H–2L: Demonstration of Four New Allelic Products and Independence of H–2D and H–2L Molecules

The H-2L molecules were detected for the first time in the Dd region products using antisera against the public specificity H-2.28. This specificity was analyzed because its presence in K as well as in D region products and its apparent allelism with another public specificity, H-2.1, indicated that these two specificities may have a special position may have a special position in the H-2 system. This was corroborated by subsequent identification of H-2L molecules in Dq and Dk products using anti-H-2.28 AND ANTI-H-2.1 sera, respectively, while none of the other previously known public or private specificities was detected on H-2L molecules. We tested the products of four D region alleles which had not been analyzed previously. In each of them we identified two distinct types of molecules: H-2D, which reacts with sera agains the D region private specificity, and H-2L, which does not react with these sera, but which is detectable either by anti-H-2.28 sera (H-2Lb, H-2Lf, H-2LS) or by anti-H-2.1 sera (H-2Ldx). This increases the number of identified H-2L alleles to seven (five H-2.8+, two H-2.1+). No association between H-2D and H-2D and H-2L molecules on the cell surface was detected in capping experiments.     

The private specificity H-2.4 and the public specificity H-2.28 of the D region are expressed on two independent polypeptide chains.

The antigenic specificities H-2.4 (private) and H-2.28 (public) in the H-2a haplotype are controlled by the D region of H-2 as defined by the available recombinants. In previous studies we have demonstrated by the antibody-induced redistribution method that the antisera against these specificities contain antibodies against at least two different polypeptide chains. We here report the results of the indirect immunoprecipitation of radiolabeled antigens after solubilization with Nonidet-P40. The antisera against the two specificities precipitated from such extracts two different and independent polypeptide chains, indicating that the products of the D region, as presently defined, comprise at least two different molecules. The molecular weight of both chains is approximately 45 000, which is similar to other molecules bearing private H-2 antigenic specificities. Consequently, the chromosomal segment presently defined by recombination studies as the D region, must contain another locus, controlling the second polypeptide chain which is detectable by anti-H-2.28 antisera, besides the H-2D locus which controls the polypeptide chain bearing the private specificity H-2.4 as well as most of the public specificities.     

Specificity of Cell-Mediated Lympholysis for Public and Private H-2 Determinants

The specificity of cell-mediated lympholysis in the mouse was investigated by studying the cross-reactivity of in vitro-generated effector cells against targets carrying H-2 haplotypes different from that of the stimulator. Congenic resistant partners of the C57BL/10 strain carrying ten different, independently derived haplotypes were tested in more than 600 combinations. Strong cytotoxicity was observed when stimulator and target cells shared a private determinant, but considerable cross-killing was also seen in combinations where stimulator and target cells shared only public determinants. Cross-killing is specific and can be blocked only by unlabeled cells of the stimulator or target strain. Although cross-killing does not correlate with the number of stimulating public determinants carried by the target individual determinants 5, 15. 25, and 27 + 28 + 29 are associated with increased cross-killing. Cross-killing can occur both at the K and the D end of H-2 and is not explained by antigens determined by the I region. We conclude that the serologically defined H-2K and H-2D molecules are the primary targets for cytotoxic T lymphocytes but that they may be perceived differently by T and B lymphocytes.     

Inhibition of killer-target cell interaction by monoclonal anti-H-2 antibodies.

The sites on target cells with which cytotoxic T lymphocytes interact were characterized using three different monoclonal BALB/c anti-CBA antibodies derived from plasma cell hybrids. The antibodies all reacted with H-2K^k and appeared to recognize public specificities H-2.5,11 and 25, respectively. Allogeneic killing directed at products of H-2K^k was inhibited by all three antibodies, irrespective of the H-2 haplotype of the responder; cytotoxicity directed at products of another allele, H-2K^d, or of the H-2 D region on the same target cell was not affected. The antibodies did not inhibit killer cells carrying H-2K^k. Cytotoxic reactions against minor histocompatibility antigens, including the male-specific antigen H-Y, were also blocked by all three monoclonal antibodies when restricted by H-2K^k, but not when restricted by H-2D on the same target cell. Cytotoxic T lymphocytes thus appear to interact with their target via the same, serologically defined H-2K^k molecule which carries public specificities, whether they recognize it as an alloantigen or as self. This argues against the existence of separate H-2 K-encoded molecules recognized by killer cells only and against H-2 specific modifications of minor histocompatibility antigens as the basis of H-2 restriction. One of the antibodies, 27 R9, which reacted with H-2K^k and H-2′ and was thought to recognize specificity H-2.25, showed a weak cytotoxic reaction but bound with a high titer to H-2 D^k, a reaction that has not previously been described. This antibody selectively and with a very high titer inhibited male-specific cytotoxicity restricted by H-2D^k, but did not significantly interfere with allogeneic killing against products of the H-2 D^k region nor apparently with H-2D^k- restricted cytotoxicity specific for other minor antigens. The results suggest the existence of at least two different restriction elements controlled by the H-2D^k region.     

