EVIDENCE OF ONLY ONE H-2D/L - RELATED GLYCOPROTEIN IN H-2dm1 MUTANT CELLS

Analyses of the H-2D/L-related glycoproteins from dm1 mutant cell extracts by sequential immunoprecipitation, by SDS gel electrophoresis and by tryptic peptide mapping indicate that dm1 cells express only a single glycoprotein with H-2D/L-related determinants. In contrast to the four H-2D/L-related antigens identified for the parental d haplotype viz. H-2D^d, H-2M^d, H-2L^d and H-2R^d, separate and distinguishable “H-2D^dm1”, “H-2M^dm1”, “H-2L^dm1” and “H-2R^dm1” glycoprotein counterparts are apparently lacking in the dm1 mutant haplotype. Only a single H-2D/L-related glycoprotein is identified in dm1 extracts by standard serological methods and this glycoprotein is designated H-2D/L^dm1 because of its H-2D^d/H-2L^d hybrid characteristics, as recently shown by Burnside and colleagues (1984). Thus, the seemingly complex phenotype of the dm1 mutant appears to originate primarily from one molecule having properties of two (or more) molecules of the parental haplotype.     

Lack of H-2Ld locus products on a BALB/c fibrosarcoma expressing H-2k-like alien antigens

The presence of H-2Ld antigens was evaluated in methylcholanthrene-induced BALB/c fibrosarcomas by a variety of approaches. Transplantation experiments showed that BALB/c-H-2dm2 mice, a mutant strain whose cells do not express H-2Ld antigens, after immunization with BALB/c normal tissues developed a resistance to the growth of two tumours (C-3 and GI-17), but not to a third neoplasm, C-1, which is known to have H-2d- as well as H-2k-like alien antigens. In vitro experiments with cytotoxic T lymphocytes generated against Ld antigens confirmed a loss of Ld antigens on C-1 but not on C-3 tumour cells. Serological experiments with an anti-Ld serum again revealed the presence of H-2Ld determinants on C-3 but not on C-1 cells. Biochemical analysis in SDS-PAGE of immunoprecipitates obtained by specific anti-H-2 sera with NP40 lysates of the tumours studied could detect H-2Kd, H-2Dd and H-2Ld antigens in C-3 fibrosarcoma cells whereas Kd and Dd were the only H-2d molecules found in C-1 lysate along with the H-2k-like specificities. The possible genetic mechanisms which may explain this apparent gain and loss modification of the H-2 profile of C-1 are discussed.     

H-2 antigens of certain t and non-t mice react with the same monoclonal antibody

Monoclonal antibody 212.i.4.2 mediated complement-dependent lysis of spleen and lymph node cells carrying the tw1 , tw12 , tw71 , t6, tw73 , and tLub1 haplotypes, while cells from mice carrying 11 other t haplotypes were not lysed. The antibody also detected an epitope controlled by genes in the H-2Dd region of non-t mice. A molecule of 46,000 molecular weight was immunoprecipitated by 212.i.4.2 from detergent extracts of 125I-labelled spleen cells of +/ tw12 and B10.D2 mice. The H- 2dm2 mutation did not alter the expression of the epitope recognized by 212.i.4.2. However, the H- 2dm1 mutation decreased the reactivity of lymphoid cells with the antibody in cytotoxicity tests, and 212.i.4.2 immunoprecipitated little or no protein from extracts of B10.D2( R106 ) spleen cells which carry the H- 2dm1 mutation.     

H-2 restriction and serotype crossreactivity of anti-reovirus cytotoxic T lymphocytes (CTL)

Murine anti-reovirus cytotoxic T lymphocytes (CTLs) were analyzed for H-2 restricted recognition of virus infected target cells and for potential cross-reactivity with cells infected by reovirus serotype 1 (T1; Lang strain) or by serotype 3 (T3; Dearing strain). Anti-reovirus CTL specifically lysed virus infected cells and lysis was shown to be H-2 restricted by the H-2Dd, H-2Ld, H-2Kd, H-2Kb, and H-2Kk antigens. No H-2 antigens were identified which failed to restrict virus recognition by anti-reovirus CTL. Anti-T1 and anti-T3 CTLs were also shown to crossreact completely with cells infected with the opposite virus serotype. Thus, anti-reovirus CTLs are restricted by a broad spectrum of H-2 antigens and they detect common rather than unique structural components of these two viral serotypes.     

