Differential induction of H-2K vs H-2D class I major histocompatibility complex antigen expression by murine recombinant interferon-gamma

The results presented here indicate that recombinant murine interferon-gamma can cause a dramatic differential induction of two distinct class I MHC molecules. Thus, IFN-gamma treatment of the murine leukemia virus (MuLV)-induced AKR SL3 tumor, a cell line that normally expresses moderate levels of class I MHC antigens, resulted in a large increase in H-2Dk expression, but no change or a slight decrease in H-2Kk expression as measured by cytofluorography. Explanations of the selective enhancement of Dk expression based on increased Fc receptor display or differential kinetics of induction were ruled out. The phenomenon was observed over a wide range of doses of IFN-gamma and with two different monoclonal antibodies to Kk, the latter finding making it unlikely that an altered form of the Kk molecule was induced. The same differential induction of the Dk antigen was observed for the LBRM.5A4 tumor cell line. Because LBRM.5A4 is also MuLV+ but of congenic B10.BR (H-2k) origin, these results were consistent with the possibility that such differential induction was associated with the H-2k haplotype and/or MuLV. The implications of these results, as a possible mechanism of tumor cell escape from an immune surveillance system monitored by class I MHC-restricted T cells and as a useful model system to dissect the mechanism of IFN-gamma induction of class I MHC antigens, are discussed.     

Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer

Among the more than 20 H-2-like genes in the BALB/c mouse genome, there are two classical transplantation antigens (H-2Dd and H-2Ld) encoded at the D-end of the major histocompatibility complex. Here we report the identification of a bacteriophage clone that encodes H-2Dd. The H-2Dd gene was identified by nucleotide sequence analysis and by characterization of the new H-2 antigen expressed when the cloned gene was introduced into mouse L cells by DNA-mediated gene transfer. The previously identified H-2Ld gene was then compared with the H-2Dd gene. The two genes appear to have the same general structure, and for the 854 nucleotides that have been compared, the two genes are 89% homologous. The H-2Ld and H-2Dd antigens expressed on mouse L cells after DNA-mediated gene transfer were examined by immunologic criteria. The stably transformed cell lines express apparently normal levels of H-2Dd and H-2Ld on the cell surface as measured by quantitative immunofluorescence by using monoclonal anti-H-2 antibodies. They synthesize H-2Dd and H-2Ld at normal rates as determined by endogenous labeling and immunoprecipitation of cell extracts. They evoke a strong specific serologic response when used to immunize C3H mice. The newly expressed antigens are able to serve as targets for alloreactive T cells. These cloned genes provide good substrates for examining the evolution of two closely linked H-2 antigen genes. Comparison of the structures of these genes provides clues to the basis for the differential expression of these antigens and their different biologic functions.     

Purification of the H-2Kk molecule of the murine major histocompatibility complex.

The intact H-2Kk antigen has been detergent-solubilized and purified using an immunoabsorbent column prepared from the 11-4.1 monoclonal antibody described by Oi et al. (Oi, V. T., Jones, P. P., Goding, J. Current Topics in Microbiology and Immunology (Melchers, F., Potter, M., and Warner, N. L., eds) Vol. 81, pp. 115-129, Springer-Verlag, New York). The mild conditions used for elution from the column, 0.5% deoxycholate in 10 mM Tris buffer, pH 8, with 0.14 M NaCl, result in recovery of 70 to 100% of the allogeneic serological activity. A murine lymphoma, RDM-4, was found to express high levels of H2-Kk; approximately 2 X 10(6) molecules/cell. Milligram quantities of H-2Kk can be purified readily using these cells.     

Role of the major histocompatibility complex in T cell activation of B cell subpopulations: antigen-specific and H-2-restricted monoclonal TH cells activate Lyb-5+ B cells through an antigen-nonspecific and H-2-unrestricted effector pathway

Monoclonal T helper (TH) cell populations were employed to study the mechanism of activation of the Lyb-5+ B cell subpopulation in T cell-dependent antibody responses in vitro. It was demonstrated that monoclonal T cell populations were sufficient to help rigorously T-depleted unprimed (B + accessory) cells for direct plaque-forming cell responses to trinitrophenyl- (TNP) conjugated keyhole limpet hemocyanin (KLH). The activation of several lines of cloned (H-2b X H-2k)F1 TH cells was antigen (KLH) specific and H-2 restricted. Individual clones were restricted to H -2b, H-2k, or unique (H-2b X H-2k)F1 encoded determinants. Under the experimental conditions employed, responses mediated by cloned TH cells were found to result in the activation of the Lyb-5+ B cell subpopulation. The activation of Lyb-5+ B cells by cloned TH cells did not require covalent linkage of carrier and hapten, and responses could be stimulated in the presence of free KLH plus TNP conjugated to an irrelevant carrier. The H-2 restriction of TH cell function was shown to reflect a requirement for T cell recognition of determinants expressed by accessory cells, whereas no requirement existed for restricted T cell recognition of B cells. These findings suggest that the help provided by monoclonal TH cells, once activated, was both antigen nonspecific and H-2 unrestricted. Consistent with this interpretation, it was found that the supernatant of antigen-stimulated TH cells provided antigen-nonspecific help to T-depleted spleen cells. Thus, these results demonstrate that the activation of Lyb-5+ B cells by antigen-specific and H-2-restricted monoclonal TH cell populations is itself antigen nonspecific and H-2 unrestricted.     

