Differential allo-response of murine thymocytes to H- 2 K region different recombinants and to H-2Kb mutants

Thymocytes used as responding cells in a mixed leukocyte culture with x-irradiated splenic stimulating cells generate highly significant proliferative and cytotoxic responses when responding and stimulating cells differ by the entire H-2 complex. On the other hand, when the genetic difference between responding and stimulating cells is only a K region, very little, if any, proliferative response is detectable and no cytotoxic response is found. In contrast, when responding and stimulating cell donors differ by a spontaneous mutation in the K region of the H-2 complex, as found in B6.C-H-2ba, B6-H-2bd and B6.C-H-2bf, highly significant proliferative and cytotoxic responses can be obtained. These results, thus, argue that the H-2 mutants cannot, with regard to their relationship to the parental strain, be readily equated with a K region difference as defined in the recombinant inbred strains.     

Inability of mice to generate cytotoxic T lymphocytes to vesicular stomatitis virus restricted to H-2Kk or H-2Dk

H-2k mice are unable to generate cytotoxic T lymphocytes (CTL) to vesicular stomatitis virus (VSV). This apparent unresponsiveness is found for both major serotypes of VSV, VSV-Indiana and VSV-New Jersey. CTL unresponsiveness occurs despite the ability of H-2k mice to generate a humoral immune response against VSV that is comparable to that found in responder (H-2b and H-2a) strains. All H-2k mice regardless of background genes, including various Ig allotypes, were found to be nonresponders. H-2k-linked unresponsiveness mapped to both H-2Kk and H-2Dk and occurred despite the presence of responder alleles in (responder x nonresponder)F1 mice. The unresponsiveness cannot be attributed to an inability of VSV-infected H-2k target cells to express viral surface antigens of H-2 molecules. Further, unresponsiveness cannot be overcome by using secondary stimulation in vivo or in vitro. H-2k-linked unresponsiveness does not appear to be due to suppression, and no complementation has been found in various (nonresponder x nonresponder)F1 mice. Thus unresponsiveness to VSV in association with H-2Kk or H-2Dk appears to represent an extensive defect of immune responsiveness that probably occurs because VSV is not a natural mouse pathogen.     

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.     

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     

Immunogeneic analysis of H-2 mutations. II. Cellular immunity to the H-2DA mutation.

The H-2da haplotype was derived from the H-2d haplotype by a mutation localized to the D end of the H-2 complex. Coculture of H-2d and H-2da spleen cells gives rise to bidirectional MLR. However, the H-2d anti-H-2da response is much stronger than that of H-2da anti-H-2d. Both haplotypes give rise to reciprocal CML. B10.D2(R103) strain spleen cells, which differ only at the D end of the H-2 complex from the H-2d haplotype, kill H-2da target cells in CML when sensitized to H-2d stimulators and vice versa. Therefore, both the mutant and strain of origin share a D end CML specificity. H-2d and H-2da reject skin grafts in both directions, although some H-2d grafts show prolonged acceptance on H-2da recipients. These data are consistent with a mutation in the D end of the H-2d haplotype resulting in gain-loss of an antigen(s) that gives rise to reciprocal MLR, CML, and skin graft rejection. Further, the mutant can be distinguished from the strain of origin on the basis of the strength of immune response in MLR.     

Spleen cells from animals neonatally tolerant to H-2Kk antigens recognize trinitrophenyl-modified H-2Kk spleen cells

A.TL mice injected with (A.AL × A.TL)F₁ cells within 24 hours after birth were rendered tolerant to H-2K^k antigens, as evidenced by acceptance of A.TL skin grafts. When spleen cells from these tolerant animals were cocultured with A.AL stimulator cells, no cytotoxic effector cells were generated in a cell-mediated lympholysis assay. However, when the A.AL stimulator cells were derivatized with trinitrophenol, effector cells that displayed a cytotoxic effect against trinitrophenyl-modified H-2K^k target cells were generated. These data indicate that animals tolerant to H-2 determinants but chimeric to only a minor extent possess cytotoxic precursor cells in sufficient frequency to mount a primary in vitro response against trinitrophenol in the context of an allogeneicH-2K region.     

Frequency of allogeneic helper T cells responding to whole H-2 differences and to an H-2K difference alone.

Limiting dilution analysis was used to determine the frequency of splenic T cells that are stimulated by alloantigen to give help in a primary antibody response to SRBC. Several haplotype combinations were tested. A semilogarithmic plot of the fraction of nonresponding culture as a function of the number of T cells added to excess B cells gave a straight line intercepting with the origin. Thus a single cell-type was limiting, which was required to help B cells respond to SRBC. The frequency of syngeneic precursors of T helper cells specific for SRBC ranged from 1/10,000 to 1/55,000 with a mean of about 1/20,000. Allohelpers generated by whole H-2 differences gave precursor frequencies that ranged from 1/1000 to 1/7000 with a mean of about 1/2500. Thus allohelpers to whole H-2 differences were approximately 8-fold more frequent than SRBC-specific helpers. When the stimulation was limited to the H-2K difference between the mutant B6.C-H-2ba and wild-type B6, frequencies of from 1/2600 to 1/7900 allohelpers were found with a mean of about 1/5000, approximately half the frequency of allohelpers to whole H-2 differences. Thus some, but probably not all, of the magnitude of allogeneic halp can be attributed to the high frequency of helper T cells that respond to a given alloantigen.     

DNA sequence analysis of the C3H H-2Kk and H-2Dk loci. Evolutionary relationships to H-2 genes from four other mouse strains

We generated nucleotide sequences for H-2Kk and H-2Dk from the C3H mouse, as well as for a genomic clone of H-2Db, in order to conduct an evolutionary analysis of the H-2 genes from three haplotypes, k, d, and b. H-2Kk from both the C3H and AKR strains, H-2Kd, H-2Kb, H-2Dk, H-2Ld, H-2Dd, H-2Db, and H-2Dp DNA sequences were aligned, and the alignments used to construct phylogenetic trees inferring the evolutionary relationships among the nine genes by two independent methods. Both approaches yielded trees with similar topologies. In addition, the sequence alignments revealed patterns of nucleotide substitutions which implicate both point mutation and recombination in the divergence of the H-2 genes. Future considerations for evolutionary analysis of class I genes are discussed.     

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.     

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.     

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).