A region of the influenza A/NT/60/68 PB2 protein containing an antigenic determinant recognized by murine H-2Dd restricted cytotoxic T lymphocytes

We have used a recombinant vaccinia virus to investigate the recognition of the PB2 protein of influenza A/NT/60/68 (H3N2) by murine polyclonal CTL populations. PB2 is recognized as a major cross-reactive target antigen. Recognition of PB2 is under strict genetic control, since BALB/c (H-2d) but not CBA (H-2k) mice are responders. We also demonstrate, by use of cell lines transfected with individual genes encoding class I molecules of the H-2d haplotype, that recognition of PB2 occurs in conjunction with the H-2Dd but not the H-2Kd or H-2Ld molecules. In contrast, recognition of the nucleoprotein of A/PR/8/34 by BALB/c-derived polyclonal CTL is restricted via the H-2Kd molecule. By using three recombinant vaccinia viruses expressing deleted forms of the PB2 protein we show that at least one epitope of the PB2 protein resides within the amino-terminal 256 amino acids. This approach offers an effective method to map the regions of large proteins containing epitopes recognized by CTL.     

Structural analyses define an additional H-2D region class I antigen that is expressed by a variant mouse strain

The genetic complexity of the H-2D region includes haplotype disparities in apparent gene and product number. To probe the genetic basis of this complexity, the products of two independently derived mouse strains (STU and B10.SAA48) that express Dw3 antigens were compared. Serologic, fluorometric and peptide map comparisons were made using monoclonal antibodies. Although both STU and B10.SAA48 mice were found to express indistinguishable Dw3 molecules, only B10.SAA48 mice were found to express an additional antigen designated Lw3. Several lines of evidence are presented that suggest the gene encoding Lw3 maps to the D region. Furthermore peptide map comparisons of Dw3 with Lw3 molecules implied that they are products of separate genes; but Dw3 and Lw3 molecules were found to be more homologous to each other than Dd and Ld molecules are to each other. Inter-haplotype comparisons of Dw3 and Lw3 molecules with other D region molecules showed no striking homologies to Dd, Ld or eight other molecules compared. However, both Dw3 and Lw3 molecules were found to be unexpectedly homologous to the Ddx and Dw25 molecules, thus defining another family of structurally related D region antigens. This so called Dw3-family was found to be quite distinct from the previously defined Ld-family of molecules, since no joint members were found. The results of these studies of Dw3 encoded antigens are discussed as evidence for intra-D region recombination or mutation.     

Intra-H-2 Recombination in H-2b/H-2t1 Heterozygotes in the Mouse I. Four Cases of Crossing Over between the S and D Regions

An examination of 1009 backcross animals produced from H-2b/H-2t1 heterozygotes resulted in the detection of four cases of intra-H-2 recombination. Three of the recombinants received the K region from H-2b and the D region from the H-2t1 parenteral chromosome. The fourth recombinant received a K region from the H-2t1 parental chromosome and the D region from H-2b. Ss typing revealed that crossing over occurred between the S and D regions of the H-2 complex in all four cases. Three of the four recombinants were developed into inbred strains and assigned the haplotype designations H-2i8, H-2i9 and H-2at1.     

Studies on the generation and expression of H-2-controlled T helper function in chimeric mice: evidence for two levels of H-2 resitriction.

F1 leads to parental, semisyngeneic/semiallogeneic and fully allogeneic bone marrow radiation chimeras were used as a source of helper T cells in the in vitro anamnestic response to dinitrophenylated keyhole limpet hemocyanin. In F1 leads to parental and in semisyngeneic/semiallogeneic chimeras, a small population of helper T cells restricted to the H-2 haplotype present in the donor only was shown to co-exist with T cells restricted to the shared haplotype. Only the population restricted to the donor H-2 could be demonstrated in allochimeras, presumably due to a lack of antigen-presenting cells of host origin during priming. Under assay conditions where, in addition to T cell/macrophage interactions, a direct T-B cell contact was necessary ("linked" cooperation), the H-2 restrictions observed were absolute. When direct T-B cell contact was made unnecessary by a special experimental protocol ("unlinked" cooperation), H-2 restriction between T and B cells was overcome. Thus, the cellular interactions during the anamnestic immune response to T-dependent antigens seem to be H-2-restricted at two levels: T cell/macrophage and T-B cell interactions.     

