Molecular basis of Qa-11 antigen and paradoxical Qa-gene expression in an H-2 recombinant

The Qa-11 Ag expressed in certain strains with the B2-microglobulin-b allele, apparently maps into the Tla region as well as into the Qa-2 region. Moreover Qa-11 has been shown to be biochemically indistinguishable from Qa-2. Genetic complementation studies combining the right Qa and Tla regions failed to lead to Qa-11 expression. To elucidate the molecular basis of this apparent paradox, we examined the expression of Qa-11 on products of transfected Q-region class I genes. Immunochemical analysis has shown that the Qa-11 Ag is expressed on class I molecules encoded by the Q7 gene from both C57BL/10 (Q7b) and BALB/c (Q7d), but not on the protein product of the Q9 gene isolated from the C57BL/10 strain (Q9b). Inasmuch as the predicted protein products of the Q7b and Q9b genes would differ at a single amino acid, a residue critical for Qa-11 expression has been identified. Based on these results it is proposed that among the beta-2-mb strains, the Qa-11+/Qa-2+ mice are likely to express at least the Q7 gene, whereas Qa-11-/Qa-2+ mice express only Q9. In support of this model, the Qa-2+/Q-11- recombinant B6.K2, essential for the apparent mapping of Qa-11 into the Tla region, expresses only Q9 but not Q7 encoded molecules on the cell surface, and only Q9 and no processed Q7 mRNA is detected in the cytoplasm. This expression pattern in B6.K2 cannot be explained on the basis of a single crossing-over event.     

A single gene encodes soluble and membrane-bound forms of the major histocompatibility Qa-2 antigen: anchoring of the product by a phospholipid tail

The H-2, Qa, and Tla genes of the murine major histocompatibility complex are related to each other by DNA sequence homology. The H-2 genes encode ubiquitously expressed transplantation antigens that serve as recognition structures for cytotoxic T cells. The identities of the Qa and Tla products, their sites of expression, and their functions are largely unknown. We report here that the Qa region gene Q7 encodes a membrane-bound as well as a secreted form of the serologically defined antigen Qa-2. The Q7 gene introduced into liver-derived cells is expressed as a membrane-bound and as a secreted molecule. In transfected L cells it is expressed only as a soluble protein. Biochemical analysis suggests that the Q7 product is anchored to the liver cell membranes by a phospholipid tail. This feature may be responsible for cell type-specific expression of the two forms of the Qa-2 molecules.     

Evolution and expression of the transplantation antigen gene family

We have cloned 26 different class I genes that are located in the major histocompatibility complex of the C57BL/10 mouse. Two of the three class I genes found in the H-2 complex encode the H-2Kb and H-2Db antigens; the other 23 class I genes map to the adjacent Tla complex. We have grouped the cosmids containing these genes into three clusters: one cluster links the H-2K and I-A regions, one cluster links the H-2D and Qa-2 regions, and the final cluster maps to the TL region. The class I gene organizations in the Qa-2 and TL regions of the C57BL/10 and BALB/c mice are generally similar, but there are several polymorphic segments. The Qa-2 region of both mice seems to have evolved by the duplication of gene pairs; furthermore, the H-2K region may have been generated by the translocation of a gene pair from the Qa-2 region. We have evidence that several of the genes in the Qa-2 region are expressed.     

TheTla region of the mouse: Identification of a new serologically defined locus,Qa-2

In cytotoxicity assays, the reactions of cells from two new congenic strains-B6.K1 and B6.K2-with antiserum prepared in B6.K1 against B6 (C57BL/6) spleen and lymph node cells identify a new locus,Qa-2, betweenH-2D andTla. This locus specifies differentiation antigens on lymphoid cells. Skin grafting of B6.K1, B6.K2, and other congenic strains on a B6 background also reveals two histocompatibility loci in the region ofTla.     

