The response of lymphocytes to H-2D and H-2K region antigens: I. Differential effect of cortisone acetate

Cell surface antigens controlled by separate portions of the H-2 region differentially stimulate lymphocytes. Cells responding to antigens controlled by loci in or near the H-2D region transform later (5 days) than cells responding to antigens controlled by loci in or near the H-2K region (3 days). Treatment of lymphocyte donors with cortisone acetate shows that lymphocytes responding to some H-2K-associated antigens are cortisone resistant and that lymphocytes responding to H-2D-associated antigens are cortisone sensitive. Parallels are drawn between these characteristics and the lymphocytes responsible for cellular and humoral immunity.     

Immunogenetic analysis of H-2 mutations. III. Genetic mapping and involvement in immune reactions of the H-2ka mutation.

Mutation M523 (H-2ka) occurred spontaneously in strain CBA/CaLacSto and was discovered during routine skin graft testing for genetic homogeneity. By linkage and complementation tests, the mutation was previously mapped in the K end of the H-2 complex. We demonstrate that the mutation occurred in the K region, without affecting the I region in the K end of the complex. The mutant antigens cause rejection of skin grafts, stimulate cells in mixed lymphocyte culture, and function as stimulators as well as targets in cell-mediated lymphocytotoxicity. Yet, they are serologically indistinguishable from the antigens of the original strain and do not induce formation of humoral antibodies upon immunization of the CBA strain. Together with the results obtained on testing of other H-2 mutants, the data strongly support the notion that classical H-2 antigens (i.e., products of the H-2K and H-2D loci) can function as lymphocyte-stimulating determinants, and that I-region differences are not required for the induction of strong cell-mediated lymphocytotoxicity.     

In vitro induction of tumor-specific immunity. III. Lack of requirement for H-2 compatibility in lysis of tumor targets by T cells activated in vitro to oncofetal and plasmacytoma antigens.

Many recent studies have demonstrated that cytotoxic T lymphocytes (CL) activated to various antigens other than those of the H-2 complex, will lyse target cells only when H-2 compatibility exists between the CL and target cell. From these observations, it has been inferred that T lymphocytes might only be capable of responding to H-2 antigen or antigens that become associated with H-2 region gene products. Our results suggest that this is not the case, and that in some situations, cytotoxic T lymphocytes can specifically lyse target cells of different H-2 types. Two in vitro systems are described where primary induction of cytotoxic T lymphocytes to oncofetal and plasmacytoma antigens results in CL capable of lysing suitable targets bearing these antigens, of either syngeneic or allogeneic derivation. Thus it is proposed that although interaction antigens involving H-2 components may preferentially activate T lymphocytes, this does not imply a restriction on the recognition potential of T lymphocytes.     

F1 hybrid-anti-parental H-2b cell-mediated lympholysis: genetic control of target antigens by the H-2D region

The immunogenetic specificity of (C57BL/6 X DBA/2)F1 anti-parental C57BL/6 cytotoxic T lymphocytes (CTL) induced in primary mixed spleen cell cultures was determined in direct lytic and competitive inhibition assays. A large panel of peritoneal exudate cells (PEC) bearing nonrecombinant and recombinant H-2-Tla haplotypes was the source of target and inhibitor cells. All PEC of H-2b, H-2bc, H-2j, and H-2ja types, irrespective of background genetic constitution, were as susceptible to direct lysis as C57BL/6 PEC, but PEC of H-2a, H-2d, H-2k, H-2q, H-2s, and H-2u types were not. The possible involvement of the Tla region in controlling target antigens was excluded by testing PEC obtained from 4 H-2/Tla or intra-Tla recombinant mouse strains. The genes controlling target antigens were mapped to the D region with the aid of 9 intra-H-2 recombinants; for target PEC to be lysed it was necessary and sufficient that Db antigens be part of the H-2 phenotype. These results were confirmed by competitive inhibition assays. Resident peritoneal cells not exposed to fetal bovine serum were also lysed by F1 anti-parental H-2b CTL, a demonstration that target antigens are expressed on normal cells.     