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.     

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 .     

The allogeneic effect: M-locus differences substitute for differences in the H-2 major histocompatibility complex.

The primary immune response against sheep red blood cells in T cell-deficient spleen cell cultures from nude mice was tested in the absence and presence of allogeneic spleen cells. The allogeneic spleen cells differed either in regard to the major histocompatibility complex (H-2) or only with respect to the M-locus. Surprisingly the M-locus different spleen cells were almost as efficient in enhancing the anti-sheep red blood cell response in nude cultures as were the cells differing on the complete H-2 complex. Evidence is presented that AKR anti-theta serum sensitive T cells are responsible for the M-locus-dependent effect edscribed. This effect is shown to be mediated by a factor released from actived T cells stimulated in M-locus different mixed lymphocyte cultures. Since almost identical parameters have been observed in both the M-locus-dependent situation as in the "classical" allogeneic situation we concluded that an allogeneic effect can be induced by T cells responding to a complete set of the major histocompatibility complex (H-2) as well as to lymphocyte-activating determinants (M-locus) alone.     

H-2 complex influences cytokine gene expression in Leishmania infantum-infected macrophages

This work aims to study the influence of H-2 locus in the control of Leishmania infantum infection by evaluating whether cytokine responses by host macrophages of different H-2 haplotype are differentially regulated, either induced or actively impaired during parasite growth and replication. This study shows that macrophages of "non-cure" phenotype (H-2(d)) are more susceptible to infection with virulent L. infantum promastigotes. Virulent parasites lead to impaired IL-12 and inhibited TNF-alpha expression. The degree of parasite virulence is an important contributing factor to differences detected in cytokine expression. Virulent parasites also induced TGF-beta, a deactivating cytokine that is known to suppress Th-1 type responses, thus allowing the parasite to subvert antimicrobial activity and increase its chances of survival. Depending on specific host haplotype, cells differentially respond to infection since TNF-alpha expression is inhibited and TGF-beta is enhanced by macrophages of "non-cure" phenotype, thus perhaps determining their degree of susceptibility in this strain of mice.     

A nonpolymorphic class I gene in the murine major histocompatibility complex

DNA sequence analysis of a class I gene (Q10), which maps to the Qa2,3 locus in the C57BL/10 (H-2b haplotype) mouse, reveals that it is almost identical to a cDNA clone (pH16) isolated from a SWR/J (H-2q haplotype) mouse liver cDNA library. Exon 5, in particular, has an unusual structure such that a polypeptide product is unlikely to be anchored in the cell membrane. Our findings suggest that the two sequences are derived from allelic class I genes, which are nonpolymorphic, in contrast to H-2K allelic sequences from the same mice, and they may encode liver-specific polypeptides of unknown function. Our previous studies indicate that the Q10 gene is a potential donor gene for the generation of mutations at the H-2K locus by inter-gene transfer of genetic information. Thus the lack of polymorphism in class I genes at the Q10 locus implies either that they are not recipients for such exchanges or that selective pressure prevents the accumulation of mutations in genes at this locus.     

Recombinants in the H-2S/H-2D interval of mouse chromosome 17 define the map position of a gene for cleft palate susceptibility

The H-2 region of mouse chromosome 17 is known to include one or more genes that affect susceptibility to cortisone-induced cleft palate. We have now studied congenic strains that possess crossovers in the interval between H-2S and H-2D and have observed significant differences in susceptibility among recombinants that had been believed to possess the same H-2 haplotypes. Pregnant mice were injected on days 11 through 14 of gestation with 100 mg of cortisone per kg of body weight. The frequency of cleft palate in B10.A(2R) was significantly greater than in B10.A(1R), despite the fact that both have H-2a/H-2b crossovers in the interval between the S and D loci and have the same alleles at all loci that have been previously characterized. Both B10.BAR5 and B10.BAR12 were significantly more susceptible than B10.A(18R), although these strains also share the same alleles at all loci that have been previously characterized. All three of these strains have H-2b/H-2a recombinant chromosomes, with crossovers in the S/D interval. Genetic linkage between H-2 and the high-susceptibility gene of B10.BAR5 was confirmed by testing H-2 homozygotes derived by intercrossing backcross animals. These data therefore suggest that a gene coding for susceptibility, which we designate Cps-1, maps in the 350-kb interval between H-2S and H-2D, and the congenic strains that we have found to be different have different crossover points within this interval. Alleles at the Cps-1 locus have embryonic effects, but no demonstrable effects on the maternal environment.     

