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.     

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.     

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.     

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).     

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.     

Study of anH-2K d mutant strain: C.B6-H-2 dm4

A new-H-2 mutant involving theH-2 ^d haplotype is described — C.B6-H- 2^dm4 (dm4). This mutant strain carries a gain and loss mutation which maps to theK ^d gene of theH-2 complex. Serological testing comparing the mutant and the parental BALB/cKh strain failed to detect any difference between the two strains and no antibodies could be produced, although a reciprocal mixed lymphocyte reaction was observed between mutant and parent.     

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.     

Immune response to the H-X antigen on P815-X2

In a preceding report, the detection of an H-2-linked immune response to the H-X ^d antigen on the P815-X2 mastocytoma was demonstrated by the significantly increased survival of (C57BL/6 × DBA/2)F₁ (B6D2F1) male hybrids (H-X ^b ) compared with female siblings (H-X ^{b/H-X d} ) after injection with the histocompatible tumor (H-X ^d ). This interpretation was supported by the absence of this sex effect in reciprocal D2B6F1 hybrids (H-X ^d and H-X ^{d/H-X b} ). Additional findings presented in this paper support the conclusion that this sex effect is due to a true immunological response to H-X ^d : (a) Reciprocal (DBA/2 × C57BL/6 H-2 mutant)F₁ hybrids, as well as D2B6F1, failed to exhibit the sex effect: (b) the demonstration of the sex effect in (BALB/c × DBA/2)F₁ and (BALB/c-H-2 ^dm2 × DBA/2)F₁ hybrids and in (C57BL/10 × DBA/2)F₁ hybrids was consistent with the known H-X incompatibilities between the strains BALB/c and DBA/2 and C57BL/10 and DBA/2, respectively, previously demonstrated by skin grafting; and (c) the sex effect was not abrogated by castration of male B6D2F1 hybrids. Variability in the presence or absence of the sex effect was observed in various [recombinant inbred (RI) × DBA/2]F₁ hybrids and may be attributed to the influence of a regulatory non-H-2 gene which is closely linked to the gene coding for mouse kidney-androgen-regulated protein (KAP) but androgen-independent, or to variability in inheritance of the H-X ^b allele among the RI lines. It is proposed that the P815-X2 model may be utilized to type RI lines derived from a cross between C57BL/6 and DBA/2 for their H-X genotypes.Abbreviations B C57BL/6 origin allele - B6 C57BL/6 - B10 C57BL/10 - B6D2F1 (C57BL/6 × DBA/2)F₁ - B6 ^m D2F1 (C57BL/6 H-2 mutant × DBA/2)F₁ - bm10 B6.C-H-2 ^bm10 - C BALB/c - D DBA/2 origin allele - D2 DBA/2 - dm2 BALB/c-H-2 ^dm2 - H-X X chromosome-determined histocompatibility antigen of the mouse - Ir gene, immune response gene - KAP kidney androgenregulated protein - MST median survival time - RI recombinant inbred - SDP strain distribution pattern     

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.     

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     

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 .     

Oxidative stress induces the expression of the major histocompatibility complex in murine tumor cells

The effect of t-butyl hydroperoxide (t-BOOH) on the induction of the Major Histocompatibility Complex (MHC) class I genes has been studied in two cell clones (B9 and G2) of the methylcholanthrene-induced murine fibrosarcoma GR9. These two clones were selected based on their different biological and biochemical behavior specially related to their tumor induction capability when injected into a BALB/c mouse. t-BOOH (0.125mM) induced the expression of H-2 molecules in both cell clones. In B9 cell clone, in which MHC basal expression is very low or absent, t-BOOH significantly induced H-2K^d, H-2D^d and H-2L^d molecules. In G2 cell clone the expression of MHC class I genes was also enhanced by the xenobiotic, the effect being especially significant on the H-2L^d molecule which is not expressed under basal conditions. H-2 molecules expression was accompanied by the activation of the transactivator factor NFκB. These results suggest that oxidative stress may modulate the antigen expression of tumor cells and thus the immune response of the host organism.

Basal levels of oxidative parameters, such as anti-oxidant enzymes, malondialdehyde (MDA) and the DNA damaged base 8-hydroxy-2′-deoxyguanosine (8-OHdG), showed differences between the two fibrosarcoma cell clones.     

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.     

Regulation of the production of murine IgG2a by an H-2-linked gene and other unlinked genes

Alleles of at least two loci (rig-1 and Rig-2) regulate the levels of serum immunoglobulin of the Igh-1b class and allotype in BALB/c Ig^b (BAB/14) and (BALB/c × BAB/14)F₁ mice. The combined effect of the BALB/c alleles at these two loci is to lower Igh-1b levels significantly below those observed in other strains and below their own levels of Igh-1a in allotype heterozygous mice. The rig-1 locus is closely linked to or within the H-2 complex. Two alleles have been defined: rig-1 ^d and rig-1 ^b in H-2 ^d and H-2 ^b haplotypes, respectively. Homozygous rig-1 ^d d animals heterozygous for the BALB/c Rig-2 allele(s) have very low levels of Igh-1b. The designation of Rig-2 is provisional since it has not been mapped or defined as a single locus.     