Induction of I region-restricted hapten-specific cytotoxic T lymphocytes.

Murine cytotoxic T lymphocytes (CTL) reactive to TNP-conjugated syngeneic target cells do lyse to a moderate but significant extent TNP-conjugated, I region compatible but H-2K or H-2D region incompatible target cells. Antibody inhibition experiments and "cold inhibition" experiments indicate that some CTL clones recognize TNP-conjugated targets in association with syngeneic I region determinants independently of H-2K or H-2D gene products. The data suggest that besides TNP-conjugated H-2K or H-2D gene products, in principle, also TNP-conjugated I region determinants do stimulate TNP-specific CTL precursor cells and act as target antigens of TNP-specific CTL.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. III. Single histocompatibility antigens dominate the male antigen

Immunization of mice with multiple non-H-2 histocompatibility antigens results in the generation of cytolytic T lymphocytes that are specific for a limited number of immunodominant antigens. The experiments presented in this communication were designed to reveal immunodominance in pairwise combinations of autosomal and sex-linked non-H-2 histocompatibility (H) antigens. Priming and boosting responders with the male antigen, H-Y, paired with the H-4.2, H-7.1, or H-3.1 antigens, resulted in the generation of cytolytic T cells specific for the autosomal H antigens but not the H-Y antigen. Furthermore, co-immunization and boosting of C57BL/6 female responder spleen cells with BALB.B male cells resulted in the generation of cytolytic T cells specific for the BALB.B immunodominant antigens but not H-Y. No dominance was observed in H-4-plus H-7-incompatible combinations. Co-immunization of three different H-3 congenic strains with H-3.1 plus H-Y demonstrated that an efficient anti-H-3.1 T cell response is required for observing H-3.1 immunodominance over H-Y. Co-expression of H-3.1 and H-Y on the same priming and boosting cells was required for immunodominance. In fact, immunization with H-3.1 and H-Y presented on different cells resulted in normal generation of H-Y-specific cytolytic T cells, but no generation of H-3.1-specific cytolytic T cells resulted unless H-Y-specific cells were stimulated in the mixed lymphocyte cultures. These observations suggest that in vitro T cell responses to paired, non-H-2 H antigens may be independent, competitive, or synergistic, depending on the identity of the antigens and the priming and boosting conditions.     

Properties of H-2L locus products in allogeneic and H-2 restricted, trinitrophenyl-specific cytotoxic responses.

The role of the recently defined L antigen (a second D region product) in allogeneic and TNP-specific syngeneic primary CML responses has been investigated. The lysis by anti-L specific cytotoxic effector cells was not inhibited when the target cells were pretreated with an antiserum directed against K and D, whereas an antiserum against L completely abrogated this response. Therefore, H-2L products are recognized on the target cell independently of H-2K and H-2D locus products. Both A.SW cells as well as B10 cells were found to respond to Ld alloantigens, in addition to Dd alloantigens when stimulated by cells differing only in the D region. The results of cold target blocking and antiserum inhibition experiments failed to detect cytotoxic cells with specificity of L antigens in association with TNP, under conditions in which TNP-specific effectors to K and D antigens were demonstrable. These findings suggest that there is a more limited involvement of H-2L locus products than the H-2K or H-2D locus products in the induction and specificity of these responses.     