Suppression of major histocompatibility complex class I and class II gene expression in Listeria monocytogenes-infected murine macrophages

Macrophage cells play a central role during infection with Listeria monocytogenes by both providing a major habitat for bacterial multiplication and presenting bacterial antigens to the immune system. In this study, we investigated the influence of L. monocytogenes infection on the expression of MHC class I and class II genes in two murine macrophage cell lines. Steady-state levels of I-Aβ chain mRNA were decreased in both resting J774A.1 and P388D₁ macrophages infected with L. monocytogenes whereas reduction of H-2K mRNA was only observed in P388D₁ cells. In addition, L. monocytogenes suppressed induction of MHC class I and class II mRNAs in response to γ-interferon as well as the maintenance of the induced state in activated P388D₁ macrophages. Exposure to the non-pathogenic species L. innocua or a deletion mutant of L. monocytogenes, which lacks the lecithinase operon, did not cause a reduction in H-2K and I-Aβ mRNA levels nor suppress expression of Ia antigens. Inhibition of MHC gene expression may represent an important part of the cross-talk between L. monocytogenes and the macrophage that probably influences the efficiency of a T cell-mediated immune response and thus the outcome of a listerial infection.     

Cytotoxic T-lymphocyte tolerance to minor H-43a alloantigen is induced exclusively in the context of the self major histocompatibility complex class I H-2Kb molecules

We elucidated previously that cytotoxic T lymphocyte precursors (CTLp) against H-43a allo-antigen, which we had discovered as a new mouse minor H antigen, were primed in H-43b mice only in the context of self H-2Kb restriction element, and that anti-H-43a CTLp tolerance was induced in H-43b mice by injection with H-43a spleen cells (SC) from H-43 congenic mice, i.e., under the condition of disparity at only the H-43 locus. The present study attempted to determine whether the H-2Kb restriction element for anti-H-43a CTLp priming is also implicated in the induction of anti-H-43a CTLp tolerance. For this purpose, we used a newly established H-43b C3W (H-2k) strain which is H-43 congenic to H-43a C3H/HeN. When (C3W X B10.MBR)F1 (H-43b, H-2Kk/b, Ik/k, Dk/q) mice were injected with H-43a-bearing (C3H/HeN X B10.AKM)F1 (H-43a/b;H-2Kk/k,Ik/k,Dk/q)SC, their selfH-2Kb-restricted anti-H-43a CTLp were were primed (cross-priming). By contrast, injection of H-43a-bearing (C3H/HeN X B10.MBR)F1 (H-43a/b; H-2Kk/b,Ik/k, Dk/q)SC, which differ from (C3H/HeN x B10.AKM) F1 SC solely at H-2K and possess H-2Kb molecules, did not prime but specifically inactivated the anti-H-43a CTLp of (C3W x B10.MBR)F1 mice. These results indicate clearly that anti-H-43a CTLp tolerance is induced exclusively in the context of the H-2Kb element expressed on the antigenic H-43a SC.     

Functional characterization of a chicken major histocompatibility complex class II B gene promoter

 A 0.7 kilobase (kb) DNA fragment from the 5′ flanking region of a chicken major histocompatibility complex (MHC) class II B gene was cloned into chloramphenicol acetyltransferase (CAT) reporter vectors and was transfected into a chicken macrophage cell line that expresses a low level of MHC class II antigens. Positive orientation-dependent promoter activity of the chicken DNA was evident in a reporter construct containing an SV40 enhancer. Deletion analysis of this 0.7 kb DNA fragment revealed a short fragment in the 3′ end that was crucial for the promoter function and negative regulatory elements (NRE) located further upstream. The conserved MHC class II X and Y boxes did not have a significant effect on promoter activity. Sequence analysis of the 0.7 kb class II B gene upstream region suggests possible involvement of interferon (IFN), E twenty-six specific (ETS)-related proteins, and other factors in regulating this promoter. A chicken T-cell line culture supernatant increased surface expression of MHC class II antigens, as well as class II promoter activity, in this macrophage cell line. This first functional characterization of a chicken MHC class II B gene promoter will aid in understanding the regulatory mechanisms that control the expression of these genes. Received: 9 July 1996 / Revised: 7 October 1996