A novel MHC class I-related molecule expressed on mouse thymic stroma cells and mature lymphocytes

A monoclonal antibody (mAb) TP-3 has been established by immunizing rats with the BALB/c mouse thymic epithelial cell line TEL-2. The TP-3 antigen is expressed on stroma cells of thymus, spleen, and lymph node in syngeneic BALB/c mice (H-2 ^d ). This antigen is also expressed at a low level on the cell surface of immature thymocytes, and at a high level on mature T and B cells. In allogeneic mice such as C57BL/6 (H-2 ^b ) or C3H (H-2 ^k ), no cells expressed the TP-3 antigen. Using H-2 congenic mice, reactivity with mAb TP-3 was found to map to a region of H-2D ^d L ^d or between D ^d and Qa, suggesting that TP-3 is a major histocompatibility complex (MHC) class I antigen. However, immunoprecipitation analysis indicated that this antigen is not identical to the classical mouse class I molecules in terms of molecular size, antigenicity, and tissue distribution.     

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

Immunoprecipitation of NP-40 lysates of 125I-labeled lymph-node cells with different anti-H-2 sera and with anti-Qa-2 serum has shown that the BALB/cByA strain (H-2d, Qa-2-negative) expresses, besides H-2Ld, another molecule that is not detectable in the BALB/c-H-2dm2 strain. Electrophoresis in SDS polyacrylamide gels indicated that this molecule, provisionally designated Lq, has an apparent molecular weight of 41000 daltons, in contrast to approximately 49000 daltons for H-2Kd and H-2Ld, and 47000 daltons for H-2Dd molecules. The anti-Qa-2 serum precipitated from the Qa-2-positive strains BALB/cHeA but not from the Qa-2-negative strains BALB/cByA and BALB/c-H-2dm2 a protein that gave a very strong band corresponding to the molecular weight 41000 daltons in the gel electrophoresis. The biochemical characteristics of the Lq molecule are thus more similar to those of Qa-2 than of H-2 antigens.     

Cytotoxic T lymphocytes recognize determinants on the BALB/c-H-2Ld molecule controlled by α1 and α2 but not α3 external domains

We have shown that cytotoxic T lymphocytes (CTL) raised in H-2 ^dmice use H-2L^d but not H-2D^d or H-2K^d antigens as restricting elements in lymphocytic choriomeningitis virus (LCMV) and vesicular stomatis virus (VSV) infections. To localize the regions of H-2L^d protein recognized by CTL, we constructed a recombinant H-2L ^d/D ^dgene encoding a hybrid antigen with 1 and 2 external domains of H-2L^d and 3, transmembrane and cytoplasmic domains of H-2D^d. The recombinant gene was transfected into mouse cells and the hybrid molecules were characterized serologically, biochemically and functionally. In all assays, H-2L^d/D^d molecules were recognized by LCMV- and VSV-specific H-2L^d-restricted CTL in a manner similar to that of wild-type H-2L^d antigens. Analogous results were obtained with alloreactive CTL. Hybrid antigens containing the 3 domain of H-2L^d fused to 1 and 2 domains of a Qa-2,3 region-encoded antigen were not used as restricting elements by LCMV-specific CTL. These results suggest that H-2L^d-restricted CTL directed against LCMV and VSV recognize determinants controlled by the 1 and/or 2 domains of the H-2L^d molecule.Abbreviations used in this paper CTL cytotoxic T lymphocytes - VSV vesicular stomatitis virus - LCMV lymphocytic choriomeningitis virus - tk thymidine kinase - HAT hypoxanthine, aminopterine, thymidine - HSV herpes simplex virus - FCS fetal calf serum - SAC Staphylococcus aureus Cowan I strain - TM transmembrane - CYT cytoplasmic     

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.     

Anti-MHC immunity detected prior to intentional alloimmunization

Cell fusion was performed between spleen cells from young BALB/cBy (H-2 ^d) mice which have never been immunized and SP2/0 mouse plasmacytoma cells. A monoclonal H-2-specific cytotoxic IgM antibody was obtained (By-1) which detected a new public biregional H-2 specificity, H-2.m210. The mcAb By-1 reacted strongly with H-2K^d, D^d, and H-2^s antigens, gave weak cross-reactions with H-2K^k, D^q, H-2^r, and H-2^v antigens and was negative with H-2^b, H-2^f, H-2^p, and H-2L^d antigens. A polymorphic reaction pattern was also observed on a panel of lymphocytes from B 10.W strains. The intriguing finding on this reaction pattern was the reactivity on H-2^d cells, including the syngeneic BALB/cBy and truly autologous cells. As shown by capping and immunoprecipitation experiments on H-2^d cells and by studies on H-2^d-transfected mouse L cells, the target molecules for McAb By-1 were H-2K^d and H-2D^d molecules. The BALB/cBy mouse, from whose spleen cells the McAb By-1 was obtained, survived after the fusion experiment, and serum was examined for the presence of cytotoxic H-2-specific antibodies during the rest of its life. At the time of the fusion, no autoreactive serum antibodies were found, but about 4 months later, we found in the serum of this mouse autoreactive H-2-specific cytotoxic IgM antibodies. The serum antibodies followed the same reaction pattern as that of the McAb By-1. As far as we know, this is the first report of autoreactive H-2-specific antibodies in serum of a mouse which has never been immunized and of the first natural autoreactive H-2-specific monoclonal antibody.Abbreviations McAb monoclonal antibody - MHC major histocompatibility complex - H-2 major histocompatibility complex of mice - CTLs cytotoxic T cells - FMF flow microfluorometry - FITC fluorescein isothiocyanate - LPS lipopolysaccharide W.E. coli 0111:134 - PBS phosphate-buffered saline - Iodogen 1,3,4,6,-tetrachloro-3,6-diphenylglycoluril - GAMIg goat-antimouse immunoglobulin - Staph-A Staphylococcus aureus Cowan I