypothesized that, within a breed, DLA-88 diversity would be restricted, and one or more dominant alleles could be identified. Accordingly, we determined allele usage in 47 Golden Retrievers and 39 Boxers. In each population, 10 alleles were found; 4 were shared. Seven novel alleles were identified. DLA-88*05101 and *50801 predominated in Golden Retrievers, while most Boxers carried *03401. In these breeds, DLA-88 polymorphisms are limited and largely non-overlapping. The finding of highly prevalent alleles fulfills an important prerequisite for studying canine CD8+ T-cell responses.

H-2 effects on cell-cell interactions in the response to single non-H-2 alloantigens. I. Donor H-2D region control of H-7.1-immunogenicity and lack of restriction in vivo.

Genes in the H-2 complex regulate the relative immunogenicity of the H-7.1 histocompatibility alloantigen, as measured by survival times of H-7.1-incompatible skin grafts in vivo. The gene controlling relative rejectability of H-7.1-incompatible grafts has been mapped to the H-2D region. H-7.1-incompatible skin grafts donated by H-2Db donors were rejected significantly more rapidly by H-2a/H-2b heterozygous recipients than similar H-7.1-incompatible grafts donated by H-2Dd donors. Further, there was absolutely no evidence of H-2 restriction in cytotoxic effector activity. In vivo cross-priming, as indicated by accelerated secondary graft rejection, was extensive. The efficiency of cross-priming was dependent upon the primary and secondary graft donor H-2 haplotypes.

H-2 effects on cell-cell interactions in the response to single non-H-2 alloantigens. II. H-2D region control of H-7.1-specific stimulator function in mixed lymphocyte culture and susceptibility to lysis by H-7.1-specific cytotoxic cells.

The relative immunogenicity of the H-7.1 alloantigen has been shown in a previous communication to be regulated by a gene in the D region of the mouse major histocompatibility (H-2) complex. The level of relative immunogenicity was inferred from survival times of H-7.1-incompatible skin grafts donated by donors with different H-2 haplotype origins of H-2D region genes. In this communication we report the results of an extension of these previous investigations into the possible role of H-2D region genes in controlling the capacity of H-7.1-incompatible lymphocytes to stimulate H-7.1-speciflc mixed lymphocyte culture proliferation and generation of cytotoxic effector cells. The results reported herein demonstrate that the H-2D genotype of H-7.1-incompatible stimulator cells determines the relative H-7.1-specific capacity of those lymphocytes to stimulate H-7.1-specific proliferation of in vivo primed responder T cells in secondary mixed lymphocyte culture. H-2D(b)-bearing, H-7.l-incompatible stimulators were significantly more effective in stimulating H-7.1-specific proliferation than H-2D(d)-bearing stimulators. As expected, H-2D(b), H-7.1-in-compatible stimulators were also more effective than H-2D(d) a stimulators in generating H-7.1- specific cytotoxic effector cells. Further, the susceptibility of (51)Cr- labeled, H-7.1-incompatible lymphoblast targets to H-7.1-specific lysis was similarly regulated by an H-2D gene. Reciprocal H-2 restriction (F(1) cells are capable of killing only the cells bearing the immunizing cell parental H-2 haplotype) observed by other investigators for cytolysis of non-H-2-incompatible targets was not observed. H-2D a-bearing, H-7.1- incompatible stimulators stimulated generation of cytotoxic effectors capable of detectably lysing H-2D(b) but not H-2D(a)-bearing, H-7.1- incompatible targets. The impact of these observations on the proposed models for H-2 restriction of non-H-2 histocompatibility antigen-specific cytolysis is discussed.

Immune mechanisms in leukemia role of the Ia antigens.

We have shown that the selective removal of cells possessing Ia determinants coded by the I-A, I-B, and I-J regions of the H-2 gene complex completely abrogates the protective capacity of nylon-wool-purified T lymphocytes against leukemic challenge. This suggests that the Ia antigen bearing T cells play an important role in tumor immunity.

Acute autoimmune encephalomyelitis in mice. II. Susceptibility is controlled by the combination of H-2 and histamine sensitization genes.