A new serologically defined locus,Qa-1, in theTla-region of the mouse

A new cell-surface antigen, specified by a gene betweenH-2D andTla is described. The provisional notationQa-1 is suggested for the locus determining this newly recognized cell surface component. Qa-1 is distinguished from known TL antigens by the following two criteria. Its expression is not confined to thymocytes — it occurs on lymph node cells (LNC) also; and the phenotypes of the new congenic recombinant strains B6.K1 and B6.K2, derived fromH-2D/Tla crossovers, are Qa-1⁺ Qa-2^–TL^– and Qa-l⁺Qa-2⁺TL^–. Qa-1 antigen is defined by reaction of the standard TL typing serum, (B6 × A -Tla ^b)F₁ anti-A strain leukemia ASL1, with lymph node cells (LNC) in the cytotoxicity assay. Qa-1 antigen evidently is expressed, at least, on a subpopulation of T cells as well as on thymocytes. The gene order isH-2D, Qa-1, Qa-2, Tla.Abbreviations used in this paper LNC lymph node cells pooled from inguinal, axillary, brachial, and mesentric nodes - BA⁺ (C57BL/6-Tla^axA)F1 - BA^– (C57BL/6 × A -Tla ^b)F₁ - PBS phosphate buffered saline pH 7.2 - Thy thymocytes - RMIg Rabbit anti-mouse immunoglobulin Please address proofs and communications concerning this paper to Dr. Thomas Stanton, Sloan Kettering Institute, 1275 York Ave., New York, N.Y. 10021     

Biochemical analysis of an MHC-linked hematopoietic cell surface antigen,Qa-2

The region of the murine 17th chromosome telomeric to H-2D encodes a group of serologically defined cell surface antigens termed Qa-1-5. These antigens are of interest because their expression is restricted to hematopoietic cells. In addition, the molecular weight and subunit structure (ie, association with β-2 microglobulin) of Qa-2 molecules are similar to H-2 and TL antigens. In the present studies, we have prepared isotopically labeled Qa-2 and H-2 molecules from mitogen-stimulated C57BL/6 spleen cells. Comparative peptide mapping of tryptic peptides from Qa-2 and H-2 molecules (K^b, D^bK^k, D^d) reveal that Qa-2 has a unique primary structure. However, considerable homology is indicated since 30–40% of the Qa-2 peptides cochromatograph with peptides derived from H-2K^b, H-2D^b, H-2K^k, and H-2D^d. Studies by other investigators have demonstrated that similar levels of structural homology are observed when H-2K, H-2D, and H-2L tryptic peptides are analyzed. We conclude from these studies that the Qa-2 alloantigen is structurally related to a class of cell surface molecules (ie, H-2) that play critical roles in immune recognition processes. These data further suggest that the genes encoding Qa-2 and H-2 molecules have arisen from a common primordial gene.     

Antigenic Heterogeneity of Qa-2 Antigens in C57BL/6

The Qa-2 antigens are class I-like molecules encoded by genes mapped telomeric to the H-2D region on chromosome 17 in the mouse. A panel of 8 new monoclonal anti-Qa-2 antibodies derived from a C3H.KBR anti-C3H. SW immunization was studied. Immunoprecipitation of¹²⁵I-labeled C57BL/6 splenocyte antigens showed that all of these antibodies precipitated 40 kDa molecules which could be completely precleared by the monoclonal antibody 20-8-4, which had previously been shown to crossreact with Qa-2. One of the monoclonal antibodies (1-12-1), however, was found not to completely preclear Qa-2 antigens precipitable by the other 7 antibodies or by 20-8-4, suggesting the existence of at least two different species of Qa-2 molecules. Cell lines transfected with Q7 or Q9 genes were reactive with all 9 antibodies and the Qa-2 antigens expressed on surface membranes of these cells were completely precleared by both 20-8-4 and 1-12-1. Therefore, the observed heterogeneity of these molecules cannot be explained by an antigenic difference between the Q7 and Q9 gene products. 2D gel analyses showed identical pI spectra between Qa-2 molecules precipitated with 20-8-4 and 1-12-1. In addition, all of the monoclonal antibodies reacted with labeled antigen preparations following treatment with Endo F or neuraminidase, indicating that carbohydrate moieties are probably not responsible for the antigenic difference between the two species of Qa-2 antigen.     

H-2 unrestricted cytotoxic T cell activity against antigens controlled by genes in the QA/TLA region.