Incompatibility at Irrelevant H-2 Specificities Augments In Vivo Stimulation of Alloaggressive Cells

Lymphoid cells of mice were sensitized in vivo either by H-2 strain-specific tumor allografts or by activation in lethally irradiated F1 hybrids and tested for cytotoxicity on 51Cr-labeled target cells. The release of 51Cr varied linearly with the logarithm to the proportion of effector lymphocytes to target cells and with the time of interaction. The release of 51Cr was immunologically specific and restricted to H-2 incompatibility. Spleen cells immune to public specificities of the target genotype were not cytotoxic. However, lymphoid cells immune to only one private specificity of a third-party target genotype were highly cytotoxic. The cytotoxicity of activated thymus cells on target cells sharing one private specificity with the genotype used for sensitization was significantly enhanced when the effector thymocytes were activated also against H-2 specificities not shared by the target strain. The results suggest that gene products that facilitate sensitization of effector cells may be determined both by the H-2K and the H-2D end of the H-2 complex. It remains to be shown whether the products of these loci, operating during sensitization in vivo, are body-wide correlated to the lymphocyte-defined specificities detectable during the mixed leukocyte culture interaction.     

Monoclonal Anti Thy 1.2 Antibodies from Hybridoma Ho13-4 do not React with Mouse Natural Killer Cells

The susceptibility of NK cells and immune cytotoxic T-cells to treatment with (a) monoclonal anti thy 1.2 antibodies from hybridoma HO13-4, (b) rabbit anti-mouse T-cell antiserum and (c) gamma globulins prepared from AKR/J anti C3H/HeJ antiserum was studied in the presence of rabbit complement. Monoclonal anti thy 1.2 antibody treatment completely abolished the cytotoxic activity of immune T-cells derived from C57BL/6J mice (H-2^b) immunized with (C57BL/6J x DBA/2)F₁spleen cells (H-2^bd) against P815 (H-2^b) target cells. The same treatment had no significant effect on the NK activity of spleen cells from unimmunized mice against YAC target cells. Rabbit anti-mouse T-cell and mouse anti theta antisera also abrogated completely the immune T cell activity of spleen cells. This treatment however also resulted in a partial loss of NK activity. These results indicate that conventional anti theta antisera contain antibodies which recognize antigenic specificities on T-cells as well as on a population of NK cells. The cross reactivity is not a result of thy 1.2 antigen expression on NK cells and T-cells as recognized by the monoclonal antibodies. The specificity recognized by the monoclonal antibody (HO13-4) is only expressed on T-cells.     

Cytotoxic T lymphocytes generated across and I-Ab mutant difference are directed against a molecule bearing Ia antigens

The recently discovered B6.C-H-2bm12 (bm12) histocompatibility mutant bears a mutation of the I-A subregion of the H-2b haplotype. We have studied cytotoxic T lymphocytes (CTL) generated in secondary mixed lymphocyte culture (MLC) between the bm12 mutant and the strain of origin C57BL/6 (B6). In both directions, strong CTL responses were generated against lipopolysaccharide-stimulated, but not against concanavalin A-stimulated target cells. Studies of the specificity of the bm12 anti-B6 CTL response, using target cells from a panel of H-2 recombinants, showed that the CTL were directed against specificities of the I-Ab subregion. This conclusion was confirmed by the observation that the bm12 anti-B6 CTL could be specifically blocked by anti-Iab, but not by anti-Kb antisera. EL4 tumor cells, which express Kb and Db but not Iab antigens, were not lysed by the CTL. The combined data confirm that bm12 is an I-A subregion mutant and indicate that the I-A subregion antigens, recognized by antibody and those recognized by CTL, are probably present on the same molecules. The CTL effector cells generated in secondary MLC across the I-Ab mutant difference had the surface phenotype Lyt 1- 2+.     