The expression of acute experimental autoimmune encephalomyelitis (EAE) in mice is controlled by several dominant genes, H-2 and histamine sensitization genes. SJL/J and SWR/J, which are H-2s and H-2q, respectively, are susceptible to EAE and sensitive to Bordetella pertussis histamine-sensitizing factor (HSF), which produces a vasoactive amine hypersensitivity. Other H-2s or H-2q strains such as A.SW, B10.Q and several others do not develop acute EAE and are not sensitive to B. pertussis HSF. One strain tested, DDD (KsIsD?) is HSF sensitive but does not develop EAE (presumably because it lacks the appropriate responder H-2 haplotype). However, F1 hybrids between B10.S and DDD are sensitive to HSF and develop EAE. The induction and effector phases of acute EAE are apparently controlled by the combination of H-2 and HSF genes. A combination of the correct H-2 hapotype and histamine sensitivity is required for the development of acute EAE.

Widespread transcription of a Qa region gene in adult mice.

The mouse MHC class I family includes genes encoded in four regions: H-2K, H-2D, Qa and Tla. While K/D genes are well characterized, relatively little is known about Qa or Tla genes. We have studied the transcription of a B10.P Qa region gene. DNA sequence comparisons of the transmembrane region, supported by Southern blot analysis of cosmid and genomic DNAs from BALB/c and C57BL/10, demonstrate the lambda 3a gene corresponds to Q4p. In both Northern blots and RNA protection experiments using probes derived from the 3' noncoding region, we found that Q4, like the H-2K and H-2D genes, is widely transcribed in B10.P tissues. These data demonstrate for the first time widespread transcription of a Qa gene.

Effects of anti-Ia sera on mitogenic responses. III. Mapping the genes controlling the expression of Ia determinants on concanavalin A-reactive cells to the I-J subregion of the H-2 gene complex.

We have shown that the Ia determinants expressed on nylon wool-purified T lymphocytes reactive to concanavalin A (Con A) in serum-free media are coded in a single I subregion of the H-2 gene complex. This region, I-J, is defined by two pairs of intra-H-2 recombinant haplotypes: H-2t3, H-2t4 and H-2i3, H-2i5, carried by B10.HTT, B10.S(9R), B10.A(3R), AND B10.A(5R), respectively. No activity against Con A-reactive T cells has been detected in any antiserum that was produced in strain combinations which shared a common I-J region. This suggests that Ia antigens expressed on Con A-reactive T cells are restricted to the I-J subregion.

Evidence of widespread binding of HLA class I molecules to peptides.

We have tested the binding of HLA class I proteins to peptides using a solid-phase binding assay. We tested 102 peptides, mostly derived from the HIV gag and HIV pol sequences. Most peptides did not bind to any class I protein tested. The pattern of binding among the three class I proteins tested, HLA-A2, -B27, and -B8, was approximately 85% concordant. Further, all five of the known HIV-1 gag T cell epitopes detected by human CTL bound at least one class I protein. Binding of class I to the peptides could be detected either by directly iodinated class I proteins, or indirectly using monoclonal antibodies specific for class I. The binding to the plates could be blocked with MA2.1, which binds in the alpha 1 region of A2, but not by W6/32, which binds elsewhere. The data presented here show that binding of class I to peptides is specific, but that many peptides bind to more than a single class I protein.

T-lymphocyte response to H-2 mutants. I. Proliferation is dependent on Ly 1+2+ cells.

We have determined the Ly phenotype of the T lymphocytes which proliferate in response to mutant H-2K and H-2D alloantigens in primary mixed lymphocyte culture. Responder T cells proliferating in reciprocal cultures of H-2d(KdDd) and H-2da(KdDda) lymphocytes were typed Ly 2+ through selective depletion with specific alloantiserum plus complement. Further, B6-Ly 1a lymphocytes proliferating in response to B6-H-2ba and B6-H-2bf stimulators were typed as Ly 1+2+ through similar analysis. These results are discussed with regard to their impact on views of lymphocyte differentiation and factors determining the identity of alloreactive lymphocytes.

Evidence for the expression of Ia (H-2-associated) antigens on thymus-derived lymphocytes.

