BALB/c-H-2 db : A newH-2 mutant in BALB/cKh that identifies a locus associated with theD region

A newH-2 mutant, BALB/c-H-2 ^db , is described. This mutant originated in BALB/c, is inbred, and is coisogenic with the parental BALB/cKh strain. The mutation is of the loss type since BALB/c-H- ^db rejects BALB/c, but not vice versa. Complementation studies have localized the mutation to theD region of theH-2 complex. A cross between BALB/c-H-2 ^db and B10.D2-H-2 ^da failed to complement for either BALB/c or B10.D2 skin grafts, indicating that these are two separate mutations at the same locus (Z2). Direct serological analysis and absorption studies revealed that, with one exception, theH-2 andIa specificities of BALB/c and BALB/c-H-2 ^db are identical. In particular,H-2.4, the H-2D^d private specificity, is quantitatively and qualitatively identical in the two strains. The exception is that of the specificities detected by antiserum D28b: (k×r)F₁ anti-h, which contains anti-H-2.27, 28, and 29. These specificities appear to be absent from theH-2 ^db mutant since they are not detected directly or by absorption. Other public specificities are present in normal amounts,e.g., the reaction with antisera to H-2.3, 8, 13, 35, and 36. The reaction with antiserum D28 (f×k)F₁ anti-s, which contains antibodies to H-2.28, 36, and 42, is the same in both strains. Antiserum made between the two strains (H-2 ^db anti-H-2 ^d ) reacts like an anti-H-2 serum, in that it reacts with both T and B cells by cytotoxicity, but is not a hemagglutinating antibody. The serum reacts as does the D28b serum in both strain distribution and in cross-absorption studies. We conclude that theH-2 ^db mutation occurred at a locus in theD region, resulting in the loss of the H-2.28 public serological specificity and of a histocompatibility antigen. Whether these are one and the same antigen is not yet known. The data, in view of other evidence, imply that the public and private specificities are coded for by separate genes.Abbreviations used in this paper are as follows CML cell-mediated lysis - MLR mixed lymphocyte reaction - GVHR graft-versus-host reaction - RFC rosette-forming cells - RAM-Ig rabbit anti-mouse IgG     

Biochemical evidence for a separate,MHC-linked locus encoding H-2.28 antigens

In comparing the tryptic peptide maps of the H-2L and H-2D glycoprotein antigens isolated from NP-40 lysates of RADA1 (H-2 ^a ) leukemic cells, no more than 37% of the observed arginine-containing tryptic peptides are found to be homologous. Thus, the primary amino-acid sequences of these two antigens are probably less than 90% homologous. This constitutes the strongest evidence to date that the MHC-linkedH-2L region encodes H-2L antigens separately from theH-2D region, even though H-2L antigens bear D-end-associated antigenic determinants of the H-2.28 family. The anti-H-2.28 alloantiserum (k×r anti h2) used to precipitate H-2L antigens in this investigation was the NIH contract antiserum D28b. As the tryptic peptide maps also surprisingly revealed, D28b precipitates H-2D antigens as well and, thus, anti-H-2.4 immunoadsorbants were employed to isolate H-2L free of H-2D antigens. In light of the dual specificity of D28b, its reactivity with BALB/c-H-2 ^dm2 mutant cells was re-examined. Even though mutant lymphocytes, which lack H-2L but not H-2D antigens, are not cytotoxically lysed by D28b (as are parental H-2^d cells), D28b appears to precipitate H-2D antigens from NP-40 extracts of mutant splenocytes.     

The relationship between theH-2 loss mutations ofH-2da andH-2db in the mouse

The mouse strain B10.D2-H-2^da carries the mutantH-2^da allele, derived after chemical induction, and this has been shown to be a gain and loss mutation involving theH-2D^d locus.BALB/c- H-2^db, derived spontaneously, is a loss mutation only, and appears not to involve theH-2D^d, but rather theH-2L^d locus. The two mutations effectboth graft rejection and serologically detected H-2 specificities (Type II mutation). In the experiments described in this study, theloss mutations in theH-2^da andH-2^db mutants have been compared by skin grafting, and by direct and absorption serological techniques: (1) By skin grafting, using the well established complementation method, it has been shown thatH-2^da andH-2^db do not complement each other, i.e., the mutation in both occurred at the same locus. However, by appropriate selection of donor and recipient, it has become clear thatH-2^da had a greater loss than didH-2^db, althoughH-2^da includes the loss found inH-2^db. (2) Serological studies have demonstrated that H-2D.4 was altered inH-2^da, but not inH-2^db; H-2.28 (detected by D-28b and D-29) was decreased or lost in both mutants;H-2^db anti-BALB/c failed to react withH-2^da; both mutants reacted similarly with D-28 sera. In addition, sera made usingH-2^da as donor did not contain an anti-H 2.28 antibody. The loss mutation involvingH-2^da therefore appears to have led also to the loss of H-2.28 as found inH-2^db. We conclude that theH-2^da strain arose after a complex mutation or recombination event which involvedboth theH-2D^d locus and the closely linkedH-2L^d locus, whereasH-2^db affects only theH-2L locus.     

