The Qa2 subregion controls the expression of two antigens recognized by H-2-unrestricted cytotoxic T cells.

B6.KI mice were immunized with spleen cells from B6.K2, a Qa2-subregion congenic strain. Cytotoxic T cells were generated that recognize two target antigens controlled by this region. One of the target antigens is Qa-2. This was demonstrated by the findings that pretreatment of target cells with monoclonal anti-Qa-2 antibody blocked lysis of target cells, and Qa-2 target antigens and serological determinants had a concordant distribution on a panel of B10.W (wild) mice. The gene controlling the Qa-2 target antigen is not polymorphic because B6.K2 and three strains of Qa-2(+) B10.W mice express the same antigens, as determined by a CTL cold target competition assay. Anti-Qa-2 CTL were H-2 unrestricted because effector cells lysed Qa-2(+) targets irrespective of their H-2 haplotype, including five B 10.W strains, and lysis was not inhibited by pretreating target cells with anti-H-2 sera. The Qa2 subregion does not act as a restricting locus for anti-minor-H antigen CTL. A second target antigen was detected that was associated with the expression of the Qa-5 determinant. However, CTL activity could not be blocked by pretreating target cells with monoclonal anti-Qa-5. Therefore, the CTL target antigen may be expressed on a Qa-5(-) molecule. Although the Qa-5 associated CTL specificity is only detected on H-2D(b) strains, it is unlikely that CTL recognition is H-2 restricted because anti-H-2(b) sera has no effect in blocking this reactivity. Qa-2 and H-2 class I antigens share a similar structure and serve as target antigens for unrestricted CTL. However, unlike class I H-2 genes, Qa-2 neither restricts antigen-specific CTL nor is polymorphie. Therefore, it is likely that Qa-2 and H-2 are derived from a common ancestral gene and have evolved to serve different functions.

Features of T cells causing H-2-restricted lethal graft-vs.-host disease across minor histocompatibility barriers.

Evidence is presented that T cells that produce lethal graft-vs.-host disease (GVHD) to minor histocompatibility antigens (minor HA) comprise discrete subgroups of H-2K- and H-2D-restricted T cells; double negative selection of T cells in irradiated H-2 recombinant mice was used to separate these two subgroups. No evidence could be found that I-restricted T cells contributed to GVHD, either as effector cells or helper cells. The (unprimed) precursor cells for GVHD expressed the Thy-1+, Lyt-1+/-2, Ia- phenotype. Studies in which H-2-semiallogeneic bone marrow chimeras were used as hosts for negative selection suggested that presentation of minor HA to T cells during the induction phase is controlled by marrow-derived cells; indirect evidence was obtained that these latter cells can "process" minor HA presented on H-2 different cells and thereby render the antigens immunogenic. Studies in which minor HA-different, H-2-compatible chimeras were re-irradiated and then injected with donor-vs.-host T cells suggested that the effector phase of lethal GVHD involves contact of antigen on non-marrow-derived cells.

Nonclassical MHC-I and Japanese encephalitis virus infection: induction of H-2Q4, H-2T23 and H-2T10.

Nonclassical MHC Class 1b antigens differ from classical MHC class 1a antigens in having a restricted polymorphism as well as varied surface expression in different cell types. They have been hypothesized to play a role in bridging adaptive and innate immune responses. We examined the effects of JEV infection on the expression of classical MHC class 1a and nonclassical MHC class 1b genes in five different cell lines. Among the nonclassical genes, H-2Q4 was induced in H-6 hepatoma, primary astrocytes, mouse embryo fibroblasts, L929 and 3T3 cells. H-2T23 and H-2T10 genes were not induced in H-6 and 3T3, respectively, but were induced in the other cell lines examined. Both H-2Q4 encoded Qb1 and H-2T23 encoded Qa-1(b) antigens were induced on the cell surface upon JEV infection in primary astrocytes and mouse embryonic fibroblasts. Classical MHC-I genes and the genes associated with antigen presentation such as Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10 as well as type 1 (alpha/beta) IFNs were induced in all cell lines. However, IFNgamma was not induced. Further, induction of H-2Q4 and H-2T23 by JEV was independent of NF-kappaB but type 1 IFN dependent while H-2T10 was dependent on NF-kappaB and type 1 IFN independent. Thus, while classical MHC genes were induced by JEV in all cell lines tested despite high levels of constitutive expression in L929 and 3T3, nonclassical genes were not inducible in all cell lines tested and involved different mechanisms of induction.

Analysis of neonatally induced tolerance of H-2 alloantigens. III. Ease of abolition of tolerance of class I, but not class II, antigens with infusions of syngeneic, immunocompetent cells.