The specificity of H-2 unrestricted cytotoxic T cells was analyzed in secondary CML responses. A/J strain effector cells, sensitized against A.Tlab lymphoid cells, lysed target cells from strains with differing H-2 haplotypes but all sharing Qa-1b/Tlab alleles; whereas, target cells from strains with Qa-1a/Tlaa were not. When B6.Tlaa animals were in vivo-primed and challenged in vitro with B6 stimulator cells, no cytotoxic effector cell activity was generated. However, if B6.Tlaa animals were primed in vivo with A.BY cells and then rechallenged in vitro with either A.BY or B6 stimulator cells, cytotoxic effector cells were generated that lysed target cells from strains with Qa-1b/Tlab alleles. This suggests that factors in addition to Qa/Tla may play a role in the generation of anti-Qa/Tla effector cell activity. It was also noted that targets from strains with Qa-1a/Tlaa alleles were killed, although to a much lesser extent than the Qa-1b/Tlab targets. SWR anti-DBA/1 efffector cells strongly lysed target cells frrom strains with Qa-1b/Tlab, lysed Qa-1a/Tlaa targets to a lesser extent, and produced no cytotoxic effect on B6.Tlaa target cells. These data suggest that in addition to a CML target antigen associated with Qa-1b/Tlab, there may be an additional specificity recognized by cytotoxic T cells controlled by a gene outside of Qa-1b/Tlab.     

Qa-2 antigen encoded by Q7b is biochemically indistinguishable from Qa-2 expressed on the surface of C57BL/10 mouse spleen cells

Qa-2 was immunoprecipitated from the surface of 125I-labeled C57BL/10 (B10) mouse spleen cells and compared with Qa-2 immunoprecipitated from the surface of R1.1 thymoma cells transfected with Q7b. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that Qa-2 glycoproteins from both of these sources have a relative molecular mass of approximately 37 kDa. After treatment with endoglycosidase F, the Qa-2 polypeptide chains derived from C57BL/10 spleen and Q7b-transfected R1.1 cells displayed identical mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis because of removal of N-linked oligosaccharide residues. Furthermore, treatment of Qa-2 proteins from both sources with cyanogen bromide or alpha-chymotrypsin resulted in identical peptide fragmentation patterns. These results therefore provide a biochemical correlation between a cloned Qa-region gene produce expressed on the surface of transfected cells, and the Qa-2 glycoprotein on spleen cells that was described a decade ago by serologic methods.     

Hybrid resistance to BALB/c plasmacytomas. I. Resistance to MPC-11 controlled by a locus not linked to H-2.

Genetic control of hybrid resistance to the BALB/c plasmacytoma MPC-11 was investigated. The results indicate that a single dominant autosomal gene or gene complex, which segregates independently of H-2 and the coat color c and b-loci, controls resistance to this tumor. This gene has the same strain distribution pattern in the CXB Bailey recombinant inbred strains as three unlinked genes, H-2, Ly-4, and Ea-4. It is possible, therefore, that it could be linked to either of the latter two loci. Strains that carry a positive allele for resistance are C57BL/10 and all of its congenic resistant partners tested, C57BL/6, C57L, C57BL/Ks, AKR, and DBA/1. BALB/c and its congenic resistant partners are presumed to carry a negative allele of the gene for resistance to MPC-11. Strains such as SJL, DBA/2, and A and its congenic resistant partners, which form susceptible hybrids with BALB/c, could carry either the negative allele of the gene for resistance, like BALB/c, or could carry both a positive allele of the gene and some other gene conferring susceptibility on the hybrids. Heterozygosity within the H-2 complex increases resistance only in the presence of this non-H-2 linked gene for resistance, and the effect maps to the left of the H-2D region.     

F1 hybrid-anti-parental H-2b cell-mediated lympholysis: genetic control of target antigens by the H-2D region

The immunogenetic specificity of (C57BL/6 X DBA/2)F1 anti-parental C57BL/6 cytotoxic T lymphocytes (CTL) induced in primary mixed spleen cell cultures was determined in direct lytic and competitive inhibition assays. A large panel of peritoneal exudate cells (PEC) bearing nonrecombinant and recombinant H-2-Tla haplotypes was the source of target and inhibitor cells. All PEC of H-2b, H-2bc, H-2j, and H-2ja types, irrespective of background genetic constitution, were as susceptible to direct lysis as C57BL/6 PEC, but PEC of H-2a, H-2d, H-2k, H-2q, H-2s, and H-2u types were not. The possible involvement of the Tla region in controlling target antigens was excluded by testing PEC obtained from 4 H-2/Tla or intra-Tla recombinant mouse strains. The genes controlling target antigens were mapped to the D region with the aid of 9 intra-H-2 recombinants; for target PEC to be lysed it was necessary and sufficient that Db antigens be part of the H-2 phenotype. These results were confirmed by competitive inhibition assays. Resident peritoneal cells not exposed to fetal bovine serum were also lysed by F1 anti-parental H-2b CTL, a demonstration that target antigens are expressed on normal cells.     

