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.     

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.     

Expression of HLA-B27 in transgenic mice is controlled by gene(s) mapping between H-2D and H-2L loci

The level of HLA-B27 transgene expression on the cell surface is dependent on the host H-2 haplotype. Mice homozygous for the H-2 ^b , H-2 ^f , H-2 ^f , H-2 ^p , H-2 ^r , and H-2 ^k haplotypes express B27 at high levels. An intermediate level of B27 expression is observed in H-2 ^v mice whereas low levels of B27 are expressed in H-2 ^q and H-2 ^d mice. The decreased expression of B27 maps to the D region of the major histocompatibility complex. Recombinant strain B10.RKDB (D^dL^b) mapped the low expression gene centromeric to H-2L. In order to determine the low expression within the H-2D region, the B27 transgene was introduced into B10.D2-H-2 ^dm1 and BALB/c-H-2 ^dm2 mice. Expression of B27 in both of these strains was high indicating that neither H-2D ^d nor H-2L ^d is responsible for the low expression. This maps the effect between the H-2D and H-2L loci. In addition, introduction of human ₂-microglobulin (₂m) into B10.D2-B27 transgenic mice caused a marked enhancement of B27 expression on the cell surface suggesting that the defect in B27 expression in certain haplotypes is due to an inability of B27 to associate with endogenous mouse ₂m. We propose that gene(s) mapping between D and L (either D2, D3, D4, or some as yet unidentified gene) may be involved in class I assembly by helping association of ₂m with class I. This putative molecule, designated Assembly Enhancer (AE) might have a negative influence in the association between human class II and mouse ₂m.     

A third class I major histocompatibility complex antigen encoded by a gene in the D region of the H-2 d haplotype recognized by cytotoxic T lymphocytes

The D region of the H-2 ^d haplotype contains five class I genes: H-2D ^d , D2 ^d , D3 ^d , D4 ^d and H-2L ^d . Although previous studies have suggested the presence of D-end encoded class I molecules in addition to H-2D^d and H-2L^d, segregation of genes encoding such molecules has not been demonstrated. In this report we have used cãtotoxic T lymphocytes (CTL) to examine the D region of the H-2 ^d haplotype for the presence of additional class I molecules. CTL generated in (C3H × B6.K1)F₁ (K ^k D ^k , K ^b D ^b ) mice against the hybrid class I gene product Q10^d/L^d expressed on L cells cross-react with H-2L^d but not H-2D^d molecules, as determined by lysis of transfected cells expressing H-2L^d but not H-2D^d. Although H-2L^d-specific monoclonal antibodies (mAb) completely inhibit H-2L^d-specific CTL from killing B10.A(3R) (K ^b D ^d L ^d ) target cells, only partial inhibition of anti-Q10 CTL-mediated lysis was observed, suggesting the presence of an additional D-end molecule as a target for these latter CTL. To identify the region containing the gene encoding the Q10 cross-reactive molecule, we show that anti-Q10 CTL lyse target cells from a D-region recombinant strain B10.RQDB, which has H-2D ^d , D2 ^d , D3 ^d , D4 ^d , and H-2D ^b but not the H-2L ^d H-2 ^d , and H-2L ^d (including D2 ^d , D3 ^d , and D4 ^d , lacks this anti-Q10 CTL target molecule. Together, these data demonstrate that a class I gene mapping between H-2D ^d and H-2L ^d encodes an antigen recognozed by anti-Q10 CTL. A likely candidate for this gene is D2 ^d , D3 ^d or D4 ^d .     

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.     

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.     