Neonatally-induced tolerance of class I H-2 alloantigens can be abolished in adult, long-term-tolerant mice by infusions of immunocompetent cells from donors syngeneic with the recipient. By contrast, neonatally-induced tolerance of Ia alloantigens can not be abolished easily, indicating that the Ia-specific tolerant state is maintained by an active process that can be impressed upon mature alloreactive lymphoid cells in the tolerant environment. This finding is concordant with previous observations that tolerance of Ia alloantigens can readily be transferred adoptively by inoculating lymphoid cells from tolerant mice into syngeneic, naive recipients. It is concluded that class II H-2 antigens induce a type of unresponsiveness that class I antigens fail to evoke, an unresponsiveness that is actively maintained among mature immunocompetent cells. It is proposed that, in addition to a central process of clonal deletion/inactivation that both class I and class II H-2 antigens induce in neonatal mice, Ia alloantigens also evoke a secondary, fail-safe mechanism that operates to prevent alloreactivity from emerging when cells with alloreactive potential escape the central mechanism, or mutate to alloreactivity at a later stage of maturation.

Structure and function of the major histocompatibility complex in domestic animals.

The major histocompatibility complex (MHC) is a genetic region that has been intensively studied for the past 2 decades. Interest in the MHC has been high because of (i) the particular involvement of the MHC in transplantation reactions, including organ allograft rejection in human beings; and (ii) the more general role of MHC gene products in the genetic control of immune responses in all mammals. The MHC has several remarkable properties that include a distinctive genetic structure which has been well-preserved through evolution, and the extreme plasticity of form of the principal MHC genes, which can coexist within a single species in 30 or more allelic forms. The genes of the MHC regulate cell-cell interactions of various types within the lymphoreticular system, and thus function as the so-called "immune response" genes that have been described in mice, rats, and guinea pigs. In human beings, the "disease associations" demonstrated between MHC alleles and various pathologic conditions are probably manifestations of abnormal functions of immune regulation governed by the MHC. Studies of the MHC in domestic species are still in their infancy. However, investigations of the MHC have been carried out in swine, cattle, horses, sheep, goats, dogs, and chickens. Further research on the MHC of domestic animals is merited, both for its contribution to the overall understanding of the biological significance of the MHC and for its practical application in clinical veterinary medicine.

[Intraspecies identification of cultured murine cells by using H-2 antigen typing]. / Vnutrividovaia identifikatsiia kul'tiviruemykh kletok myshi s pomoshch'iu tipirovaniia H-2-antigenov.

17 mouse cell lines have been screened with specific sera against H-2 antigens. All the cell lines tested expressed H-2 antigens characteristic of the donor haplotype. The data obtained indicate that H-2 typing of cultured mouse cells can be used as an approach to control their intraspecies diversity.

H-2 antigen frequencies among wild mice from Chile.

Thirty-five wild mice (Mus musculus L.) were captured at four different localities in Chile. The mice were typed for the presence of 15 K-, 11 D-, 14 A-, and two E-region H-2 antigens, using the complement-antibody microcytotoxicity assay. The mice from the sample representing the largest locality had a characteristic antigenic profile distinguishing them from other mouse populations thus far studied. With respect to class I H-2 antigens, the profile was characterized by the presence of antigens H-2K.16, 31, 103, 19, 108, 21, 33, 26, 115, and H-2D.4, 106, 30, 32, 111, 114, and 18 (in order of decreasing antigen frequency), and by the absence of antigens H-2K.15, 17, 20, 113, 116 and H-2D.2, 9, 110, and 112. The profile was, furthermore, characterized by the relatively high frequency of antigens H-2K.16, 31, and H-2D.4 and 106. The profile of class II antigens was also unique to the Chilean population but less conspicuously so than that of the class I antigens. Analysis of antigenic associations suggested that among the 34% blank H-2K alleles there were at least two coding for relatively frequent but as yet unidentified K antigens. Similarly, among the 57% blanks at the H-2D locus there were at least two frequent alleles encoding unidentified D antigens. Analysis of genetic distances suggested similarity between South American mice and mice from coastal regions of Europe.

Analysis of neonatally induced tolerance of H-2 alloantigens. I. Adoptive transfer indicates that tolerance of class I and class II antigens is maintained by distinct mechanisms.

Neonatal inoculation of mice with semi-allogeneic lymphohematopoietic cells produces a state of highly specific allograft tolerance. Phenotypically, by both in vivo and in vitro criteria, antigen-reactive cells specific for the tolerated antigens appear to be clonally deleted from intact, tolerant mice. However, a series of adoptive transfer experiments using mice rendered tolerant of various H-2 alloantigens revealed that tolerance of Ia (class II) antigens is maintained by a different mechanism than tolerance of K/D (class I) antigens. Long-term acceptance of Ia-disparate grafts by recipients of Ia-tolerant lymphoid cells suggested that an active process (rather than passive clonal deletion) mediates and maintains this type of tolerance. No comparable success was achieved when tolerance of isolated class I or entire H-2 haplotype disparity was examined, suggesting that clonal deletion might be operative in these combinations. Modest prolongation of skin-graft survival was observed in adoptive transfer recipients of lymphoid cells from donors tolerant of I-JECSD disparity. These data are compatible with the hypothesis that the central I region (JE) promotes tolerance induction to associated strong IA- and D-region alloantigens by activating a suppression mechanism.