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.     

Ly-m11: the H-3 region of mouse chromosome 2 controls a new surface alloantigen

Spleen cells from an SJL mouse immunized with B10.S spleen cells were fused with the nonsecretor myeloma line NS.1. One established hybrid cell line continuously secreted antibody that recognized a new antigenic specificity, tentatively called "Ly-m11." This newly found antigen is detectable on nearly 100 percent of spleen and lymph-node cells, 70 percent of bone-marrow cells, and 20 percent of thymus cells by direct cytotoxicity assays, and on the cells derived from kidney and liver. Strains that are Ly-m11 (+) include C57BL/6, C57BL/10J, B10.S, C57BR/cdJ, C57L/J, and C57BL/KsJ. Other mouse strains so far tested are Ly-m11 (-). The strain distribution pattern distinguished Ly-m11 from any known murine lymphocyte alloantigens, but it follows the H-3 alpha haplotype which is defined by skin transplantation. Linkage tests of nine congenic strains of H-3 and/or H-13/alpha loci and five recombinant inbred lines including CXB, BXH, AKXL, SWXL, and BXD revealed no recombinations between H-3 and Ly-m11 loci on chromosome 2. This newly discovered Ly-m11 alloantigen could itself constitute a minor histocompatibility antigen detectable by serological means.     

The minimal length of the differential segment in H-2 congenic lines

One hundred and four H-2 congenic lines were typed for alleles at seven loci, Qa-1, Qa-2, Tla, C3, Ce-2, Pgk-2, and Upg-1, residing distal to the H-2 complex. The results of the typing were used to estimate the length of the segment of chromosome 17 derived from the donor strain of each line—that is, the minimal length of the differential segment. The results indicate that only lines derived by intra-H-2 crossing-over in such a way that they inherited the right-hand portion of H-2 from the inbred partner have the telomeric half of chromosome 17 identical with that of the inbred-partner strain. In other lines the differential segment is at least 3 to 10 cM long. It is argued that in some lines the entire telomeric half of chromosome 17 might be of donor-strain origin.     

Genes of the H-2 complex regulate the antibody response to murine leukemia virus

The influence of the major histocompatibility complex of the mouse (H-2 complex) on the antibody response against murine leukemia virus (MuLV) was investigated after 3 different ways of virus presentation (milk transmission of virus, spontaneous virus activation, and immunization with inactivated virus). The antibody response against MuLV was measured in the sera of H-2 congenic C57BL V+ sublines (V+ denotes positive for milk-transmitted MuLV), (B10.AV+ X C57BL)F1 mice, (C57BL X AKR)F1 mice and of C57BL animals after immunization with inactivated AKR virus. The pattern of immune responsiveness of the different C57BL strains to MuLV was independent of virus replication and was similar for the 3 ways of virus presentation. Serum antibody levels were controlled by 2 genes within the H-2 complex. The first gene (Ir-MuLV-1) was located in the I-A region and was complemented by a second gene (Ir-MuLV-2), which was situated in the regions to the right of the I-B region. Presence of 2 responder alleles (Ir-MuLV-1+,2+) led to an optimal antibody response (H-2b haplotype). Animals that only expressed a responder allele in the I-A region (Ir-MuLV-1+,2-) were intermediate responders. Animals lacking a responder allele in the I-A region (Ir-MuLV-1-,2+ or Ir-MuLV-1-,2-) were low responders.     