The blocking activity of antisera raised to either tumor antigens or alloantigens in cytotoxicity assays using syngeneic or allogeneic killer cells on the P815 murine mastocytoma target

Methods have been published whereby a tumor-specific antigen associated with membranes of the P815 mastocytoma of DBA/2J mice was purified. Antiserum, raised in rabbits, to this material demonstrated specificity for P815 as opposed to other cells or materials of DBA/2J origin when tested by either complement-mediated target cell lysis or the enzyme-linked immunosorbent assay ELISA. This antiserum was tested for its ability to block killing by in vitro raised syngeneic lymphocytes cytotoxic for P815. It was found that this antiserum as well as antiserum raised in rabbits to normal DBA/2J membrane components and anti-H-2^d antiserum (raised in congenic mice) were all able to block killing when ⁵¹Cr-labeled P815 targets were pretreated with these antisera. On the other hand, only the anti-DBA/2 serum and the anti-H-2^d serum were capable of slightly blocking syngeneic killing of L1210 cells. Similarly, C57B1/6 cytotoxic lymphocytes raised against DBA/2 cells were blocked by pretreatment of ⁵¹Cr-labeled P815 targets with the rabbit anti-DBA/2 serum and the anti-H-2^d serum but not by the anti-P815 serum. The implications of these observations are discussed.     

Molecular heterogeneity of D-end products detected by anti-H-2.28 sera

In capping experiments with peripheral T lymphocytes, two anti-H-2.28 sera (AKR anti-AKR.L, anti-K^b, and C3H anti-0H.B10, k anti-b) that do not contain any Qa-2-specific antibodies are able to redistribute not only the H-2.28-positive H-2 molecules, but also Qa-2 molecules. This is due to the capacity of these sera to react with Qa-2 molecules because on cells where all known molecules of the H-2 ^d haplotype were capped (K1^d, K2^d, D^d, M^d, L^d, L2^d), both antisera still reacted when the cells came from a Qa-2 positive D^d strain (B10.A) but not when the cells were of Qa-2 negative strain (BALB/cByA). The reaction with la and non-H-2 antigens was excluded in these experiments. These data show that Qa-2 and H-2 antigens share some specificities of the H-2.28 family. Other anti-private and anti-public anti-H-2 sera failed to react with the Qa-2 molecules.     

Antigen-specific helper T cells recognize determinants encoded in the H-2D region of the H-2 gene complex

Observations have frequently been interpreted as showing that the helper T cells which collaborate with alloantigen-specific cytotoxic T-cell precursors can only recognize antigens encoded in the I region of the H-2 gene complex. An experimental system is described here that allows analysis of the recognition repertoire of these helper cells. CBA helper T-cell precursors can be primed in vitro to antigens encoded in the H-2 ^b gene complex. These helpers can then be tested for the existence of a subset of helper cells which recognize antigens encoded in the D region of H-2 ^b haplotype. CBA thymocytes were used as a source of cytotoxic T-cell precursors that respond poorly in the absence of exogeneous helper activity. The source of alloantigen was varied by using irradiated spleen cells from various (BALB/c × recombinant)F₁ hybrid mice as stimulator cells. When the stimulator cell bears BALB/c determinants recognized by the cytotoxic T-cell precursor and also bears only the D region antigens of the H-2 ^b haplotype, an anti-BALB/c cytotoxic response is generated only if the anti-H-2^b helper population contains cells able to recognize H-2D^b. A positive cytotoxic response was obtained, indicating that helper cells are not limited to recognition of I region antigens and can efficiently recognize antigens encoded in the D region of the H-2 gene complex. This was confirmed by the demonstration of helpers specific for H-2D^d. We were unable to detect any evidence for Ia-restricted recognition of the H-2D alloantigens, suggesting that, as for cytotoxic T lymphocytes (CTL), helper cell recognition of class I alloantigens is an unrestricted event.     