A monoclonal antibody recognizes a subset of the H-2Dd mouse major class I antigens.

Binding studies and competition experiments have shown that a monoclonal antibody (mAb) named 28-8-6 recognizes only 5 to 10% of the cell surface Dd molecules. The molecules detected by 28-8-6 mAb appear to be genuine H-2Dd antigens on the basis of their MW and isolectric points. In addition, the detectability of the subset of cell surface Dd molecules by 28-8-6 does not depend on their degree of glycosylation nor on the presence of mouse beta-2-microglobulin. Several interpretations are discussed. mAb 28-8-6 might detect a particular conformation or a particular chemical derivatization of otherwise normal H-2Dd molecules. Also, because the epitope recognized by 28-8-6 lies close to the peptide binding site, it is possible that mAb 28-8-6 recognizes a subset of Dd molecules bearing a certain category of self peptides.

On naming H2 haplotypes: functional significance of MHC class Ib alleles.

George D. Snell began defining and naming the H2 haplotypes many years ago by histogenetic typing. Since then, a few haplotypes have been given an additional letter, such as bc for strain 129, to show that they are minor variants from the prototype (b). But by and large, differences in nonclassical class I antigens have been known (only?) to those in the field without being acknowledged by a separate haplotype symbol. Thus, strains BALB/c and NZB/BlNJ are both considered H2d and strains C3H/HeJ and B10.BR are both called H2k, although each pair differs in the TL and Qa1 antigens. In parallel with the interest in nonclassical class I antigens, the need for an appropriate haplotype nomenclature is growing. The haplotypes that require splitting are b, d, k, q, and s; the symbol bc should be retained and used, and, for the other haplotypes, the suffix 2 denotes a Qa1a haplotype with highly TL-positive thymocytes.

The mouse primed lymphocyte typing (mPLT)test. II. Detection of variant H-2 K and D molecules in the typing analysis of the class I-associated lymphocyte-stimulating (LS) determinants, using the B10.W lines.

The mouse primed lymphocyte typing (mPTL) assay, based on the sequential reactivation of specific immunocompetent, alloantigen-reactive T blast cells in secondary mixed leucocyte culture (MLC), has been utilized to define the major histocompatibility complex (MHC) Class I-associated lymphocyte-stimulating (LS) determinants. The test panel of secondary stimulating cells has been expanded to include 25 B10.W lines (mouse strains carrying MHC regions derived from wild mice), thus permitting examination of more than thirty 'independently derived' MHC haplotypes. Data obtained using mPLT cells generated in primary MLC between H-2 K- and/or DL-disparate strain combinations indicate that each allelic Class I product expresses a unique set of LS determinants recognized by alloreactive T lymphocytes. In addition, we have observed that Class I products, which are apparently serologically identical but which are encoded for by genes of independently derived haplotypes, can express mutually distinct LS determinants. These data suggest that T lymphocytes possess the capacity to distinguish between Class I antigens that are serologically similar (or even identical) and support the concept that LS determinants recognized by T cells are distinct from the majority of specificities recognized by B cells. This concept has marked importance in interpretation of data in the human system, for which there is now an attempt to correlate serology and PLT.

Anti-Thy-1 response of H-2f/H-2r heterozygotes: an unusual case of genetic control.

Anti-Thy-1 responsiveness of H-2r homozygous and H-2f/H-2r heterozygous mice was studied. Good responsiveness appeared to be independent of H-2 phenotype of responder but was influenced by the phenotype of the donor. These results were incompatible with the concept of Ir-Thy-1 genes controlling the response to cell-free Thy-1 in these mice. In contrast the results were indicative of the response to the cell-bound form of the Thy-1 antigen. It is proposed that good anti-Thy-1 response may reflect the presence of clones capable of recognizing the Thy-1 antigen in the context of or in association with incompatible class I H-2 molecules.

The crystal structures of K(bm1) and K(bm8) reveal that subtle changes in the peptide environment impact thermostability and alloreactivity.

The K(bm1) and K(bm8) natural mutants of the murine MHC class I molecule H-2K(b) were originally identified by allograft rejection. They also bind viral peptides VSV8 and SEV9 with high affinity, but their peptide complexes have substantially decreased thermostability, and the K(bm1) complexes do not elicit alloreactive T cell responses. Crystal structures of the four mutant complexes at 1.7-1.9 A resolution are similar to the corresponding wild-type K(b) structures, except in the vicinity of the mutated residues, which alter the electrostatic potential, topology, hydrogen bonding, and local water structure of the peptide binding groove. Thus, these natural K(b) mutations define the minimal perturbations in the peptide environment that alter antigen presentation to T cells and abolish alloreactivity.

