SEROLOGICAL AND IMMUNOCHEMICAL STUDIES OF H-2 ALLOSPECIFICITIES ON k36, A SYNGENEIC TUMOUR OF AKR

Expression of H-2 antigenic specificities on K36, a spontaneous leukaemia originating from AKR (H-2^k) mice, was studied by serology and immunochemistry. Two ascites lines of the tumour, as well as a tissue culture adjusted and cloned tumour line, were used in these studies with similar results being obtained. K36 expresses on its cell surface D-region encoded H-2K antigens but does not express K-region encoded H-2K alloantigens. It also expresses on its cell membrane, H-2 specificities of foreign haplotypes not present on normal AKR lymphoid cells. The molecular basis of the H-2D^d specificity on K36 (H-2^k was analysed by immunoprecipitation and polyacrylamide gel electrophoresis. The specificity was shown to be present on a glycoprotein of apparent molecular weight 45,000. However, antisera against the H-2D^d private specificity (H-2.4) precipitate additional glycoprotein of 45,000D and also 70,000D. In tryptic peptide maps of the isolated 45,000D fraction precipitated by anti-H-2.4 serum from radiolabelled K36 glycoprotein, all H-2D^d specific peptides were present in the same quantitative ratio. This is consistent with the structural identity of the foreign H-2D^d from the K36 tumour with normal H-2D^d and supports the hypothesis of a regulator system controlling the H-2 allelism. Under certain circumstances such a system could cause suppression of one and derepression of the other H-2 gene products.     

EXPRESSION OF FOREIGN H-2-LIKE ANTIGENS BY A CHEMICALLY-INDUCED MURINE TUMOUR (MCG4)

Alien H-2^k-like antigens were found to be expressed by a methylcholanthrene induced tumour of BALB/c (H-2^d) origin. H-2 specificities of the k haplotype were detected on this tumour by a variety of serological techiques, including ⁵¹Cr-release cytotoxicity, microradioassay and absorption. The antisera employed were conventional polyspecific alloantisera, typing sera with restricted specificty and monoclonal hybridoma-derived anti-H-2^k antibodies. The tumour has a low expression of the private specificty 31, which characterizes K^d molecules, and does not seem to express the private specificities of D^d, K^k and D^k molecules. It appears to express predominantly alien H-2-like antigens which are very similar to but not identical with normal H-2^k molecules.     

Cell-mediated anti-tumor response in the RLmale 1 system. I. T nature, H-2Dd involvement and antigenic specificity of the effector cells.

After in vivo priming and in vitro secondary stimulation by the radio-induced RL♂ 1 lymphoma cells, syngeneic BALB/c splenic lymphocytes evidenced a specific but very weak cytolytic activity against RL♂ 1 target cells. Under the same experimental conditions, spleen cells from (B6 x BALB/c)F₁ were at least 100 times more efficient. In both cases, the effector cells were cytolytic T lymphocytes (CTL). Normal H-2D^d antigens of the tumor cell surface were involved in the effector-to-target cell interaction since anti-H-2 or anti-H-2D^d antisera abolished the RL♂ 1 cytolysis by immune syngeneic CTL, whereas anti-H-2K^d were devoid of blocking activity. The testing of a series of lymphoma cells, either virus-induced or not, belonging to different H-2 haplotypes, show that the immune CTL were highly specific for RL♂ 1 target cells. Only with the P815 mastocytoma cells was a weak cross-reactivity detected by both direct tests and competition experiments. Allogeneic (H-2^k) AKR lymphoma cells which share the X1 antigen with RL♂ 1 do not react, possibly due to an H-2 restriction of the CTL activity. The CTL-reacting antigen cannot be definitely identified, since several specificities, including the serologically defined X1 antigen, are possibly involved simultaneously. The Hh antigens which could have accounted for the F₁ anti-parent reaction, appear irrelevant.     

ABSENCE OF FOUR H-2d ANTIGENIC SPECIFICITIES IN AN H-2d SARCOMA

MCG4 is a BALB/c sarcoma induced as a solid tumour with 0.2 mgs of methylcholantrene. The primary tumour was serially transplanted subcutaneously in syngeneic mice. The ascites form obtained was used to study the expression of H-2 antigeneic specificities in a postlabelling radioassay. MCG4 did not express H-2D.4 (private specificity of H-2^d haplotypes) as well as H-2.3, H-2.8 and H-2.13 (public specificities). In addition it expressed H-2.5 (a public specificity not present in H-2^d cells). These results were confirmed by quantitative absorption analysis using MCG4 and positive-negative normal lymphoid cells for a particular specificity. Results are discussed with regard to the control of expression of H-2 antigens by regulatory genes.     