Expression of alien histocompatibility antigens on SJL/J tumors detected by cell-mediated and serological analyses

Summary Reticulum cell sarcoma (RCS) cells of SJL/J (H-2^s) mice have been shown to express antigens that are cross-reactive with allogeneic cells of the H-2^d and H-2^b haplotypes by cell-mediated cytotoxicity, antibody-mediated cytotoxicity, immunofluorescence, and quantitative absorption assays. These alien antigens have been detected on both spontaneous and in vivo- and in vitro-passaged RCS cells to varying degrees.The in vitro cell lines were able to stimulate a syngeneic cytotoxic T cell response detected in a 4-h ⁵¹Cr release assay. The cytotoxic cells reacted with in vitro RCS tumor targets but not with in vivo or spontaneous RCS tumors. Furthermore, the cytotoxic cells lysed H-2^d and to a lesser extent H-2^b target cells, but not H-2^k, H-2^p, or H-2^r cells. The cross-reactivity was also observed with SJL/J anti-BALB/c cytotoxic cells, which can lyse in vitro RCS targets effectively. The in vivo tumors were not stimulatory in cytotoxic responses and did not serve as targets.H-2^d specificities were also detected in cultured RCS tumor cells by cytotoxic antibody. Both allogeneic SJL/J anti-BALB/c, C57B1/6 anti-BALB/c sera reacted with RCS tumor cells and not normal SJL/J cells. Furthermore, monospecific D^d sera were also cytotoxic against RCS lines. The cytotoxic activity could be absorbed by BALB/c cells and RCS cells but not with normal SJL/J cells. The H-2^d specificities were also detected on the in vivo lines by indirect immunofluorescence. The majority (60%) of spontaneously arising tumors expressed either H-2^d or H-2^b allospecificities in the immunofluorescence assays. Although these antigens may not be inappropriate for the SJL/J strain, their differential expression on tumor cells may be significant in the etiology of the tumor.     

Biochemical evidence for structurally distinct H-2Dd antigens differing in serological properties

H-2D^d antigens, as defined by the private H-2.4 determinant, exist as two immunochemically distinct populations in H-2^a and H-2^dm2 splenocytes and in the transformed cell line, RADA1(H-2 ^a). The two populations are distinguishable by the anti-H-2.28 serum, k/r anti-h2, which is directed, in part, against the H-2.28 family of public determinants encoded by the D end of the b haplotype. Sequential precipitates of lentil-lectin-purified glycoprotein extracts metabolically labeled with radioactive amino acids reveal that approximately one-quarter to one-third of the H-2D^d antigens, designated H-2D^d (b28 ⁺), react with this antiserum, whereas two-thirds to three-quarters, designated H-2D^d(b28^–), do not. Paired-label tryptic peptide maps in this and a previous study indicate that H-2D^d(b28⁺) and H-2D^d(b28 ^–) are closely related structurally and are more likely to represent modified forms of the same gene product rather than products of different genes, although the existence of closely related genetic loci is not rigorously excluded. Together, H-2D^d(b28⁺) and H-2D^d(b28^–) have a radioactivity level seven times higher than H-2L^d, which also reacts with the anti-H-2.28 serum but which lacks the H-2.4 determinant. As yet unresolved, however, is the question of whether the observed quantitative differences between these three antigens reflect actual molar differences at the cellular level, or whether the variation is the result of metabolic or compositional factors. In any case, a complex serological and structural relationship is found to exist between antigens encoded by the D/L end of the MHC.     

Complex genetic effect of B10.D2 (M504) (H-2dm1) mutation

Serological and capping experiments show that the strain B10.D2 (M504) carrying the mutant haplotypeH-2 ^dm1 has two molecules in the products of theD region: H-2D^dm1 and H-2L^dm1 which are detectable by anti-H-2.4 and by anti-H-2.28 sera, respectively. Both these molecules differ serologically from the H-2D^d and H-2L^d molecules of the original (nonmutant) strain B10.D2. A third molecule, different from H-2D and H-2L, was detected inH-2 ^d ,H-2 ^dm2 but not inH-2 ^dm1 products.     

Anti-H-2 antibodies induced by syngeneic immunization

Alloreactive cytotoxic antibodies were induced in BALB/c mice by syngeneic immunization with normal lymphoid cells. Sixteen out of 41 mice produced antibodies with distinct anti-H-2 specificity. Anti-K^k antibodies were present in all positive sera, but the individual sera produced different reactivity patterns when tested on a panel ofH-2 haplotypes. Absorption and immunoprecipitation experiments confirmed the H-2 specificity of the syngeneic sera. We hypothesize that virus-modified H-2^d structures have triggered alloreactive B-cell clones to produce anti-H-2 antibodies.     