We have demonstrated in an anti-Ia serum the presence of specific antibodies reacting with T cells, as well as with B cells, using a highly sensitive dye exclusion test. This antiserum reacts with both spleen and lymph node in a characteristic biphasic titration curve killing up to 70% of these cells. It also reacts with cortisone-resistant thymocytes. The A.TH-alpha-A.TL serum can be absorbed with spleen, lymph node, cortisone-resistant thymus, or normal thymus cells. Further in vivo absorptions in BALB/c nude cannot remove all of the cytotoxic activity for normal BALB lymph node lymphocytes, while completely removing the activity for nude cells. A Thy-1 positive cell line derived from a C57Br leukemia is reactive with this anti-Ia serum.

Role of self carriers in the immune response and tolerance. V. Reversal of trinitrophenyl-modified self suppression of the B-cell response by blocking of H-2 antigens.

Trinitrophenylated syngeneic spleen cells (TNP-SC) are potent tolerogens of the anti-TNP plaque-forming cell (PFC) response in vivo and in vitro. This unresponsive state requires T cells for both its induction and maintenance. Because H-2K/D-restricted cytotoxic T cells are also induced by exposure to TNP-SC, we determined the role(s) of histocompatibility antigens (K, I, and D) in the suppression of the PFC response by TNP-SC. We treated syngeneic TNP-modified stimulator cells with antiserum directed at K-, I-, or D-region determinants and found that blocking of H-2K or D antigens on TNP-SC transformed these tolerogens into immunogens capable of eliciting an anti-TNP PFC response in the absence of extrinsic immunogens like TNP-polymerized flagellin. In H-2k or H-2a(k/d) mice, only H-2Kk needs to be blocked on the stimulator cells, whereas H-2K or D recognition was apparent in B10.A(4R) mice. These observations indicate that suppression of the PFC response by TNP-SC shows the same restriction in recognition as does the cytotoxic T-cell response. Furthermore, our results suggest that TNP-I-A is recognized by the helper cells in this system as the intrinsic antigen. When both TNP-K and TNP-I-A are present and available on the same stimulator cell, suppression (via modified K recognition) is dominant over help.

New family of exon-shuffled recombinant genes reveals extensive interdomain interactions in class I histocompatibility antigens and identifies residues involved.

The specificities of an extensive panel of anti-H-2Dd monoclonal antibodies, which had been previously characterized using exon-shuffled H-2Dd/H-2Ld molecules and a number of anti-H-2DP antibodies, were examined using H-2Dd/H-2DP recombinants. The use of this new family of recombinant antigens revealed extensive interaction between the membrane-distal (N and Cl) domains of class I molecules. 20 out of 48 mAbs recognize complex epitopes formed by the interaction of these two domains. These antibodies exhibit a number of distinct patterns of crossreactivity with other class I proteins, revealing the presence of multiple epitopes within the region of domain interaction. Comparison of the data presented here with those from previous work allowed the identification of a small number of residues in the Cl domain that participate in the generation of complex epitopes involving both the N and Cl domains. The results are discussed in terms of the structural information available for these two domains.

Effects of anti-Ia sera on mitogenic responses. II. Differential expression of the Ia marker on phytohemagglutinin and concanavalin A-reactive T cells.

Genes mapping in the I region of the H-2 complex control a system of lymphocyte alloantigens (Ia) which are expressed on a subpopulation of T cells and on most B cells. Specific anti-Ia serum in the presence of rabbit complement removed the splenic T-cell subpopulation responsive to Con-A, but did not affect the response to phytohemagglutinin (PHA) or Leucoagglutinin. Antibodies specific for Ia, H-2K, or H-2D membrane antigens were used without complement to pretreat spleen cells. These antibody pretreated cells responded normally to Con-A and PHA.

Structure of murine Ia antigens. Two dimensional electrophoretic analyses and high pressure liquid chromatography tryptic peptide maps of products of the I-A and I-E subregions and of an associated invariant polypeptide.

We demonstrate that an invariant polypeptide, first described by Jones et al. (21), co-immunoprecipitates with our Ia molecules, that its interaction with Ia polypeptides varies with haplotype, and that it is not a precursor of the Aalpha, Abeta, Ealpha, or Ebeta. polypeptides. We also show that the polypeptides that we have previously characterized are contaminated with very little, if any, invariant protein. Further, we have used our high-pressure liquid chromatography tryptic peptide map technique to formally map the genes encoding Aalpha, Abeta, and Ebeta to the I-A subregion using recombinant and F1 hybrid mice.