Biochemical evidence for a separate,MHC-linked locus encoding H-2.28 antigens

In comparing the tryptic peptide maps of the H-2L and H-2D glycoprotein antigens isolated from NP-40 lysates of RADA1 (H-2 ^a ) leukemic cells, no more than 37% of the observed arginine-containing tryptic peptides are found to be homologous. Thus, the primary amino-acid sequences of these two antigens are probably less than 90% homologous. This constitutes the strongest evidence to date that the MHC-linkedH-2L region encodes H-2L antigens separately from theH-2D region, even though H-2L antigens bear D-end-associated antigenic determinants of the H-2.28 family. The anti-H-2.28 alloantiserum (k×r anti h2) used to precipitate H-2L antigens in this investigation was the NIH contract antiserum D28b. As the tryptic peptide maps also surprisingly revealed, D28b precipitates H-2D antigens as well and, thus, anti-H-2.4 immunoadsorbants were employed to isolate H-2L free of H-2D antigens. In light of the dual specificity of D28b, its reactivity with BALB/c-H-2 ^dm2 mutant cells was re-examined. Even though mutant lymphocytes, which lack H-2L but not H-2D antigens, are not cytotoxically lysed by D28b (as are parental H-2^d cells), D28b appears to precipitate H-2D antigens from NP-40 extracts of mutant splenocytes.     

Generation of cytotoxic lymphocytes in mixed lymphocyte reactions II. Importance of private and publicH-2 alloantigens on the expression of cytotoxicity

MLC were established to test for the generation of specific cytotoxic effector cells in CML. The target cell used to assay for CML in the five combinations tested was of a differentH-2 haplotype from the stimulating cell population. Cytotoxicity was observed against this target only when it shared private alloantigens (antigens that are specific for theH-2D andH-2K region of differentH-2 haplotypes) with the stimulating cell population. Very weak or no Cytotoxicity was found when such alloantigens were not shared, although cross-reactive publicH-2 specificities were. These findings indicate that T cells display a cytotoxic potential against privateH-2 antigens in a primary response in vitro and are not capable of responding to publicH-2 specificities to the same level.BSS balanced salt solution - CML cell-mediated lympholysis - GPC guinea pig complement - ¹²⁵IUdR ¹²⁵I-iodo-deoxyuridine - MLC mixed lymphocyte culture - SE standard error     

Expression of the I-E target antigen for T-cell killing requires two genes

The H-2Ik region encodes at least two different target antigens for unrestricted T-cell mediated killing. The first is controlled by the I-A region alone and the second depends on a pair of alleles, one located to the left of I-B (presumably in I-A) and the other to the right of I-J (presumably in I-E). Hence, effector cells nominally specific for a product of the I-E region do not kill target cells with the same I-E region as the stimulator unless the I-A region is also shared. Some effectors specific for H-2Ik, such as A.TH anti-A.TL and B10.A(4R) anti-B10.A(2R), cross-react with B10.A(3R) and B10.A(5R) target cells. A product of the H-2b haplotype was shown to complement products of the H-2d or H-2k haplotypes in forming this cross-reactive determinant. The results are consistent with recent biochemical data on the component chains of Ia antigens.     

Synergistic interactions between lymph node and thymus cells in response to antigenic differences limited to selected regions of the H-2 complex.

Thymus and lymph nodes from the A.TL recombinant line were utilized as sources of responding cells in MLR (mixed lymphocyte response) assays to MHC-determined (major histocompatibility complex) antigenic differences. Cells from both sources were stimulated to proliferate by antigenic determinants controlled by the H-2K region alone, H-2D region and the H-2I-H-2S regions. Nylon-fiber-adherent splenic cells from each of the stimulating cell strains stimulated T-cell-dependent responses. Synergistic interactions between A.TL thymus and lymph node cells were initiated by antigenic products limited to single H-2 regions. Antigenic differences determined within the H-2I region were not required for synergistic responses to H-2K-controlled products or for the generation of cytotoxic killer cells to H-2D-associated antigens. The H-2I-region-associated products also were very effective in stimulating T-cell synergy. These data demonstrate that the two responsive T-cell subpopulations can both be stimulated by alloantigens coded within a single known H-2 region.     

Synthesis and immunochemical study of the antigenic determinant of the H-2Db molecule of the murine major histocompatibility complex

Primary structure of murine class I histocompatibility antigens has been analysed to select possible antigenic determinant. Hexapeptide Leu-Gln-Gln-Leu-Ser-Gly, homologous to the region 95-100 of the H-2Db antigen heavy chain, was synthesised by stepwise elongation of peptide chain beginning from the COOH-terminal Gly. Rabbit anti-hexapeptide antibodies were obtained and shown to interact specifically with purified H-2Db antigen as well as with the native antigen on cell surface. These antibodies bind to lymphocytes of H-2b haplotype (C57BL/6 mice) but not H-2d (BALB/c) or H-2k (CBA). These data suggest that the region 95-100 is responsible for serologic differences between the alleles of H-2 antigens, i.e. it may be a xenotypic as well as an allotypic antigenic determinant. The latter was confirmed by study of interaction of the hexapeptide with allogeneic monoclonal antibodies specific to H-2Db antigen.     