H-2d-LIKE SPECIFICITIES ON GARDNER (H-2k) TUMOUR DETECTED IN A RESTRICTED ANTI-TUMOUR REACTION

Cytostatis of the H-2^d tumour LSTRA by H-2-restricted effector lymphocytes was inhibited by antisera against H-2.4 and H-2.31 but not by antisera against public specificities or non-H-2 antigens. The unexpected reaction of the same effector cells against Gardner tumour (H-2^k) was also shown to be inhibited by a combination of antisera against H-2.4 and H-2.31 but not by each antiserum used separately. The inhibitory capacity of these antisera was removed by absorption with B10.D2 but not with B10 lymphocytes. This indicated the presence of H-2^d-like specificities on gardner tumour which could function as self-recognition structures in an H-2K^d and H-2D^d restricted system.     

Fine structure of receptors for H-2 antigenic specificities as measured in the PAR assay

Recognition of a distinct antigenic specificity, H-2 transplantation antigen 32, could be demonstrated in a congenic strain of mice, using the PAR test. In another congenic strain of mice, recognition of a group of 15 such H-2 specificities was demonstrated. Both recognition processes could be blocked either by treating target cells, representing the appropriate antigens, with corresponding alloantisera or by treating aggressor cells, carrying receptors for these antigens, with the appropriate anti-receptor (anti-RS) antibodies. Antigens recognized in the two strain combinations were of different H-2 specificities. No cross-reactivity of inhibitory antisera was found. This indicates that lymphoid cells possess highly specialized receptors which enable competent cells to recognize individual antigenic specificities.     

Education of human lymphocytes against mouse cells: specific recognition of H-2 antigens.

Xeno-sensitization of human peripheral blood lymphocytes (PBL) against mouse lymphoid cells has been studied in vivo in a local graft-vs. -host (GVH) assay and in vitro in a mixed lymphocyte culture-cell-mediated lympholysis system. Human PBL were injected into the footpads of mice rendered unresponsive by total-body irradiation, and these were subsequently tested for the presence of cytotoxic cells in the draining popliteal lymph node (LN). In spite of a definite PBL-induced LN proliferation, no cytotoxic activity was detected against mouse target cells. In contrast with the GVH assay, human PBL collected after 7 days of in vitro culture in the presence of irradiated mouse cells, were strongly cytotoxic against mouse target cells. The antigenic specificities recognized by in vitro educated cells were primarily those coded for by the H-2 complex. Only target cells with an H-2 haplotype identical to that of the sensitizing mouse strain or with at least an H-2 D end in common with that strain were killed by xeno-sensitized lymphocytes. Mouse target cells derived from congenic resistant strains remained unaffected. It was verified that in vitro educated PBL depleted of B cells retained their cytotoxic effect, indicating that non-B cells and probably T cells were involved in the response.     

The H-2 Restriction Specificity of Cytotoxic T Cells Derived from Intrathymic Precursors

Both cortical and medullary thymocytes could give rise to both allospecific and hapten-specific cytotoxic T lymphocytes (CTLs) if the cultures contained the T-helper-cell-replacing hormone, interleukin 2 (IL-2). The H-2 restriction specificity of these CTLs was tested by using three different specificity determinants that are known to be influenced by the H-2 genotype of the thymus (rather than by the H-2 of the precursor cell itself): the cross-killing of trinitrophenyl (TNP) coupled allogeneic targets in anti-TNP self responses; the dominance of H-2k-restricted CTLs over H-2d-restricted CTLs in the response of (H-2k X H-2d)F1 mice to TNP-self; and the capacity of alloreactive CTLs to cross-reactively lyse TNP-coupled syngeneic cells. The experiments show that, with regard to all of these specificity determinants, CTLs derived from these intrathymic populations are similar to CTLs derived from the adult mouse spleen. These data suggest that the H-2 restriction specificity is dictated before or at the same time that the cell acquires the capacity to respond to allogenic or haptenated syngeneic cells.     