DNA polymorphism of MHC III genes in inbred and wild mouse strains.

Genes encoding the second component (C2), factor B, and complement protein C4 and Slp (sex-limited protein) are members of the major histocompatibility complex class III gene cluster. In this report we describe isolation of a mouse C2 cDNA clone and its use together with factor B and C4 cDNA clones to examine the S region in a panel of 42 haplotypes in laboratory and wild mice representing 5 species and subspecies of Mus. Conservation of the C2 factor B gene duplex was evidenced by relatively limited polymorphism associated with speciation and nucleotide sequence homology between mouse and human C2 and factor B. The C4-Slp gene duplex, on the other hand, showed extensive polymorphism by DNA blot analysis. This polymorphism correlated poorly with the C2/factor B restriction fragment length polymorphism, suggesting independent evolution of these two segments of the S region. Taken together, these data will be of particular importance in studies of mouse strains with abnormal regulation of immune effector systems since the class III gene products are essential for activation of the complement cascade.

Suppressive effects of soluble histocompatibility antigens on the in vitro generation of cytotoxic T cells to D-end alloantigens.

Murine histocompatibility antigens were solubilized from the spleens and lungs of C57BL/6 (H-2b) animals with hypertonic salt (3 M KC1). Aggregate-free soluble antigens were incubated with nonadherent lymph node cells from BALB/c (H-2d) mice for 18 hr prior to their use as responder cells in the mixed-lymphocyte reaction (MLR). It was found that the generation of cytotoxic cells was suppressed while the proliferative response was not affected. The observed suppression was not due to a shift in the kinetics of the generation of cytotoxicity as determined throughout a 10-day culture period. The suppression was specific in that the response in MLR to unrelated H-2f stimulator cells and the subsequent generation of cytotoxic cells were unchanged. Using various H-2 recombinant strains as target cells in the assay of cell-mediated lympholysis, suppression of cytotoxicity was observed when the D end, but not the K end, was shared with the C57BL/6 strain from which the antigens were derived.

Recognition of influenza-infected cells by cytolytic T lymphocyte clones: determinant selection by class I restriction elements.

The fine specificity of virus recognition by influenza A/PR8/34(H1N1)-specific cytolytic T lymphocyte (CTL) clones was analyzed with the use of a panel of syngeneic target cells infected with five heterologous influenza A strain viruses. Forty-five H-2 D-end-restricted CTL clones from B10.A(5R) responders (Dd,Ld) demonstrated 14 different patterns of recognition. Many of these clonotypes were able to distinguish between closely related viruses of the same subtype. Such discriminatory capacity, however, was often accompanied by cross-reactivity against a distantly related viral subtype. This supports the contention that virus-specific CTL see different structures than do virus-specific antibodies. A similar analysis of the fine specificity of 60 Db-restricted clones from C57BL/6 responders was performed. The vast majority of this response was composed of clonotypes not observed in the B10.A(5R) response. In addition, the hierarchy of relatedness between the virus strain used for immunization and the various heterologous viruses was different in C57BL/6 and B10.A(5R). In contrast, the D-end-restricted response of Balb/c (Dd,Ld) demonstrated clonotypes similar to those found in B10.A(5R). These data suggest that determinant recognition in an anti-viral CTL response is a function of the H-2 restricting elements, and this is discussed in the context of determinant selection by class I molecules.

A comparison of the neonatal tolerance-inducing capacities of H-2 class I and class II antigens.

We have investigated the abilities of murine major histocompatibility complex-encoded antigens to induce in vitro hyporeactivity of T lymphocytes when these antigens are injected neonatally. Class I molecules, presented on F1 donor cells having an H-2 K or D region difference from recipients, can readily induce tolerogen-specific cytotoxic T cell hyporeactivity; as few as 1 X 10(6) neonatally injected donor cells suffice. In contrast, class II molecules, presented on F1 donor cells having an H-2 I region difference from recipients, can induce tolerogen-specific helper T cell hyporeactivity only when at least 1 X 10(7) neonatally injected donor cells are used, and then only in some of these recipients. Results from another in vitro assay system, taken in conjunction with these data, indicate that the molecular class of the tolerizing disparity, rather than the effector function of the responding cell type assayed, may be the most important factor in controlling the ease with which neonatally induced alloantigen tolerance can be achieved. In each type of tolerance described here, the hyporeactivity seen is antigen specific, in its induction and its expression; the implications of this fact for considerations of possible mechanisms of tolerance maintenance are discussed.