Bone-marrow-cell grafts involving theH-2D andH-2L mutant haplotypes

TwoH-2 ^d mutants,H-2 ^dm2 (H-2L loss mutation) andH-2dm1 (gainplus-loss mutation involving bothH-2L andH-2D) were evaluated for any change in the immunogenicity of marrow stem cells. Grafts of 2 or 4 × 10⁶ BALB/c(C) or BALB/c-H-2^dm2 (C-H-2 ^dm 2) marrow cells were accepted by lethally irradiated B10.D2(H-2 ^d ) recipients and were rejected by irradiated B10(H-2 ^b ) recipients. Moreover, both (B6 × C)F₁ and (B6 × C-H-2 ^dm 2)F₁ mice, as irradiated recipients, resisted the growth of parental-strain B6(H-2 ^b ) marrow cells but accepted grafts from C or C-H-2 ^dm 2 parental-strain donors. Thus, theH-2 mutation involvingH-2L but notH-2D did not affect the expression ofH-2 ^d -associated Hemopoietic or Hybrid(Hh) antigens of marrow stem cells. Grafts of 2 to 8 × 10⁶ B10.D2 or B10.D2-H-2 ^dm 1 marrow cells were rejected by B10.BR(H-2 ^k ) and B6 hosts and were accepted by B10.D2 hosts. However, B10.D2-H-2 ^dm 1 marrow cells grew to a much greater extent than B10.D2 cells in irradiated (B6 × B10.D2)F₁ or (B6 × B10.D2-H-2 ^dm 1)F₁ host mice. Therefore, theH-2 ^dm 1 mutation has altered the expression of Hh antigens at least quantitatively, resulting in a relative loss of hybrid resistance with the retention of Hh determinants recognized by allogeneic recipient mice which are notH-2 ^d . Since the Hh determinants of B10.D2 marrow cells have been mapped 16 cM to the right ofH-2, this mutation atH-2D/H-2L may have affected a regulatory gene.     

Synthesis of a defective H-2 histocompatibility antigen by mutant mouse strain BALB/c-H-2 dm2

Mutant mouse strain BALB/c-H-2 ^dm2 (dm2), which fails to express the H-2L^d histocompatibility antigen associated with the wild type, BALB/c, synthesizes instead a smaller molecule that is structurally related to H-2L^d but does not carry detectable alloantigenic determinants. This new protein, p40, is a membrane glycoprotein found in dm2 cells but not in BALB/c. p40 was detected by electrophoresis of dm2 glycoprotein preparations and by immunoprecipitation with heterologous H-2-specific antibodies. The p40 molecule is found associated with intracellular membranes but was not detected at the cell surface. Peptide mapping studies suggest that dm2 carries an alteration in the H-2L ^d structural gene, which prevents proper maturation of the protein product.     

Fine specificity of cytotoxic T lymphocytes directed againstH-2L d

The fine specificity of cytotoxic T lymphocytes (CTL) directed againstH-2L ^d was analyzed by studying the lytic activity of BALB/cH- 2^dm2 (H-2L ^d loss mutant) anti-BALB/c-H-2 ^d CTL, generated in secondary mixed lymphocyte culture (MLC) against a panel of target cells of differentH-2 haplotypes. Target cells of allH-2 haplotypes tested, except that of the MLC responder, were lysed by anti-L^d CTL, although to a widely varying extent. The genes coding for antigens detected by anti-L ^d CTL were mapped to distinct regions in theH-2 ^d ,H- 2^dm1,H-2 ^q ,H-2 ^k , andH-2 ^b haplotypes. The sequence of lysis intensity against the variousH-2 haplotypes and theH-2 regions involved were as follows:L ^d ,D ^q L ^q ,D ^dm1 L^dm1,K ^k ,D ^b L ^b ,r, p, f, s, C3H.OH (K ^d D ^k L ^k ), strong lysis occurring againstL ^d and weak lysis againstH-2 ^s and C3H.OH.By monolayer adsorption and cold target inhibition experiments, it was shown that anti-L ^d CTL contained a CTL subset directed against a private L^d specificity, hitherto undetected by anti-L ^d antibodies. This subset of CTL was separate from the CTL subsets reacting againstH-2 ^q and against the mutant haplotypeH- 2^dm1. The reactions against the latter two haplotypes were also mediated by separate CTL subsets. It is concluded that the L^d molecule, to a varying extent, shares target antigens for CTL with K- and/or D-end H-2 molecules of all haplotypes tested. These antigens are detected by multiple subsets of anti-L ^d CTL. One CTL subset is directed against a target structure unique forL ^d (L^d private specificity).     

Consequences of a single Ir-gene defect for the pathogenesis of lymphocytic choriomeningitis