RELATIONSHIPS BETWEEN H-2 AND VIRAL ANTIGENS IN MURINE ONCORNAVIRUS-INDUCED TUMOURS*

Cytolytic T lymphocytes (CTL) from murine sarcoma virus (MSV) or Friend leukaemia virus (FLV) inoculated mice lyse syngeneic much more efficiently than allogeneic FMRGi+ lymphoma cells. By comparing the cytolysis of various H-2 different ⁵¹Cr lymphomas by CTL from several inbred and congenic lines differing at H-2, and by competition experiments using unlabelled cells, one can demonstrate that this phenomenon is due to an H-2 barrier. H-2^b/H-2^d hybrid-anti-MSV-CTL immunized by H-2^b, H-2^d or H-2^b/H-2^d tumours lyse only FMRGi+ lymphomas of the same H-2, and their activity for a given target is inhibited only by H-2-identical competitive cells. H-2 antigens are therefore directly involved in the interaction between tumour cells and immune CTL which probably react with an 'H-2 modified' antigen of the tumour cells surface. The use of CTL from intra-H-2 recombinant lines shows that H-2D and probably H-2K molecules are involved, but vary according to the tumour cells. A possible role of the I region is discussed as well as the implications of these results in immunosurveillance against viral neoplasia.     

Original H-2d and alien H-2k-like antigens of a BALB/c fibrosarcoma as defined by in vitro and in vivo studies of cell-mediated immunity

The present study examines the immunosensitivity and the immunogenicity of both original H-2^d and alien H-2^k-like antigens of the BALB/c (H-2^d) fibrosarcoma C-1 as detected by in vitro and in vivo cell-mediated cytotoxicity (CMC) assays. It was found that ⁵¹Cr-labeled C-1 cells were lysed in vitro by C 57 BL/6 anti-H-2^d lymphocytes. The specificity of this reaction was shown by cold inhibition experiments in which the anti-H-2^d cytotoxic activity on YC8 (H-2^d) targets was inhibited by unlabeled YC8 or C-1 but not by C3UR11 (H-2^k) tumor cells. Both D^d- and K^d-encoded antigens were recognized by appropriate cytotoxic effectors. The immunogenicity of H-2^d antigens of C-1 was revealed by the ability of C 57 BL/6 anti-C-1 lymphocytes to lyse YC8 targets. The expression of H-2^k-like alien alloantigens on C-1 was indicated by the finding that anti-H-2^k cytotoxic T lymphocytes (CTL), generated by culturing BALB/c spleen cells immune to BALB.K (H-2^k), C3Hf (H-2^k) or A (H-2^a = H-2^k/d) tissues with the cells of the same strain used for immunization, lysed C-1 targets. The cytotoxicity of these anti-H-2^k CTL against C 3 UR11 (H-2^k) targets could be specifically inhibited by cold C 3 UR 11 or C-1 cells but not by two other BALB/c tumors. Using recombinant H-2-congenic mice, it was shown that both D^k and K^k antigens were recognized by CTL on C-1 cells. The immunogenicity of the H-2^k-like antigens, however, could not be detected in vitro. In fact, effector spleen cells from BALB/c mice immune to C-1 did not develop any detectable cytotoxicity against C 3 UR 11 targets as assayed either by a direct in vitro test or after in vitro restimulation with C-1 sarcoma cells. A similar experimental design was adopted in Winn assays carried out by mixing spleen cells of BALB/c immune mice with either C-1 or C3 UR 11 targets and injecting the mixtures in BALB/c or hybrid recipients. These in vivo tests revealed the presence of both H-2^d (K^d and D^d) and H-2^k-like (K^k and D^k) antigens on C-1. At variance with the in vitro CMC assays, however, the Winn assay also detected the immunogenicity of the H-2^k alien antigens, since BALB/c anti-C-1 spleen cells were able to significantly reduce the growth of C 3 UR 11 lymphoma cells in (BALB/c × C 3 Hf)F₁ hosts.     