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.     

The specificity and significance of the inhibition of Fc receptor binding by anti H-2 sera

The possibility that Ia antigens are unique among H-2 antigens in their relationship to the Fc receptor was investigated in an EA rosette assay. Antibody specific for antigens in various regions of theH-2 complex was incubated with mouse cells, and the ability of the cells to form rosettes with antibody-coated chicken erythrocytes was tested. Antibody raised against the H-2 antigens of Ia-negative tumor cells was highly effective in inhibiting rosette formation. A variety of antisera againstK-, I-, andD-region antigens tested in recombinant mice inhibited EA rosette formation, suggesting that antigens in each of these regions could be detected in rosette inhibition. The F(ab′)₂ fragments of all antisera tested also produced specific EA rosette inhibition. Finally, antibody against Ia antigens failed to inhibit bone marrow RFCs, although antibody against H-2K and H-2D antigens did inhibit. Although H-2 serology is in a state of rapid change at present, it must be concluded that in this assay, antibody against antigens in theK andD regions as well as theI region can inhibit EA rosette formation. Inhibition of these rosettes by anti H-2 sera is therefore not due to a special association of Ia antigens with Fc receptors.     

Epitopes associated with the MHC restriction site of T cells. I. Selective expression of Iat epitopes on H-2-restricted helper T cells

We previously established monoclonal antibodies (mAb) that are putatively directed to the I region of H-2k but are reactive only with T cells. Because of their specificity to the unique epitopes different from class II antigens, they are designated as anti-Iat reagents. The present study demonstrated that these anti-Iat inhibit the H-2k-restricted helper T (Th) cell function by acting on the very H-2 restriction site of both H-2k and H-2kxb F1 T cells. This was determined by both the cytotoxic treatment and blocking of antigen-primed Th cells. In the F1 Th population, only those restricted to H-2k were eliminated, leaving the H-2b-restricted Th cells uninhibited. The inhibition of the response was not due to the induction of suppressor T cells, but to the elimination of the function of radioresistant Lyt-1+,2- Th cells. Iatk epitopes were also found on an H-2k-restricted but not on H-2b-restricted Th cell clone established from the same H-2kxb F1 animal. None of the anti-Iatk were reactive with class II antigens on B cells. These results indicate that Iat epitopes are not directly encoded by the I region genes, but are associated with the H-2 restriction site of T cells, which see the self class II polymorphism. Thus, Iat epitopes are expressed clonally in high frequency on H-2k-restricted Th cells of F1, being excluded from the H-2b-restricted Th population. The relationship between Iat and T cell receptor molecules is unknown.     

Characterization of Ia antigens in mouse serum.

Sera obtained from normal B10.BR mice were shown to inhibit selectively a specific anti-Ia alloantiserum.Partial purification of the Ia antigenic activity was accomplished by isolation of the high density lipoproteins from these sera by fractional precipitation with sodium phosphotungstate and MgCL2. Both H-2.23 and Iak antigens present in this high density lipoprotein fraction were completely adsorbed by rabbit anit-rat beta2-microglobulin immunoadsorbents, whereas specific anti-H-2.23 immunoadsorbents removed only the H-2 activity. These data deomnstrate that Ia antigens, like H-2 antigens in the sera of B10.BR mice are associated with high density lipoproteins and further suggest that both H-2 and Ia antigens are associated with a beta2-microglobulin-like molecule.     