The H-2L ^d allele has been identified by others as the sole Ir gene in the H-2 ^d haplotype for the cytotoxic T lymphocyte (CTL) response to mouse lymphocytic choriomeningitis virus (LCMV). The BALB/c-H-2 ^dm2 (C-H-2 ^dm2 ) mutant lacks H-2L ^d , and thus should be ideal for assessing the contribution of virus-immune CTL to LCM immunopathology. Comparison of the C-H-2 dm2 mice with congenic BALB/c mice revealed that there is a delay of about 24 h in the onset of severe inflammatory process and symptoms in the mutant strain, but the absence of H-2L ^d did not prevent the later development of fatal disease in mice injected intracerebrally (i.e.) with neurotropic LCMV. This could indicate that virus-immune CTL are not the major mediators of clinical LCM. Spleen cells from LCMV-primed BALB/c mice did not show CTL activity for LCMV-infected C3H.OH, C-H-2 ^dm2 , or (CBA × C-H-2 ^dm2 )F₁ target cells. However, immune lymphocytes from both the mutant and the F₁ strains lyse virus-infected BALB/c cells. Furthermore, BtO.HTG and, in some experiments, B10.A(5R) mice generated CTL lytic for LCMV-infected BALB/c, C-H-2 ^dm2 , and (CBA × CH-2 ^dm2 )F₁ macrophages. Apparently H-2L ^d is immunodominant in the H-2^d restricted response to LCMV. However, in the absence of H-2L ^d , it seems that H-2K ^d and, to a lesser extent, H-2D ^d also serve as Ir genes for the CTL response in this infection. Even so, the absence of the H-2L^d-restricting element results in a disease process which is either delayed in onset or less severe.     

Serological characterization of previously unknown H-2 molecules identified in the products of theK d andD k region

The molecular relationship between H-2 private and some public specificities was studied in C3H.OH (H-2 ⁰² ) mice using surface-antigen re-distribution methods. Besides the K^d- and D^k-region antigens, which can be capped by antisera against the private and public specificities characteristic for a given allele, a previously unknown type of molecule was found in the products of both theK ^d andD ^k regions. These can be capped by the respective anti-private serum but not by antisera against some public specificities. The two K^d-region molecules are provisionally named H-2K1^d and H-2K2^d. We detected them onH-2 ⁰² (K ^d ,I ^d ,S ^d ,D ^k ) and also onH-2 ^dx (K ^d ,I ^f ,S ^f ,D ^dx ) T lymphocytes. Similarly, the two types of molecules detected on the products of theD ^k region are provisionally named H-2D1^k and H-2D2^k. The serological characteristics of these molecules are described. When compared with the products of theD ^d region, in which we previously described three different molecules (H-2D^d, H-2M^d, and H-2L^d), the mutual relationship between H-2K1^d and H-2K2^d as well as between H-2D1^k and H-2D2^k appears to be similar to that between H-2D^d and H-2M^d. In the absence of relevant recombinants or informative biochemical data, it is, however, difficult to establish homology between molecules produced by differentK- andD-region alleles.     

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.     

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.     

Immunogenetic analysis ofH-2 mutations

A.BY, B10.LPa, and B10.129(5M) mice were presensitized in vivo against B10.A(5R) cells and then restimulated in vitro by the same cells in the standard CML assay. The effector cells thus generated lysed not only B10.A(5R), but also C57BL/6 targets, indicating that, in addition to anti-H-2D_d response [measured on the B10.A(5R) targets], response to minor histocompatibility (H) antigens (measured on the C57BL/6 targets) also occurred. The latter response was directed against multiple minor H antigens in the case of the A.BY effectors, and against H-1 and H-3 antigens in the case of B10.129(5M) and B10.LPa effectors, respectively. The sensitization against minor H antigens occurred in the context of H-2K^b H-2D^d antigens, but by testing the response on C57BL/6 targets, only cells reacting with minor H antigens in the context of H-2K^b were assayed. The same effector cells were then tested against H-2^b mutant strains, in which theH-2K ^b allele was replaced by a mutant one. All three effector types [A.BY, B10.LPa, and B10.129(5M)] behaved in a similar way: they all reacted with theH-2 ^bg1 mutant to the same degree as withH-2 ^b, they did not react at all or reacted only weakly with theH-2 ^bd andH-2 ^bh mutants, and they reacted moderately or strongly with theH-2 ^ba mutant. The degree of crossreactivity with the mutants reflects, with one exception, the degree of relatedness of these mutants toH-2 ^b, as established by other methods. The one exception is theH-2 ^ba mutant, which is the most unrelated toH-2 ^b, and yet it crossreacted strongly. Further testing, however, suggested that in this instance the crossreactivity was probably directed against H-2 antigens: the anti-H-2D^d effectors apparently crossreacted with the H-2K^ba antigens. This finding is an example of cell-mediated crossreactivity between the products of two differentH-2 genes (H-2K andH-2D). It is also an example of anH-2 mutation generating an antigenic determinant known to be present in another strain.     