Genetic control of immunity to Trichinella spiralis: influence of H-2-linked genes on immunity to the intestinal phase of infection

The time course of expulsion of adult Trichinella spiralis from the intestine was determined in B10 background, H-2 congenic and recombinant mice. Non-H-2 genes exerted the major influence on worm expulsion (i.e. determined rapid or slow response phenotype) but marked time course differences were seen among the slow responder B10 background strains, implying that H-2-linked genes also influence this parameter of immunity. Independent H-2^q haplotype mice showed the most rapid expulsion, H-2^k and H-2^b the slowest. Data from H-2 recombinant mice carrying the q allele suggested that alleles at H-2K loci have a strong influence in immunity, but showed also that H-2D alleles exert a significant modulating effect. The q allele in otherwise susceptible k haplotype mice (B10.AKM) gave increased resistance; the d allele at H-2D in mice carrying the q resistance allele elsewhere [B10.T(6R)] gave decreased resistance. Adoptive transfers using immune mesenteric node lymphocytes (IMLNC) from a series of donors were used to identify how the modulating influence of H-2D^d was expressed in B10.T(6R) mice. IMLNC from this strain transferred immunity to recipients of other (histocompatible) strains, but IMLNC from such strains failed to accelerate expulsion in B10.T(6R) recipients as did homologous B10.T(6R) cells. Two alternative models are proposed to explain these results: either that H-2D^d influences the response of myeloid precursors to lymphocyte-derived factors, and thus the generation of intestinal inflammatory changes necessary for expulsion, or, on the assumption that the generation of intestinal inflammation requires initial cooperation between helper and effector lymphocytes, that H-2D^d is associated with a restricted ability of effector cells to respond to the helpers present in IMLNC.     

ORIGINAL H-2d AND FOREIGN H - 2k - LIKE ANTIGENS ARE INDEPENDENT ENTITIES ON A CHEMICALLY INDUCED SARCOMA

We have previously shown that a methylcholanthrene-induced sarcoma of BALB/c strain (C-1) expressed, in addition to its original H-2^d antigens, foreign H-2^k-like determinants. In the present study the relationship between H-2^d and H-2^k-like antigens was examined by in vitro and in vivo assays. Cultured tumour cells were exposed in the cold to either a monospecific (D-23, D-25, D-1) or polyspecific (BALB/c anti-C3Hf) alloantiserum directed to H-2^k specificities and then used to absorb the cytotoxic activity of either monospecific (D-31) or a polyspecific (C3Hf anti-BALB/c) alloantisera to H-2^d antigens (blocking test). The opposite was also done, i.e. tumour cells were coated with anti-H-2^d sera and used to absorb the cytotoxicity of anti-H-2^k sera. No reciprocal interference was found between the two H-2-different antigens in the absorption of related alloantisera. Suspensions of irradiated C-1 tumour cells were exposed in vitro to either anti-H-2^d or anti-H-2^k antisera and then used to immunize either syngeneic BALB/c or allogeneic C3Hf mice. The coating of immunizing neoplastic cells with BALB/c anti-C3Hf serum prevented anti-C3Hf (anti-H-2^k) antibody production in BALB/c mice without affecting anti-BALB/c (anti-H-2^d) antibody development in C3Hf animals; coating of H-2^d antigens on tumour cells strongly reduced anti-BALB/c but not anti-C3Hf antibody production. Both in vitro and in vivo experiments indicated that foreign H-2^k-like determinants are physically separated from the wild H-2^d antigens on the C-1 sarcoma cells.     

Cytotoxic T cell response against lymphoblasts infected with Moloney (Abelson) murine leukemia virus. Methodological aspects and H-2 requirements

A method for infection of lymphocytes with Moloney(Abelson) murine leukemia virus [M(A)-MuLV] is described. Only lymphoblasts obtained after stimulation of normal spleen cells by the B cell mitogen lipopolysaccharide (LPS) were satisfactory targets for virus-specific, secondary cytotoxic T lymphocytes (CTL), whereas spleen cells stimulated by the T cell mitogen concanavalin A were not. The secondary CTL response against M(A)-MuLV could be efficiently measured using M(A)-MulV-infected LPS blasts as stimulating cells for secondary in vitro restimulation and as target cells for virus-specific destruction. Cold target inhibition demonstrated virus specificity of CTL. The T cell character of the cytotoxic cells was demonstrated by their sensitivity to anti-Thy-1.2 treatment. Using syngeneic virus-infected LPS blasts as target and stimulator, CTL responses were measured with effector cells from C57BL mice of the H-2^b haplotype and of recombinant haplotypes sharing either K or D alleles with H-2^b. In analogy with previous studies on Moloney virus-specific CTL. it was observed that C57BL/6 (H-2^b) effector cells predominantly lysed D^b-compatible, virus-infected target cells; B10.A(5R), (K^bD^d) effector cells showed a poor CTL response against syngeneic, virus-infected target cells. The combined findings indicate the existence of an Ir gene in the H-2D region regulating the CTL response against Moloney leukemia virus.     