Herpes-simplex-virus-specific,H-2Dk-restricted T lymphocytes bear receptors for H-2Dd alloantigen

Cytotoxic T lymphocytes generated in the course of an HSV-infection of CBA (H-2 ^k ) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected target cells expressing the D^d alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2D^k determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for D^d alloantigen could be positively selected on H-2^d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing D^d alloantigens and syngeneic HSV-infected target were lysed with equal efficiency. The results imply that the phenomenon of H-2-restricted versus nonrestricted T-cell reactivity is not due to distinct T-cell subsets, but rather is dependent on the antigeneic determinants recognized.     

MHC epitopes of Ks and Dd restrict the same population of cytolytic T lymphocytes

When A. SW (H-2 ^s) mice are immunized with B10.S (H-2 ^S) epidermal cells, cytolytic T lymphocytes are evoked that efficiently lyse B10.D2 (H-2 ^d) as well as B10.S target cells. Immunogenetic analysis of this apparent H-2-unrestricted killing revealed that the most plausible explanation was a sharing of an H-2-restricting epitope by H-2K^S and H-2D^d molecules.     

H-2 antigen variants in a cultured mouse leukemia cell line

We have investigated the effect of immune selection against a single gene product on a cultured mouse Friend leukemia cell line. The clonal cell line used is heterozygous at theH-2 complex and expresses theH-2 ^d andH-2 ^b haplotypes. The genes selected against were theH-2K locus alleles. Variants were obtained after a single-step selection using either antiH-2K^b or anti-H-2K^d serum. The phenotypes of the variants obtained showed an interesting asymmetry between the two haplotypes. Selection against theH 2K ^b allele resulted in the isolation of the two expected types of variant-those that had lost only H-2K^b and those that had lost both H-2K^b and the linked H-2D^b. Selection against H-2K^d yielded, exclusively, variants that had lost both the selected antigen and the linked H-2D^d. None of the variants showed an alteration in expression of antigens intrans configuration. Karyotypic analyses of the variants revealed that all the cells had retained both copies of chromosome 17 present in the wild-type cells. The results suggest that the variants did not emerge through chromosome loss.     

Isolation and characterization of an isoantigenic variant from a heterozygous mouse lymphoma in culture

A cell line from a mouse lymphoma heterozygous at the chromosome region for the H-2^d and H-2^k alleles was originally obtained from a transplantable lymphoma in the (C3H × DBA/2)F₁ hybrid (H-2^d/H-2^k) and cultured in vitro. The original cultured line, termed parent line, was susceptible to the cytotoxic action of antibodies directed against antigenic components of both the d and k alleles. The parent line also absorbed hemagglutinins from both anti-d anti-k antisera. A resistant, variant subline was selected from the original population by immunoselection in vitro with anti-H-2^d antibody and complement in a cytotoxic system. After one year in continuous culture in the absence of selecting antisera, the variant subline was still resistant to the cytotoxic action of anti-H-2^d antibody. Serologic analysis of the variant indicated that it had lost the D antigenic component of the d allele, had a reduced amount of the H component, controlled by both the d and k alleles, and had retained the K component of the k allele. Possible genetic mechanisms that might account for the emergence of the variant line are discussed. While the results do not necessarily support an analysis based on mitotic recombination, ascribing other mechanisms is also difficult because of aneuploidy in the cell line. Finally, the experiments point out the advantages of using in vitro immunoselective methods in the genetics of mammalian somatic cells.     

Dissociation ofH-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2 ^b /H-2 ^b ). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (allH-2 ^b /H-2 ^b ), FY7 failed to induce the primary in vitro generation of anti-H-2^b CTL by (B10.A x A)F₁ (H-2 ^a /H-2 ^a or (B10.D2 x BALB/c)F₁ (H-2 ^d /H-2 ^d ) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2^b CTL. Quantitative absorption tests with H-2K^b and H-2D^b antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2K^b product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2K^b CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2K^b-gene product expressed by FY7.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MHC major histocompatibility complex - MLC mixed lymphocyte culture - PAGE polyacrylamide gel electrophoresis     

Unexpected complexity of the dm1 mutation revealed in the structure of three H-2D/L-related antigens