H-2 haplotypes carrying identical D but different L alleles

The B10.SAA48 congenic line was derived by transferring the H-2 haplotype of a wild mouse onto the background of the inbred strain C57BL/10Sn (abbreviated as B10). The line carries the Dw3 allele in combination with an L allele different from that present in other Dw3 strains. One possible explanation of this finding is that crossing over occurred between the D and L loci. The determinants H-2.m64 and H-2.m65 represent an inclusion doublet in which the latter never occurs without the former. Typing of B10.W lines with Ld-specific CTLs reveals the absence of this allele in all the lines, including those carrying an allele serologically similar to Ld.     

D2d, a D-End class I gene: tissue expression and alternative processing of the pre-mRNA

The H-2D region in the major histocompatibility complex (MHC) of the BALB/c mouse includes at least five class I genes: Dd, D2d, D3d, D4d, and Ld. The pattern of expression and functions of the D2d, D3d, and D4d genes are not known. To examine the expression of D2d we obtained mouse L cells transfected with a wildtype D2d gene or an exon shufled D2d/D2d/Dd gene that contained exons encoding the alpha 1 and alpha 2 domains of D2d and the alpha 3 domain of H-2Dd. Analysis of the mRNA in transfected cells suggested that two forms of the message were generated at approximately equal abundance by alternative splicing. Several tissues were analyzed and shown to express both forms of the D2d mRNA although the highest levels were found in lymphoid organs. Tumor lines also expressed D2d mRNA but exhibited differing ratios of alternative versus normally spliced message. This ratio was also affected by treatment of cells with Con A supernatants that contain interferon or infection with vesicular stomatitis virus (VSV).     

Immunogenicity of H-2 positive and H-2 negative clones of a mouse tumour, GR9

The GR9 tumour was induced with methylcholanthrene in a BALB/c mouse, adapted to tissue culture and cloned without any passage in vivo GR9 clones were typed for H-2 with three monoclonal antibodies that define H-2Kd + Dd, Kd and Dd antigens. A great heterogeneity of H-2d expression was found from clones which were Kd and Dd positive to clones Kd and Dd negative. These results were confirmed for A7 and B9 clones using immunoprecipitation with anti-H-2D.4 and anti-H-2K.31 alloantisera and SDS-PAGE analysis. In addition, the number of chromosomes per cell was heterogeneous amongst the clones, ranging from 38 +/- 2 to pseudotetraploid clones which have 75 +/- 2 chromosomes. GR9 clones were injected into syngeneic BALB/c mice to measure local tumour growth. We found that the growth correlated with the amount of H-2 antigen expressed, i.e. clones with low H-2d expression were highly malignant while clones with normal H-2d expression were highly immunogenic. Finally we found that BALB/c mice immunized against A7 (Kd, Dd-positive) protected against A7, as expected, but surprisingly also against B9 (Kd, Dd-negative).     

Molecular analysis of H-2 class I molecules expressed on the UV-induced tumour 1591

We have biochemically characterized by 2D (two-dimensional) electrophoresis three novel class I molecules called A166, A149 and A216 expressed by 1591, a UV-induced fibrosarcoma, and have compared them to class I molecules expressed by mice of the H-2q and H-2s haplotypes. A166 and A149 are very similar if not identical to Dq and Lq respectively. We have shown, using HPLC (high-pressure liquid chromatography) tryptic peptide mapping, that the expression of A166 is approximately three fold greater than A149, reminiscent of Dd compared to Ld. In addition A216 possess an identical isoelectric point to that of the Ks molecule. We demonstrate that outbred Swiss Webster mice express an analogous constellation of class I molecules and we conclude that our results can be most easily interpreted in terms of an allogeneic origin for the novel class I molecules expressed on 1591.     