IMPAIRED H-2 EXPRESSION IN B 16 MELANOMA VARIANTS

We studied the expression of H-2^b alloantigens in three different B16 melanoma lines cultured in vitro, Cell lines were B16-F1 and two cell cultures (named B16-A and B16-B) newly derived from two different in vivo sublines of B16 melanoma. The assays used were in vivo tumour growth in allogeneic (BALB/c and B10.BR) as compared to syngeneic mice, in vitro cell-mediated cytotoxicity by anti-H-2^b immune lymphocytes and absorption of anti-H-2^b antisera activity. The B16-F1 line was able to efficiently kill allogeneic hosts, could not be lysed by anti-H-2^b cytotoxic effectors and did not express any serologically detectable amount of H-2^b alloantigens. The B16-A line was H-2 positive during the early in vitro passages, then, at the 8th–10th passages, it acquired the capacity to kill allogeneic hosts, lost the sensitivity to anti-H-2^b cytotoxic effectors and the H-2K^b antigens became undetectable. The expression of H-2D^b was reduced, although at a lower degree. Similar data were obtained with B 16-B cells, which after 10 in vitro passages grew and killed allogeneic hosts, showed a decreased sensitivity to cytotoxic anti-H-2^b effectors and a very low expression of the K region antigens. The results indicate that H-2 expression is altered in B 16 melanoma lines and this may influence the different metastatic capacity of such cells.     

CHANGE OF H-2 ANTIGENS' EXPRESSION ON MOUSE LEUKAEMIALBN/a-2 AND LBN/b-3 CELLS IN THE COURSE OF SERIAL TRANSPLANTATION

The inbred mouse strains BN/a and BN/b have haplotype H-2^bp characterized by H-2.33 and lacking any other private specificity known in inbred strains. Two transplantable B cell leukaemias which originated in BN/a and BN/b mice treated with anti-lymphocyte globulin were tested serologicaly for H-2 antigens. Tests during passages 169-181 revealed several quantitatively different reactions with sera against public specificities, some of these being due to Ia antibodies. No change in expression of the private specificities was seen. On the other hand, during the later passages (239-258) a number of qualitative differences were seen, the most remarkbale being the loss of H-2.33 and gain of H-2.4, 31. The overall serological pattern of cells resembled that of H-2^d rather than of H-2^bp haplotyes and this was confirmed by absorption tests. The changes reported here may be due to alterations of repression and derepression pattern of the presumed multiple structural H-2 genes present in the genome.     

INACTIVATION OF SPECIFIC ANTI-H-2 SUPPRESSOR T CELLS BY ANTISERA TO I-J AND I-C SUBREGION PRODUCTS OF THE H-2 COMPLEX

Two antisera to Ia antigens, products of the H-2 complex I-C^d and I-J^kE^k subregions, respectively, have been obtained by immunization of the F₁ hybrids of recombinant strains of mice. These antisera are shown to display a 50% cytotoxic effect in vitro, in the presence of complement, upon lymphocyte populations immune to the H-2-complex antigens and enriched for specific suppressor T cells (SSTC) by fractionation on a monolayer of target cells. The specificity of anti-Ia cytotoxins is shown by cross-antibody absorption with T and B cells of mice originating from the recombinant H-2 haplotypes and bearing either particular I-C^d, I-J^k and I-K^k antigens, or their combinations. Anti-I-C^d cytotoxins were found to react with both B and T cells, but at a different rate, and the anti-I-J^kE^k serum contains two antibody types directed to I-E^k and I-J^k products, respectively, the latter being able to react preferently with T cells. Although both antisera do inactivate the in vitro SSTC function in the presence of complement to a similar degree, the inactivating action of the anti-I-C^d serum, but not that of the anti-I-J^kE^k serum, occurs without complement. SSTC are shown to bear both Ia-antigens, I-J and I-C, as shown by both inactivation of the anti-suppressor effect of the antisera absorbed with spleen cells of different H-2 origin, and variation of the H-2 origin of SSTC pretreated with the intact antisera. It is suggested that these two markers, located on the same SSTC, function differently in SSTC immune to the H-2 antigens, and I-C antigen expression on the SSTC surface is presumed to be required for their interaction with the inhibited responder T cells proliferating in MLC.     