Specificity of anti-HLA-B27 cytotoxic T lymphocytes

Sub-types of HLA-B27 were detected by cytotoxic T lymphocytes (CTL) generated between HLA-A, -B- and -C-identical B27-positive individuals. We now report the specificity of six independent CTL's generated by mixed lymphocyte culture (MLC) of HLA-A, -B and -C serologically identical B27-positive responder and stimulator cells. Three CTL's recognize one sub-type, and three the other. The combined reactivity of all CTL's allows unequivocal "typing" of B27-positive cells for the two different sub-types B27K and B27W. The specificity of two CTL's was analysed by cold-target inhibition. The results indicate that (1) no further sub-types of HLA-B27 can be detected by the CTL's raised in these combinations; (2) the majority of the CTL's is directed against the B27 antigens; and (3) "extra reactions" on B27-negative cells are caused by a subset(s) of CTL's recognizing unknown antigens shared between stimulator and target cells. CTL's raised by stimulation of HLA-B27-negative responder cells with B27-positive cells of either sub-type lysed all B27-positive target cells indiscriminately. In cold-target inhibition, however, B27-positive cells, carrying the sub-type of B27 different from that of the stimulator, could not inhibit the lysis of cells bearing the stimulator sub-type of B27. This indicates the activation, in B27-negative responders, of at least two different groups of CTL clones, one directed against shared determinants of HLA-B27, and one against the HLA-B27 sub-type. Heterogeneity of the HLA-B27 antigen may have implications for studies on the well-known association between this antigen and various diseases.     

Cell-mediated cytotoxicity against ectromelia virus-infected target cells. III. Role of the H-2 gene complex

The role of the H-2 gene complex in expression of cytotoxicity exerted by specific ectromelia-immune thymus-derived (T) cells against ectromelia-infected target cells was examined. A repertoire of inbred mouse strains (some congenic) including the H-2 haplotypes k, d, b, s, q, the recombinant H-2^a (k|d) and F₁ hybrids (k/b and d/b) were immunized with virus and their spleen cells tested 6 days later, at the peak of the primary response, against H-2^k, H-2^d and H-2^b target cells. Significant specific cytotoxicity occurred only when the immune cell donors and the target cells shared all or part of the same H-2 gene complex. For example, H-2^a (k|d) immune cells killed both H-2^k and H-2^d target cells. There wasno detectable effect of the non-H-2 genetic background, H-2 public specificities, or the M-locus Target cells infected with ectromelia virus exhibited quantitative or qualitative changes (or both) in expression of normal H-2 antigens as indicated by reduced susceptibility to killing by T cells activated against H-2 antigens in mixed lymphocyte culture. These data are consistent with the hypothesis that T cells in this system are responding to virus-induced, specific changes in antigens on infected cells which are controlled by genes in the H-2 complex; these genes seem likely to be those coding for H-2 private specificities, or genes closely linked to them.     

Recognition by major histocompatibility complex class I-restricted cytolytic T lymphocytes of cells expressing vaccinia-encoded viral and class I proteins

Target cells expressing influenza virus hemagglutinin (HA) could be recognized by cytotoxic T lymphocytes (CTL) in conjunction with the murine major histocompatibility complex class I antigen, H-2Kd, when both antigens were encoded by recombinant vaccinia virus. This recognition occurred if HA and H-2Kd were encoded by separate vaccinia viruses following dual infection of target cells or if HA and H-2Kd were encoded by a single recombinant virus. In contrast, target cells expressing nucleoprotein (NP) were only recognized by H-2Kd-restricted CTL if both NP and H-2Kd were encoded by the same vaccinia virus. These results show that the requirements for association of H-2Kd with different viral antigens derived from HA or NP can vary. Possible factors contributing to this difference are discussed.     

Type-specific reovirus antiserum blocks the cytotoxic T-cell-target cell interaction: evidence for the association of the viral hemagglutinin of a nonenveloped virus with the cell surface.

It has previously been demonstrated that spleen cells from mice immunized with reovirus type 1 or 3 generate virus-specific cytotoxic T lymphocytes (CTL) after in vitro restimulation. Such cytotoxic T cells lyse H-2 identical targets that are infected with the appropriate reovirus type. Viral recombinants were used to demonstrate that the S1 gene is the predominant viral gene determining the specificity of the CTL. Reoviruses are nonenveloped, non-membrane-maturing viruses; therefore, it was important to determine whether viral products were being recognized by CTL on the surface of target cells. Antiserum blocking was utilized to investigate this issue. Using viral recombinants and antisera to reoviruses types 1 and 3, we were able to demonstrate that the major viral antigen recognized by the CTL on the target cell surface is the sigma 1 polypeptide encoded by the S1 genome segment. Thus, viral antigens on the target cell membrane seem to be important in the CTL response to a nonenveloped, non-membrane-maturing virus.     

The Detection of Ia Antibodies in Polyspecific H-2 Alloantisera Absorbed with Erythrocytes

H-2 alloantisera contain Ia antibodies whose detection is obscured by the presence of serodominant K, D specificities. A method is described whereby Ia antisera can be prepared by absorbing conventional H-2 sera with donor-strain erythrocytes. The analysis of such absorbed antisera has led to the identification of the new specificity Ia. 16, and the redefinition of the specificities H-2.34 and H-2.46 as complex Ia specificities which map in the I-A region of the H-2 complex. A number of other so-called H-2 specificities also appear to have Ia-like properties.     

