Structural analysis of H-2Kf and H-2Kfm1 by using H-2K locus-specific sequences

The H-2Kf allele and the spontaneous mutant Kfm1 have been cloned using locus-specific sequences. The mutation consists of a cluster of four nucleotide changes, resulting in amino acid substitutions at positions 95 (Leu----Ile) and 97 (Val----Arg). This finding has structural, genetic, and technical implications. The amino acid substitutions are located on the beta-strands of the antigen recognition site. Their influence on the allogeneic properties of the Kf glycoprotein is consistent with the hypothesis that alloreactivity results from alterations in the spectrum of peptides presented to T cells. These substitutions would not, however, be predicted to be directly accessible for binding to antibodies. Nonetheless, the fm1 mutant binds anti Kf alloantisera and mAb much less strongly than the parent molecule, suggesting some indirect effect of these residues on serologic phenotype. The mutant is also interesting genetically because the sequence of the mutated region is identical to the sequence of the Df gene. This implies that there is a gene conversion-like mutational mechanism operating in the H-2f haplotype. Finally, the strategy used to obtain these K-locus cDNA should prove generally useful for isolating other MHC alleles.     

Intra-H-2 recombination in the mouse. III. I-Region characterization of haplotypes H-2at2, H-2at3 and H-2a2

Serological characterization of three K-S interval recombinant strains, TBR2 (H-2at2), TBR3 (H-2at3) and AIR 1 (H-2a2) was performed using anti-H-2, Ia, Ss and Slp antisera. The data presented here reveal that the crossover events in both TBR2 and TBR3 occurred between the I-A and I-E subregions. In both cases, the H-2K and I-A subregions were derived fron the H-2t1 of chromosome, while the I-E, S and H-2D regions were derived from the H-2b chromosome (KsAkEbSbDb). The H-2a2 chromosome resulted from a crossover event between the H-2a1 and H-2i9 chromosomes. Ia and Ss typing of AIR 1 suggested that the K to I-E regions originated from H-2a1 and the S and D regions originated from H-2i9 (KkAkEkSbDd).     

Serological detection of H-2K- and H-2D-gene products

Using the fluorescence-activated cell sorter (FACS II), we have analyzed the expression of H-2K- and H-2D-gene products on the membrane of various cellular components of the murine immune system. Using this serological technique we show a basic difference between T and B lymphocytes. Whereas all cellular components analyzed — hydrocortisone-resistant thymocytes, splenic T and B lymphocytes, macrophages and bone-marrow cells — expressed H-2K-subregion-encoded alloantigens at a high density, it seems that the high density expression of H-2D-encoded alloantigens is restricted mainly to B cells and to macrophages. Hydrocortisone-resistant thymocytes, splenic T lymphocytes and bone-marrow cells, on the other hand, showed significant expression of the H-2D alloantigens only at low membrane density. These results, then, provide evidence for the existence of an imbalance in serologically detectable expression of H-2K- and H-2D-region-gene products on the cell membrane of various cells comprising the murine immune system.Abbreviations usedin this paper DTH delayed type hypersensitivity - FCS fetal calf serum - FITC fluorescein isothiocyanate - HrT hydrocortisone-resistant thymocytes - Ig immunoglobulins P. De Baetselier is an EMBO and Euratom postdoctoral fellow     

Anti H-2Dd alloreactivity mediated by herpes-simplex-virus specific cytotoxic H-2k T lymphocytes is associated with H-2Dk

Herpes-simplex-virus (HSV) specific, H-2^k-restricted, immune cytotoxic T lymphocytes also lyse noninfected H-2^d target cells. Genetic mapping studies revealed that HSV-specific D^k-restricted CTL cross-react with allogeneic targets expressing D^d alloantigens. Cold target inhibition experiments indicate that only a minority of HSV-specific CTL mediate cross-reactive cytolysis. The data give an example of where the phenomenon of H-2-restricted versus nonrestricted responsiveness is not due to distinct subsets of T cells but solely depends on the antigenic determinants recognized.This work was supported by the SFB 107 and the Stiftung Volkswagenwerk.     