THE H-2 COMPLEX: IMMUNOGENICITY AND ENHANCEMENT STUDIES OF H-2K REGION ALLOANTIGENS

Antigenic differences arising from incompatibilities at the K region of the H-2 complex of the mouse (including the H-2K locus) were studied in different combinations to determine the immunogenicity of K^k, K^q and K^s region products. Only congenic strains and selected recombinant strains were used, so that the only known differences arose from the K region of the H-2 complex. In males, first grafts were rejected in 12–13 days so that, as immunogens, K region antigens were equivalent to those of H-2I. If the H-2 histocompatibility loci are ranked in decreasing order of the immunogenic strength of their antigenic specificities: H-2K = H-2I > H-2D > H-2IC. Second grafts were rejected more rapidly than first skin grafts. After immunization by either lymphocytes or a single skin graft, high cytotoxic antibody titres were achieved, e.g. 1/512, after lymphocyte immunization. However, it was not possible by the passive administration of antiserum to delay graft rejection and produce enhancement by any antisera directed only against K region specificities. In several different combinations studied, it was proved that antigenic differences arising from the I region but not the K region are essential to produce an enhancing antiserum for K + I region differences. In particular, differences incorporating IA and IB are more important than IC differences. However, when differences arise only from the K region, enhancement cannot be produced by any antiserum, whether it contains anti-Ia antibodies or not, presumably as there is no I region incompatibility.     

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.     

Influence of glycosylphosphatidylinositol-linked H-2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)-linked H-2D^d molecule. The GPI-linked H-2D^d molecule is recognized by H-2D^d-specific antibodies and cytotoxic T lymphocytes. Expression of the GPI-linked H-2D^d molecule on H-2^b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H-2^b, H-2D^d) from rejection by NK cells. In addition, NK cells from mice expressing the GPI-linked H-2D^d molecule as a transgene were able to kill nontransgenic H-2^b lymphoblast target cells. The GPI-linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.     

MEMBRANE ANTIGENS RECOGNIZED BY HETEROANTISERA AGAINST MOUSE TUMOURS,TESTED IN CELL LINES OF DIFFERENT H - 2 HAPLOTYPES

The pattern of reactivity of two heteroantisera against mouse tumour cells (rabbit anti-Meth A sarcoma: RAMA; and rabbit anti-RBL-5 leukaemia: RAR-5) has been studied in various cell lines of different H-2 haplotypes in complement-dependent cytotoxicity tests using a microadioassay. RAMA was cytotoxic not only for Meth A (H-2^d) but also for MCG4, P815Y, LSTRA, YC8 (H-2^d), L929, TLC5, Gardner (H-2^k), RBL-5 and TLX/9 (H-2^b). RAR-5 similarly killed RBL-5, as expected, as well as Meth A, P815Y, MCG4, SL2, YC8, LSTRA (H-2^d), L929 (H-2^k), TLX/9, MBL2, Gil IV, EL4 (H-2^b) and YAC (H-2^a). The cross-cytotoxicity RAMA-RBL-5 and RAR-5-Meth A was abolished when RAMA or RAR-5 were absorbed with allogeneic tissues from C57B1/6, BALB/c, CBA/H, C57B1/10 (B10), B10.BR, B10.D2, but not with rat lymphoid cells. A species-specific antigen present in these strains of mice thus appeared to be responsible for this cytotoxicity. The direct killing obtained with RAMA on Meth A and RAR-5 on RBL-5 was not absorbed by: (a) allogeneic cells of C57B1/6, CBA/H, AKR, B10, B10.BR, B10.D2 for RAMA, and CBA/H, BALB/c, B10, B10.BR, B10.D2 for RAR-5; or (b) syngeneic normal lymphoid cells (BALB/c and C57B1/6 respectively). This suggests the presence of a heteroantibody in the sera recognizing structures at the tumour cell surface that are absent in normal lymphoid cells—a tumour-specific antigen (TSA). Finally, the TSA of Meth A (recognized by BALB/c-absorbed RAMA) did not cross-react with eight mouse cell lines of different aetiology and origin: MCG4, P815Y, Gardner, TLC5, L929, TLX/9, LSTRA and RBL-5. However, the TSA of RBL-5 (recognized by C57B1/6 absorbed RAR-5) reacted not only with RBL-5 but also with tumour cells induced by viruses antigenically related to Rauscher, belonging to the group FMR Gi. It reacted also with EL4, a tumour cell line not virus induced, and weakly with SL2, a spontaneous tumour cell line. It did not react with TLX/9 (X-ray induced) or Meth A (chemically induced). From these results it is concluded that heteroantisera against mouse tumour cells recognize at least species-specific antigens and tumour-specific antigens.     