Kidney alloantigens determined by two regions of the rat major histocompatibility complex

Recombination inH-1, the major histocompatibility complex (MHC) of the rat, has defined two regions,H-1A andH-1B, which determine antigens apparently homologous to the KJD and Ia antigens of the mouse, respectively. Alloantisera directed at these antigens have been absorbed with kidney homogenates. The results showed that cells in the kidney express serologically detectable MHC antigens determined by both theH-1A andH-1B region. Control absorptions indicated that to account for these results in terms of recirculating lymphocytes, two perfused kidneys would need to contain more than 60 percent of the recirculating lymphocyte pool. It appears likely, therefore, that H-1B antigens are expressed by cells resident in the kidney.     

Detailed analysis of the mouse H-2Kb promoter: enhancer-like sequences and their role in the regulation of class I gene expression

Sequencing and deletion analyses of the H-2Kb promoter have suggested that several regions may be important for expression and regulation of this gene. Two of these regions are conserved inside the promoter of several genes coding for classical transplantation antigens, but not in the promoter of class I genes located in the Qa region. They display enhancer-like activity in cells that express H-2 genes, but show some tissue specificity in that they function very poorly in undifferentiated embryonal carcinoma cells in which H-2 genes are not expressed. They also have been shown not to be the target of the adenovirus-12 induced repression of class I gene expression recently demonstrated by Schrier et al. The promoter of the beta 2-microglobulin gene also contains a sequence with enhancer-like activity, but shares no homology with the H-2Kb promoter region.     

Effect of thymocyte-stimulating factor-containing supernatants on the surface antigens of murine thymocytes.

Incubation of murine thymocytes in thymocyte-stimulating factor (TSF)-containing supernatants causes a four- to fivefold increase in the expression of the H-2^k and H-2^d antigens and a similar decrease in the expression of the TL antigen (in TL⁺ strains) on the surface of these cells. Experiments with antisera directed toward the private H-2K and H-2D antigens showed that TSF-containing supernatants cause approximately the same increase in the expression of the H-2K and H-2D antigens of thymocytes of the d and b haplotypes. With thymocytes of the k haplotype, only an increase in the expression of the H-2D antigens takes place, while no significant increase was found for the H-2K antigens. TSF-containing supernatants cause no significant change in the expression of the following antigens on the surface of thymocytes: Thy-1.1, Thy-1.2, Ly-1.2, Ly-2.2, Ly-6.2, Th-B, Ia-1,2,3,7, and G_IX. A factor similar to murine TSF, produced by human peripheral blood leukocytes, does not affect appreciably the expression of the H-2 antigens on the surface of murine thymocytes. The factor(s) causing the increased expression of the H-2 antigens on the surface of murine thymocytes appears to be produced by T lymphocytes. The factor(s) is eluted from a Sephadex G-100 column in at least two broad peaks with molecular weights of 300,000 and 90,000-25,000. Most of the activity enhancing the expression of the H-2 antigens is lost at pH 2, while most of it is maintained at pH 11.5 and at 56 °C. On the basis of these properties, it is concluded that the factor under study is probably different from the factor enhancing the phytohemagglutinin responsiveness of thymocytes.     

F1 hybrid anti-parental H-2k cell-mediated lympholysis. I. Stimulator and target determinants controlled by the H-2K region.

H-2k-heterozygous F1 hybrid mouse spleen cells cultured with irradiated H-2k-homozygous stimulator cells generated specific anti-parent cytolytic effectors. The parental antigenic determinants recognized by responder cells during induction (afferent arm) and by effector cells during cytolysis (efferent arm) were coded for, or regulated by, the H-2K-Hh3 region of the MHC, according to recombinant analysis. There were no detectable influences by other linked or unlinked genes on the phenotypic expression of parental antigens; however, the anti-parent responsiveness was modulated by background genes of responder cells. These experiments establish that the K end of H-2 controls determinants of F1 anti-parental H-2k CML, like the D end controls those of F1 anti-parental H-2b CML, thus confirming the basic symmetry of the H-2 complex. The relationship of this primary in vitro cell-mediated response with natural in vivo resistance to parental and allogeneic bone marrow grafts is discussed.