Major histocompatibility complex--dependent T cell epitopes of lymphocytic choriomeningitis virus nucleoprotein and their protective capacity against viral disease

In mice the immune response to infection with lymphocytic choriomeningitis virus (LCMV), a member of the arenavirus family, is mainly based on the activity of cytotoxic T cells. The immunogenic epitopes of the viral nucleoprotein recognized by cytotoxic T cells in various inbred strains of mice were defined. These epitopes were located in H-2d and H-2q mice in the amino-terminal region and in H-2b mice in the carboxy-terminal region of the nucleoprotein. A detailed analysis with synthetic peptides allowed the definition of a common epitope of 9 amino acids in H-2d and H-2q mice and of about 15 amino acids in H-2b mice. These T cell epitopes were all recognized in association with H-2 D or L transplantation antigen. The protective capacity of recombinant vaccinia viruses expressing these epitopes was documented by assaying prevention of virus replication, protection against LCM and prevention of the local footpad swelling reaction. Thus, distinct T cell epitopes on the same internal viral protein mediate protection in a major histocompatibility complex-restricted manner.     

Specificity of cell-mediated transplantation reactions: II. Studies with a sensitive assay of cell-mediated cytotoxicity

To study the specificity of transplantation immunity in vitro the capacity of two aggressor cell populations from allografted mice to destroy target cells obtained from a panel of strains having different H-2 types was assessed. ⁵¹Chromium-labelled macrophages were found to be suitable target cells to allow for quantitative comparisons of cytotoxicity. Spleen cells from skin-grafted mice lysed only target cells of strains which shared at least one H-2 region (K or D) with the donor. By contrast, non-adherent peritoneal exudate cells, produced as a consequence of ascites tumour allografts, exhibited a cytotoxic potential approximately 16- to 32-fold greater than the above spleen cells and were capable of lysing third-party target cells of strains having disparity with the graft donor at both H-2 regions. The difference between the two systems appears to be quantitative. These findings appear to reconcile previously conflicting observations, and suggest minimal or no participation of public H-2 specificities in cell-mediated reactions.     

H-2 Private Specificity of In VivoSensitized Lymphocytes Studied by Adsorption on Fibroblast Monolayers

Spleen cells or thymocytes immune to one H-2 genotype are cytotoxic to allogeneic target cells of a third-party genotype, provided the target shares private specificities determined by either the H-2K or the H-2D locus of the H-2 complex with the H-2 genotype used for sensitization in vivo. The present results on elimination of cytotoxicity to specificities determined by one of the H-2 loci after adsorption of the immune lymphoid cells onto fibroblasts bearing this specificity suggest a state of diversity among effector lymphocytes reactive to the antigens determined by either the H-2K or the H-2D locus of the H-2 complex. Thymus cells activated in vivo were less specifically adsorbable than spleen cells of tumor-allografted mice.     

Differences in the expression of histocompatibility antigens on mouse lymphocytes and tumor cells: immunochemical studies.

Immunochemical studies have shown that labeled, detergent-solubilized extracts of SL2 (H-2d) lymphoma cells contain components reactive with several anti-H-2 alloantisera of restricted specificity. Anti-H-2k and anti-H-2ja as well as anti-H-2d sera precipitated labeled polypeptides of a molecular weight similar to that of H-2 heavy chains. In addition, all antisera tested precipitated a component of 70000 daltons molecular weight, which is antigenically related to gp 69/71 of Friend murine leukemia virus. Reactions with antisera directed against haplotypes other than H-2d could be blocked by addition of unlabeled, detergent-solubilized extracts of H-2d lymphocytes, or by H-2 antigens against which the antiserum was directed. Sequential immunoprecipitations initially using antisera against the K, D, or L region gene products to remove individual known H-2d antigens have made possible the identification of some molecules responsible for these reactions. The results show that antisera against haplotypes other than H-2d which react with SL2 cells, cross-react with normal H-2d antigens. Quantitative absortion of these antisera with intact or solubilized cells has shown that lymphocytes and tumor cells differ in their expression of some H-2 determinants. The antibodies bind only weakly to intact H-2d lymphocytes, but strongly to the corresponding detergent-solubilized antigens. These results do not, therefore, support the derepression hypothesis put forward earlier.