Public H-2 specificities are target determinants for alloreactive cytotoxic T lymphocytes

The specificity of the cross-killing exerted by cytotoxic T lymphocytes (CTL's) generated against H-2 region products was investigated in a 51Cr release assay on a panel of target cells from a number of different H-2 haplotypes. Their pattern of reaction shows that: (1) The target cells, expressing public specificities (H-2.28, H-2.1, H-2.3 or H-2.8) which should, theoretically, be recognized by the CTL's were killed, while those expressing no public specificities, recognized according to the H-2 chart by the CTL's were not killed. (2) The CTL's generated against the H-2.28 specificity expressed on the D region products cross react with target cells expressing this specificity in their K region products and vice versa. The same phenomenon was observed with the H-2.1 specificity. These results provide evidence that public specificities are targets for CTL's. Antiserum reacting against the public specificity recognized by the CTL's was found to block the cross-killing, however, to the same extent as antisera directed against any specificity (private or public) expressed on the same molecule as the target determinant. Finally both the inhibition studies using anti-H-2 antisera and the direct cytotoxic assays showed that the public specificities of the H-2.28 family carried by the H-2.D and H-2.L molecules were recognized by different subpopulations of CTL's.     

ANALYSIS OF THE B10.D2-H-2d m l MUTANT WITH MONOCLONAL ANTIBODIES

We compared H-2D products of B10.D2 and the mutant B10.D2-H-2^dml spleen cells using two monoclonal antibodies designated D3.179 and S11.7. D3.179 reacts with public specificities and appears to define more than one product as shown by cytotoxicity and binding studies. Strong reactivity was obtained in both binding and cytotoxicity and binding studies. Strong reactivity was obtained in both binding and cytotoxicity to B10.D2 and marginal cytotoxicity and low binding were observed with B10.D2-H-2^dml cells. The second monoclonal antibody S11.7, in contrast, reacts only with H-2D^d bearing strains and gave strong binding to B10.D2-H-2^dml and weaker binding to B10.D2. Taken together, the observations indicate that the mutant not only expresses H-2D, encoded antigens but bear quantitatively greater amounts of some specificities than the parent. A Product of approximately 46,000 daltons was immunoprecipitated from B10.D2 cells by D3.179. Very small amounts of this product were seen in B10.D2-H-2^dml immunoprecipitates.     

α1 / α2 domains of H-2Dd,but not H-2Ld,induce “missing self” reactivity in vivo – No effect of H-2Ld on protection against NK cells expressing the inhibitory receptor Ly49G2

Introduction of the MHC class I transgene H-2D^d on C57BL / 6 (B6) background conveys NK cell-mediated “missing self” reactivity against transgene-negative cells, and down-regulates expression of the inhibitory receptors Ly49A and Ly49G2 in NK cells. We here present an analysis of transgenic mice expressing chimeric H-2D^d / L^d MHC class I transgenes, and show that the α1 / α2 domains of H-2D^d were necessary and sufficient to induce “missing self” recognition and to down-modulate Ly49A and Ly49G2 receptors. In contrast, transgenes containing the α1 / α2 domains of H-2L^d induced none of these changes, suggesting that not all MHC class I alleles in a host necessarily take part in NK cell education. The lack of effect of the α1 / α2 domains of H-2L^d on NK cell specificity was surprising, considering that both H-2L^d and H-2D^d have been reported to interact with Ly49G2. Therefore, the role of H-2L^d for protection against NK cells expressing Ly49G2 was re-investigated in a transfection system. In contradiction to earlier reports, we show that H-2D^d, but not H-2L^d, abolished killing by sorted Ly49G2⁺ NK cells, indicating that H-2L^d does not inhibit NK cells via the Ly49G2 receptor.     

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.