Maternally derived transferrin in pigeon squabs.

With the use of genetically marked transferrin, a major portion of circulating transferrin from a newly hatched squab was found to be derived from the mother through the egg. The transfer is not through the parental crop milk. The squab does not accumulate enough transferrin of its own making to be detectable until it is about 8 days old. The maternally derived protein remains detectable until 14 days after hatching. The squab actively synthesizes a portion its own transferrin from hatching onward.

Inhibition of immune responses in vitro by specific antiserums to Ia antigens.

Mouse antiserums prepared against Ia antigens, which are products of I (immune response) region genes of the H-2 complex, can inhibit both primary (immunoglobulin M) and secondary (immunoglobulin G) immune responses in vitro by mouse spleen cultures to heterologous erythrocytes. Antiserums directed specifically at products of either the H-2K or H-2D loci have no effect on this response.

LCMV-specific, class II-restricted cytotoxic T cells in beta 2-microglobulin-deficient mice.

Intracranial infection of normal mice with lymphocytic choriomeningitis virus (LCMV) causes meningitis and death mediated by CD8+ major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs). beta 2-Microglobulin-deficient mice (beta 2M-/-) do not express functional MHC class I proteins and do not produce significant numbers of CD8+ T cells. When beta 2M-/- mice were infected with LCMV, many died from LCMV disease and produced a specific response to LCMV mediated by CD4+ CTLs that were class II-restricted. In these mice, CD4+ CTLs may compensate for the lack of CD8+ CTLs.

Identification of a BALB/c H-2Ld gene by DNA-mediated gene transfer.

Gene transfer and immunoselection were used in the identification of a BALB/c genomic clone containing an H-2Ld gene (clone 27.5). Transformation of thymidine kinase-negative C3H mouse L cells with the cloned 27.5 DNA together with the herpes simplex virus tk gene produced transformants expressing Ld molecules detected by radioimmune assay with monoclonal hybridoma antibodies to Ld antigens. The foreign Ld gene products expressed by cloned mouse L cell transformants were shown to be virtually indistinguishable from BALB/c spleen Ld molecules by two-dimensional electrophoretic analysis of H-2Ld immunoprecipitates. These results indicate that the genomic clone 27.5 contains a functional BALB/c H-2Ld gene and demonstrate the usefulness of this approach for identifying the gene products encoded by cloned genes which are members of a multigene family. Furthermore, the ability to place cell-surface recognition molecules on the surfaces of foreign cells provides a powerful opportunity for functional analyses of these molecules.

Construction of microcell hybrid clones containing specific mouse chromosomes: application to autosomes 8 and 17.

Fibroblast cultures prepared from mice homozygous for a Robertsonian translocation (centric fusion) between autosomes 8 and 17 [Rb(8.17)] were used as donors in microcell-mediated chromosome transfer experiments. By using hamster recipient cells deficient in adenine phosphoribosyltransferase (APRT-) and selecting for expression of murine APRT (a chromosome 8 marker), microcell hybrids were isolated which retained only the mouse Rb(8.17) translocation in addition to the hamster chromosome complement. The translocation was stable in cells maintained under APRT+ selective pressure, and mouse marker traits encoded by genes on both chromosomes 8 and 17 segregated concordantly. A second family of hybrid clones was constructed by fusing microcells derived from wild-type mouse fibroblasts with APRT- hamster cells. Four of six clones analyzed retained only mouse chromosome 8. These studies demonstrated that microcell hybrids containing specific Robertsonian translocations as the only donor-derived genetic material can be obtained. Furthermore, a number of Robertsonian translocations between chromosomes which carry selectable markers (chromosomes 3, 8, and 11) and other autosomes have been described. By using fibroblast cultures prepared from mice containing these translocations as donors in microcell fusions, 18 of the 20 mouse chromosomes could be selectively fixed in different hybrid clones. Thus, a collection of 20 hybrid clones, each containing a single, specific mouse chromosome, can be constructed by using the strategy described in this report. The potential utility of such a monochromosomal hybrid panel is discussed.