Analysis of the H-2Kbm8 mutant: correlation of structure with function.

The gene for H-2K class I major histocompatibility antigen on the bm8 variant was cloned and the DNA sequence compared with the parental gene. Sequence analysis demonstrated that seven nucleotides were changed with respect to the parental gene sequence spanning 24 nucleotides. These changes represent an alteration of four amino acids from the parent protein. As this mutation occurred in a single generation, a potential donor gene for such a complex mutation was suggested and identified. The Q4 gene class I-like molecule has a stretch of 95 nucleotides of identity in the region of the bm8 mutation. Genomic Southern analysis of the mutant and parental DNA with a gene-specific oligonucleotide demonstrated that the potential donor gene Q4 is a likely candidate sequence for such an event. The amino acid alterations for the H-2Kbm8 mutation are discussed in consideration of hte three-dimensional structure of the characterized human class I glycoprotein.     

Biochemical analysis of a public H-2 specificity revealed by an anomalous reaction of an alloantiserum with a chemically induced C57BL/10 fibrosarcoma

D-25 is a H-2 alloantiserum produced in (B10.D2 X C3H.NB) (H-2d X H-2p)F1 mice after immunization with B10.RIII(H-2r) cells, and which is known to recognize the H-2.25 public antigen on H-2k haplotypes. The "anomalous" reaction of D-25 with a partially purified deoxycholate-solubilized glycoprotein of B10-1 (H-2b) fibrosarcoma was studied with various biochemical techniques. The 125I-labelled precipitates were analysed both in one- and two-dimensional SDS-PAGE and by a partial proteolysis peptide mapping (Cleveland's mapping). The results indicate first that the D-25-related antigen was a genuine H-2 antigen normally associated with the B2-microglobulin, second that this antigen was borne by the Kb but not Db gene products, and finally that D-25 was able to precipitate the same antigen on normal H-2b spleen cells. We conclude that this serum recognized a normal H-2 specificity shared by H-2b and H-2r haplotypes and tentatively identified as the public antigen H-2.54 which for the first time could be assigned to the K but not to the D region of the H-2b haplotype.     

Class I H-2Db determinants are not involved in hybrid resistance to parental H-2b/Hh-1b bone marrow allograft

Hybrid resistance to parental H-2b bone marrow grafts is directed to a cell surface structure controlled by the Hh-1 locus in or near the H-2D region. The nature of this surface structure is not known. Since homozygosity at the class I H-2D locus or loci in this haplotype would seem a necessary but not sufficient condition for the grafts' susceptibility to resistance, we tested whether the expression of this phenotype is dependent on the expression of class I H-2Db determinants. Cloned variants of H-2b tumor RBL-5 were obtained by immunoselection for the absence of H-2Db expression, as determined by the inability to bind specific antibody and to induce or react with alloreactive cytotoxic T lymphocytes. The three clones used in this study were H-2Db negative but H-2Kb positive and were natural killer cell resistant. When tested in vivo as competitive inhibitors the variant cells were capable of blocking hybrid resistance to parental H-2b bone marrow grafts as were unselected H-2Db-positive parental line cells. Therefore, H-2Db expression is irrelevant for Hh-1b expression. An incidental observation was that YAC-1 cells, a non-H-2b tumor with pronounced susceptibility to natural killing, were able to block hybrid resistance. This reactivity, not observed in our previous studies, raises the possibility that at least some of the effector cells are cross-reactive or capable of dual recognition.     

Analysis of the extracellular processing of HIV-1 gp160-derived peptides using monoclonal antibodies specific to H-2Dd molecule complexed with p18-I10 peptide