Further biochemical data on Qa-2

The Qa-2 differentiation alloantigen is coded by a gene situated between the D and Tla loci of the murine major histocompatibility complex (H-2). Qa-2-bearing protein was isolated by immunoprecipitation and found to be composed of subunits of 40 000 and 12 000 daltons by SDS polyacrylamide gel electrophoresis (PAGE). The 12 000 dalton material was identified as 2-microglobulin (2M) by its molecular weight (SDS PAGE), charge (isoelectric focusing), antigenicity (reactivity with xenogenic anti- 2M), and genetics. The 40 000 dalton mol. wt. of Qa-2 heavy chain is 5 000 daltons less than that of D and K molecules (45 000 daltons). The quantity of Qa-2 isolated by immunoprecipitation was found to vary in a strain-specific fashion and as much as a 15-fold difference was observed.Abbreviations used in this paper B6 C57BL/6 strain mice - B10 C57BL/10 mice - 2M beta 2-microglobulin - IEF isoelectric focusing - K 1000 daltons - MHC major histocompatibility complex - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TL thymusleukemia antigen     

The differentiation of cytotoxic T cells in vitro. IV. Interleukin-2 production in primary mixed lymphocyte cultures involves cooperation between Qa-1+ and Qa-1- "helper" T cells

Studies with C57BL/6-TIaa mice have established that both Qa-1+ and Qa-1- helper T cells are required for the optimal production of Interleukin 2(IL-2) activity in primary MLC. This was established both by depletion of Qa-1 bearing cells by treatment of responder cells with anti-Qa-1 serum in the presence of complement and by positive selection (using FACS II analysis) of those cells displaying Qa-1. Furthermore, microfluoremetry revealed that the great majority of C57BL/6-TIaa and of A/J splenic T cells bore Qa-1 alloantigen but that only those with the highest antigen density were susceptible to complement-mediated lysis.     

Studies of two H-2Db mutants: B6. C-H-2bm13 and B6.C-H-2bm14

Two new C57BL/6 H-2 mutants, B6.C-H-2bm13 and B6.C-H-2bm14 are described. They arose independently in C57BL/6 as spontaneous mutations of the gain and loss type. Complementation studies map the mutations in both bm13 and bm14 to the H-2Db gene. However, these two mutant strains are not identical, but occurred as independent mutations at the same locus, as shown by reciprocal graft rejection and by the inability of the (bm13 X bm14)F1 hybrid to accept C57BL/6 grafts. Serological studies by direct testing (cytotoxicity and hemagglutination) and by quantitative absorption demonstrated a decrease in the H-2Db private specificity H-2.2 in both bm13 and bm14 when compared to C57BL/6. This was confirmed by SDS-PAGE analysis using antisera detecting the H-2.2 specificity. Attempts to produce antibodies to either the gained or lost specificities of the two mutant strains failed.     

Identification of a new CML target antigen controlled by a gene associated with theQa-2 locus

BALB/cBy anti-BALB/cJ spleen cells were tested in a secondary cellmediated lympholysis assay. The effector cells generated displayed a positive cytotoxic effect against Con A lymphoblasts from only those strains that were typed serologically as having theQa-2 ^a allele. Confirmation that the target antigen is controlled by a locus closely associated with or identical toQa-2 was obtained by the findings that target cells from B6.K2 (Qa-2 ^a,Qa-3 ^a) mice were lysed by the effector cells, while those from theQa-2, 3 congenic strain B6.K1 (Qa-2 ^b,Qa-3 ^b) were not. The fact that target cells from aQa-2-positive/Qa-3-negative strain (DBA/1,Qa-2 ^ai,Qa-3 ^b) were killed indicates that the target antigen is controlled, at least in part, by theQa-2 locus, not the Qa-3.There is no observedH-2 genetic restriction for this cytotoxic effect, since target cells which have theQa-2 ^a allele but differ from the stimulator cells at theH-2K, D, andI regions were lysed efficiently.     

The Tla locus: a new allele and antigenic specificity.

Four of 23 H-2 alloantisera screened for anti-TL activity contained such activity. One of these alloantisera, D-35, defined a new TL specificity, TL.5, and a new Tla allele, Tlad. TL.5 has all the characteristics of a TL antigen and has a different strain distribution than previously known ones. This new complexity at the Tla locus and the previous finding of other serologically defined genes in the Tla region indicate that this genetic region cannot be ignored in analyzing antisera produced in strains made congenic for the major histocompatibility complex.