The H-2L^dm1 and H-2D^dm1 MHC antigens of the B10.D2 (H-2 ^dm1 ) mutant mouse strain (formerly known as M504 or H-2 ^da ) have been compared to the H-2L^d and H-2D^d antigens of the B10.D2 (H-2 ^d ) mouse strain. L^dm1 and L^d are 45 000 M_r antigens and both are reactive with anti-H-2.28 (k/r anti-h2) serum and unreactive with anti-H-2.4 (k/b anti-a) serum which detects private determinants of the D^dm1 and D^d antigens. However, the tryptic peptide compositions of these two antigens are different and, based on the number of major tryptic peptides which coelute during ion-exchange chromatography, the estimated peptide homology between L^dm1 and L^d is 80 percent. A newly defined antigen (M_r = 39 000), designated gp39^dm1, was found in glycoprotein extracts of the ^dm1 strain but not of the d strain. This antigen coprecipitates with L^dm1 but does not coprecipitate with D^dm1 indicating that it lacks the H-2.4 determinant. In comparison with L^dm1, gp39^dm1 appears to contain far fewer Arg and Lys residues and is most likely not a simple proteolytic fragment of L^dm1. Finally, peptide maps of the D^dm1 antigen show that the majority of its Arg peptides are identical to D^d Arg peptides, whereas at least five of its Lys peptides and three of its Arg peptides correspond not to D^d peptides but to L^d and L^dm1 peptides. These data raise the possibility that the D^dm1 antigen is a hybrid molecule and they have also revealed an unexpected level of complexity in the dm1 mutant phenotype.     

Polymorphism of minor histocompatibility genes in wild mice

H-2^b-restricted cytolytic T lymphocytes (CTL) were generated against H-1, H-3, and H-4 antigens and tested against target cells of F₁ hybrids between wild mice and inbred H-2 ^b mice. The congenic strain combinations for the CTL production were such that they tested one allele each at the H-1 and H-4 loci and four alleles at the H-3 locus. Most of the wild mice tested came from Southern Germany, but a few mice came from other European countries and Egypt and Israel. Virtually all wild mice typed as positive with CTL directed against H-3^b and H-4^b antigens; 32% of the F₁ hybrids tested reacted with anti-H-1^cCTL and 9% reacted with anti-H-3^d CTL. The positive results were not caused by cross-reaction with allogeneic H-2 antigens controlled by the major histocompatibility complex (Mhc) genes of the wild mice. At least some of the H-3 and H-4 antigens detected by the CTL in the F₁ hybrid were not identical with antigens of the immunizing strains. These results suggest a relatively low degree of polymorphism of the tested minor H loci in wild mice and further support the notion that minor H loci are unrelated to the Mhc.     

Interacting genetic factors controlling the antibody response against the H-2.2 specificity

The antibody response against the H-2.2 specificity has been studied in three H-2 ^d strains, B10.D2, DBA/2, and BALB/c, and their hybrids (B10.D2 × DBA/2)F₁ and (B10.D2 × BALB/c)F₁. The genetic control of the response appears to be complex: The three pure strains are responders, whereas both hybrids when immunized with C3H-HTG are nonresponders. Individual analysis of N3 offspring is compatible with the idea that, in this combination, an Ea-4 incompatibility between donor and immunized strain is necessary for the anti-H-2.2 response to occur. H-2 ^d /H-2 ^k hybrids (B10.BR × B10.D2)F₁ or (B10.BR × DBA/2)F₁ are responders when immunized with C57BL/10 (H-2 ^b ) but not with B10.A(2R) (H-2 ^h ), indicating that simultaneously recognized H-2 specificities are necessary for the anti-H-2.2 response.     

Teratocarcinoma transplantation rejection loci: Genetic localization of the Gt-1 locus on chromosome 17 and the expression of alternate alleles

A series of congenic mice on the BALB/c genetic background have been employed to localize a teratocarcinoma transplantation rejection locus, Gt-1, to the K side of the H-2 locus on chromosome 17. Previous studies have placed the Gt-1 ^sv allele about 8 centimorgans away from the H-2 ^b or H-2 ^bv1 locus. Teratocarcinomas derived from 129/sv mice, Gt-1 ^sv (H-2K ^bv1/H-2D ^bv1), are rejected by BALB/c (H-2K ^d/H-2D^d) and BALB-G mice (H-2K ^d/H2D-^b, but form tumors in BALB-B (H-2K ^b/H2D ^b) and BALB/5R5 mice (H-2K ^b/H2D ^d). In the reciprocal tumor-rejection test, a BALB/c teratocarcinoma was rejected by immunized BALB·B mice, but formed tumors in the immunized isogenic BALB/c mouse. These studies demonstrate the reciprocal expression of two Gt-1 alleles, one Gt-1 ^c, in BALB/c mice, and the other, Gt-1 ^sv, in the congenic BALB·B mice. Shedlovsky and co-workers have placed the Gt-1 locus in a similar location on the K side of the H-2 locus on chromosome 17.     

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.