The immunodominant peptide of human immunodeficiency virus 1 gp 160 for murine cytotoxic T cells of H-2d haplotype, has been originally identified as a 15 amino acid residue peptide P18IIIB (RIQRGPGRAFVTIGK) (Takahashi et al., 1988). Further studies have indicated that a more active form of the peptide is generated by removal of the C-terminal dipeptide by angiotensin-I-converting enzyme (ACE), and additional detailed studies have shown that the actual immunodominant peptide is a decamer P18-I10 (RGPGRAFVTI) (Kozlowski et al., 1993). The effect of proteolytic processing on the antigenicity of P18IIIB peptide and its analogs was investigated by functional T cell assays based on the ability of T cell receptor (TCR) to recognize a specific major histocompatibility complex class I (MHC-I)/peptide complex. Recently we described a new monoclonal antibody (MAb) KP15 directed against the MHC-I molecule H-2Dd complexed with the 10-mer peptide P18-I10. Using this MAb, the cell surface H-2Dd/P18-I10 complex can be easily detected by flow cytometry (Polakova et al., 2000). Here we examined whether peptides longer than P18-I10 decamer form H-2Dd complexes recognized by KP15 MAb. Further we also analyzed how the ACE processing of P18IIIB-related peptides of different length affects their ability to form complexes with H-2Dd recognized by MAb KP15. These experiments confirmed that the ACE digestion of 15-mer peptide P18IIIB is the most effective in the production of a peptide capable of forming complex with H-2Dd recognized by KP15 MAb. The ACE digestion of longer peptides (16-mer to 19-mer) did not produce a significant quantity of peptides, capable of forming H-2Dd complexes recognizable with by MAb KP15. Peptides shorter than P18IIIB (13-mer to 10-mer), notably the optimally sized P18-I10 peptide lost most of their capacity to form H-2Dd complexes recognized by KP15 MAb. Our results show that the extracellular processing of MHC-I-restricted peptides, which cannot be overlooked in designing peptide-based vaccines, can be also studied by as simple and rapid assay as flow cytometry, provided MAbs specific to a particular MHC-I/peptide complex are available.     

H-2 restriction and H-2 phenotypes of spleen cells from thymus-grafted radiation chimeras: evidence consistent with extrathymic suppression

As an approach to analyzing the factors that contribute to determining H-2 restriction specificities of cytotoxic T cells, thymectomized semiallogeneic radiation chimeras were given transplants of fetal thymus from parental strain or F1 hybrid donors. A pronounced preference for lysis of infected targets of the same H-2 haplotype as the thymus was observed, the bias ranging from an undetectable preference through to absolute restriction to the thymic H-2. Virus-immune Tc cells could also be restricted to H-2 antigenic determinants of haplotypes expressed only by donated stem cell progeny lymphomyeloid cells. T cells from chimeras that had received thymuses of both parental strains were less effective than normal syngeneic F1 hybrid T cells in lysing allogeneic cells and both infected and uninfected parental targets. Variation in H-2 restriction bias from one chimera to another was not reflected in modulations to the class I H-2 phenotypes of the chimeric spleen cells.     

Demonstration of a murine cell surface component with affinity for exogenous beta 2-microglobulin.

Murine and human beta 2-microglobulin (beta 2m) bind to various types of mouse cells. The binding is saturable and displays a single association constant of about 1 x 10(9) liter/mol. The binding of beta 2m to splenocytes was not affected by a variety of metabolic inhibitors but was temperature-dependent. It is suggested that the beta 2m "receptor" exhibits a temperature-dependent conformational change since the "receptor", whether integrated into the membrane or solubilized by the detergent Triton X-100, binds beta 2m poorly at low temperatures. Spleen T and B lymphocytes display more binding sites than thymocytes, kidney, liver and brain cells. The relative amounts of the beta 2m-binding "receptor" on these cell types are strongly correlated to the relative amounts of H-2 antigens. This correlation is also obvious for the teratocarcinoma cell line F9, which lacks both beta 2m "receptor" and H-2 antigens, but spermatozoa, which express very small amounts of H-2 antigens, have an appreciable amount of the beta 2m "receptor". The latter observation, together with the fact that alloantisera directed against H-2 K and D antigens do not measurably affect the binding of beta 2m to the "receptor", may argue against the notion that the beta 2m "receptor" represents H-2 antigens which have lost their endogenous beta 2m. Normal mouse serum contains a component which inhibits the binding of beta 2m to splenocytes. It is likely that this serum protein is identical to a newly discovered H-2 antigen-like glycoprotein. The beta 2m "receptor" appears to be under the control of the major histocompatibility complex as splenocytes of the H-2f haplotype bind considerably more beta 2m than splenocytes of other haplotypes.