The nucleotide sequence and comparative analysis of the H-2Dp class I H-2 gene

We present the complete nucleotide sequence and the deduced amino acid sequence of the H-2Dp class I gene. This gene, which was cloned from a B10.P genomic DNA library, encodes and intact, functional H-2Dp molecule. Comparative analysis of the Dp sequence with other class I sequences reveals both similarities and differences. This analysis also shows that these genes exhibit D region-specific, locus-specific, as well as allele-specific sequences. The H-2Dp nucleotide sequence is greater than 90% homologous to the H-2Ld and H-2Db genes and only approximately 85% homologous to the H-2Dd gene. The K region and Qa region genes are less homologous. The 3' noncoding sequences appear to be region-specific. All of the previously described D region genes, Db, Ld, and Dd, possess the B2-SINE Alu-like repetitive sequence, as does Dp. Thus, this B2 repeat is a region-specific marker present in all D region genes studied so far. The additional polyadenylation site found in the H-2Dp gene starting at nucleotide 4671, which is homologous to non-D region sequences, as well as unique protein Dp coding sequences, make this gene an interesting model for studying the evolution of polymorphism and structure/function relationships in the class I gene family.     

Anti H-2Dd alloreactivity mediated by herpes-simplex-virus specific cytotoxic H-2k T lymphocytes is associated with H-2Dk

Herpes-simplex-virus (HSV) specific, H-2k-restricted, immune cytotoxic T lymphocytes also lyse noninfected H-2d target cells. Genetic mapping studies revealed that HSV-specific Dk-restricted CTL cross-react with allogeneic targets expressing Dd alloantigens. Cold target inhibition experiments indicate that only a minority of HSV-specific CTL mediate cross-reactive cytolysis. The data give an example of where the phenomenon of H-2-restricted versus nonrestricted responsiveness is not due to distinct subsets of T cells but solely depends on the antigenic determinants recognized.     

Clonal analysis of H-2Kb + TNP recognition by T cells with the use of H-2Kbm mutants and H-2Kb-specific monoclonal antibodies

Eleven long-term cytotoxic T lymphocyte (CTL) clones derived from C57BL/10 T cells sensitized in vivo and in vitro with trinitrobenzene sulfonate- (TNBS) treated syngeneic cells were all restricted to the K end of H-2b. The fine specificity of these CTL clones was analyzed by using H-2Kbm mutant target cells and H-2Kb-specific monoclonal antibodies (mAb). Seven distinct patterns of reactivity of the T cell clones could be observed with the use of six H-2Kbm mutant target cells. Further heterogeneity could be detected in terms of the ability of anti-Lyt-2 mAb to inhibit CTL activity. Cross-reactivity between H-2Kb + TNP and H-2Kbm + TNP was observed for all clones tested for bm5 and bm6, but less frequently for bm3 (8/11), bm8 (7/10), bm4 (4/11), and bm1 (3/11). It was further observed that amino acid substitutions located in the first domain only (one clone), or in the second domain only (six clones), or in either the first or the second domain (three clones) of the H-2Kb molecule could affect target cell recognition by a given T cell clone. the latter type of reactivity suggested that some clones recognized "conformational" determinants of the H-2 molecule, or that amino acid substitutions in one domain might influence the structure of the next domain. One H-2Kb + TNP-reactive clone exhibited a heteroclitic behavior with decreasing avidities for target cells expressing H-2Kbm8 + TNP, H-2Kb + TNP, and H-2Kbm8, which further extends the various patterns of T cell cross-reactions observed within a given class of MHC products. The use of H-2Kb-specific mAb in blocking studies as an attempt to define further the H-2Kb epitopes recognized by CTL clones indicated that: a) TNBS treatment may affect the antigenicity of the H-2Kb molecule as assessed by some mAb; and b) that the T cell clone-target cell interaction may or may not be inhibited by a given mAb, depending on structural variations of the H-2Kb molecule (use of H-2Kbm mutants) that do not affect the interaction itself. These results indicate that this type of analysis does not permit correlation of serologic- and T cell-defined epitopes.     

Discrimination between mammalian RNases H-1 and H-2.

The two principal RNases H in mammalian cells, H-1 and H-2, differ in their responses to sale, divalent metal, and sulfhydryl inhibition. Specific reaction conditions that provide unambiguous discrimination between RNases H-1 and H-2 with only two assays are described. The assays were used for identification in a new purification procedure for RNases H-1 and H-2.     

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.     

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.     

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-2k) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected taraget cells expressing the Dd alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2Dk determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for Dd alloantigen could be positively selected on H-2d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing Dd 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.