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     

Multiple CTL subsets generated by H-2.L locus products stimulation: evidence for an antigenic determinant private to H-2.Ld

Analysis of the fine specificity of CTL subpopulations raised by an H-2.L locus products stimulation (H-2dm2 anti-H-2d) was performed by absorption experiments by using monolayers of macrophages of H-2m, H-2q, H-2b, and H-2k haplotypes. The results show the existence of four CTL subsets. The pattern of reactivity of three of them could be correlated with that of antibodies present in H-2dm2 anti-H-2d antisera (anti-H-2.64, anti-H-2.65, and anti-H-2.Kk). The fourth CTL subset reacted with a specificity unique to H-2.Ld molecules (a private specificity?), absent on cells from H-2m, H-2q, H-2b, and H-2k haplotypes, and undescribed as yet by serologic methods. These data support the hypothesis that the H-2.L locus products are comparable in their antigenic properties to those of the H-2.K and H-2.D loci.     

Quantitative variation in H-2-antigen expression

Minor differences in the expression of individual H-2K and H-2D antigens were detected on mouse spleen cells. The method involved the use of an¹²⁵I-protein A radioimmunoassay using highly specific anti-H-2 sera to make estimates of the number of cell-bound antibody molecules. The maximum number of antibody binding sites varied for each H-2 antigen reflecting differences of between 10 and 70 percent in the expression of any two antigens. The order of magnitude of expression was D^b>(K^d)=K^k=K^b=D^q>D^d>K^q>D^k. Minor background differences were detectable, but antigen expression was allele-specific and independent of the expression of other K, D or I antigens expressed on the same cell.     

Cytolytic T cells activated by H-2-controlled E molecules cross-react with A molecules

Cell-mediated lymphocytotoxicity was generated in four strain combinations differing only by the cell-surface expression of the class II E molecule controlled by the H-2 complex. The four combinations were: B10.D2(R107) anti-B10.A(3R), B10.A(4R) anti-B10.A(2R), B10.GD anti-B10.D2(R101), and B10.S(7R) anti-B10.S(9R). In all four of these combinations, the stimulator expresses E molecules on the cell surface, while the responder does not. The cytolytic T lymphocytes generated in the B10.D2(R107) anti-B10.A(3R) and B10.A(4R) anti-B10.A(2R) combinations reacted not only with the stimulator but also with strains that do not express cell-surface E molecules, in particular, strains carrying the H-2 ^f and H-2 ^q haplotypes. The cross-reactivity with E-negative strains could be blocked by monoclonal antibodies specific for the A^f or A^q molecules but not by antibodies recognizing determinants on E or class I (K) molecules. The anti-H-2^f cross-reactivity could be inhibited by H-2 ^q cold targets and, reciprocally, the anti-H-2^q reactivity could be blocked by H-2 ^f cold targets. These findings are interpreted as indicating that the cytolytic T lymphocytes stimulated by E molecules can recognize and lyse cells lacking E molecules but expressing A molecules. The observed E-A cross-reactivity supports the notion of structural and functional relatedness between the A and E molecules and suggests a common evolutionary origin of the A- and E-encoding loci.     

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     

The spatial relationship of the viral and H-2 antigens recognized by anti-viral CTLs

With the use of monospecific rabbit anti-G protein and mouse monoclonal anti-H-2K^k, we have analyzed the spatial relationship of the serologically defined H-2K^k antigens and the major surface glycoprotein (G protein) of vesicular stomatitis virus (VSV) to those antigens recognized by B10.A (k, d) anti-VSV cytotoxic T lymphocytes (CTLs). The ability of monoclonal anti-H-2K^k or rabbit anti-G protein to inhibit specifically the cytolytic activity of B10.A anti-VSV CTLs indicates that the G protein and the H-2K^k molecules are in close proximity to the viral and H-2K^k antigens recognized by the anti-VSV (CTLs. By the method of sequential immunoprecipitation, we also demonstrated that only 10–30 percent of the serologically defined G and H-2K^k molecules are in theG-H-2K ^k complexes.Abbreviations used in this paper Con A Concanavalin A - cpm counts per minure - CTLs cytotoxic T lymphocytes - E: T ratio effector: target ratio - G major surface glycoprotein of VSV - MHC major histocompatibility complex - MOI multiplicity of infection - NP40 Nonidet-P40 - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - SaCI Staphylococcus aureus, Cowan I strain - SDS sodium dodecyl sulfate - UV ultraviolet light