Evidence for three carbohydrate prosthetic groups on mouse histocompatibility antigens H-2Kd and H-2Db

Previous studies have determined that H-2K^b molecules have two glycosyl units which are attached at asparagine-86 and asparagine-176. Investigations concerning the number and location of carbohydrate groups in the H-2D^b and,H-2K^d antigens were carried out on [³H]glucosamine-labelled material isolated from EL-4 (H-2^b) and C14 (H-2^d) tumor cells, respectively. The present study, employing CNBr cleavage, indicates that H-2K^d and H-2D^b have, in addition to those reported for H-2K^b, a third glycosyl unit located between residues 229 and the papain cleavage site near residue 281.     

Amino acid sequence of the cytoplasmic segment of the h-2kd (h2.31) murine major histocompatibility antigen

The primary structure of the carboxy-terminal portion of the H-2K_d murine major histocompatibility antigen has been determined using radiosequencing methodology. The two peptides encompassing the entire cytoplasmic portion of the H-2K^d molecule were isolated from cyanogen bromide digests of the detergent solubilized molecule. These two peptides are not present in CNBr digests of papain-solubilized H-2K^d. Alignment of the two CNBr peptides was deduced from tryptic overlap peptides derived from the whole molecule. Alignment with the corresponding region of the H-2K^b antigen shows 90% homology and supports the assignment of this segment of H-2K^d as the C-terminal. The sequence obtained in this study is (Met)-Arg-Arg-Asn-Thr-Gly-Gly-Lys-Gly-Val-Asn-Tyr-Ala-Leu-Ala-Pro-Gly-Ser-Gln-Thr-Ser-Asp- Leu-Ser-Leu-Pro-Asp-Pro-Lys-Val-Met-Val-His-Asp-Pro-His-Ser-Leu-Ala. These data allow extensive comparisons with the protein sequences deduced from the 3' ends of H-2^d haplotype cDNA and genomic clones as well as with the homologous regions of H-2K^b and H-2D^b.     

Isolation,characterization and amino acid sequence studies of the cyanogen bromide fragments of the H-2 Dd glycoprotein

The papain-solubilized fragment of the H-2D^d antigen, representing the NH₂-terminal 80% of the native glycoprotein, can be fragmented into five polypeptides using CNBr cleavage. These fragments have been isolated, characterized and subjected to amino acid sequence analysis using radiochemical microtechniques. It was possible to align these CNBr fragments by comparison of their NH₂-terminal amino acid sequence with the known amino acid sequence of the H-2 K^b molecule. Thus, the NH₂-terminal fragment, D, contains 23 residues and is followed by a glycopeptide, fragment C, of 75 residues. Following fragment C are three fragments held together by disulfide bonds: fragment b 4, which has 40 amino acid residues, a second glycopeptide, fragment b 2, of 90 residues, and fragment b 3, a peptide of approximately 55 residues which terminates at the site of papain cleavage. The complete amino acid sequence of the NH₂-terminal CNBr fragment, D, has been determined, and this is presented here along with sequence data for the NH₂-terminal 30 residues of each of the other four CNBr fragments of the papain fragment of H-2D^d. A total of 146 positions have been examined in the approximately 280-residue H-2D molecule, and a total of 127 residues have been positively determined. Most of the unassigned positions may be tentatively assigned as Asp or Asn, the only amino acids so far not incorporated in radiolabeled form by our metabolic labeling procedures into the H-2 D^d molecule. Comparison of the sequence data obtained for the H-2 D^d molecule to data available for other histocompatibility antigens revealed homologies of approximately 90% with H-2 K^b and H-2 L^d, and approximately 75% with HLA-B 7 and HLA-A2.     

Biochemical purification of detergent-solubilized H-2 alloantigens

A multistep Chromatographic fractionation scheme is described for purifying the H-2K^b and H-2D^b major histocompatibility antigens as isolated from the murine lymphoblastoid cell line EL4 (H-2^b haplotype). The membrane-integrated antigen molecules were solubilized with the non-ionic detergent NP-40 and were purified by gel filtration chromatography, ion exchange chromatography and affinity chromatography with lentil lectin conjugated to Sepharose. During the latter procedure, use of a linear gradient monosaccharide elution effected partial separation of the H-2K^b and H-2D^b antigens. At this stage the H-2 glycoproteins were highly purified based on several criteria. Upon polyacrylamide gel electrophoresis in SDS the major band migrates with a mol. wt of approximately 45,000 daltons corresponding to the mol. wt of antigens obtained by immunoprecipitation. Moreover, near identity of the profiles of the arginine-containing tryptic peptides from chromatographically-purified and immunoprecipitated H-2K^b preparations suggests that a high degree of homogeneity has been achieved in the Chromatographic purification. As is demonstrated, mg quantities of the H-2K^b and the H-2D^b antigens can be isolated and partially separated from each other by this purification scheme thereby opening the way for structural studies of the H-2K and H-2D molecules by a variety of biochemical methods.     

Structure of murine major histocompatibility complex alloantigens: Identification of cysteine residues involved in two intrachain disulfide bonds of H-2Kb

Two disulfide bonds are present in the antigenic portion of H-2K^b. Four of the five cysteine residues have been shown to be involved in these intrachain linkages. Isolation of the disulfide-bonded residues was accomplished by trypsin digestion of H-2K^b_pap∥ and subsequent gel filtration of the digested material. Reduction, alkylation and further separation of each of the pairs of disulfide-bonded peptides resulted in the separation of individual cysteine-containing peptides. Sequence analysis of each of these separated peptides indicated that Cys 101 is linked to Cys 164, and Cys 203 is linked to Cys 259. Cys 121 is not involved in intramolecular disulfide linkages.The disulfide bonds found in H-2K^b are homologous to those found in HLA-B7 and, most probably, in H-2D^b, H-2K^d, H-2D^d and H-2L^d. This pattern of disulfide linkages suggest that this feature is common to all major histocompatibility complex class I antigens.     

Further structural analysis of the murine H-2Kb glycoprotein using radiochemical methodology.

This report documents progress towards determination of the complete amino acid sequence of the murine MHC2 H-2 K^b molecule, using radiolabeled material obtained by immune precipitation of H-2.33 bearing glycoproteins from non-ionic detergent lysates of lymphoid tumor cells (EL4) cultured in the presence of radiolabeled amino acids. All amino acids including Gln but with the exception of aspartic acid and asparagine have been successfully incorporated in isotopically labeled form in the K^b molecule at levels sufficient to allow assignment of 153 residues in 170 positions. Automatic sequence analysis was carried out on the intact molecule, on CNBr fragments, and most recently on peptides isolated by ion exchange chromatography from tryptic digests of CNBr fragments. Analysis of our recently obtained data on the amino acid sequence of the H-2K^b antigen reveals 73% homology to human HLA molecules for the 124 positions available for comparison at this time. The majority of differences between the molecules occur within discrete regions.     

EQUIVALENT DECREASE OF H-2Kk AND H-2Dk EXPRESSION AFTER VACCINIA VIRUS INFECTION

Expression of H-2K^k and H-2D^k molecules was studied by indirect immunofluorescence on vaccinia virus-infected L_s cells (derived from the L929 cell line), using D-23^b and ASA-21 monospecific alloantisera, respectively directed against the H-2.23 and H-2.32 private specificities of the H-2^k haplotype. Our study demonstrates that the increase in vaccinia virus-induced antigens on the membrane of infected cells (as a function of the multiplicity of infection) is associated with a concomitant decrease in the expression of both H-2K^k and H-2D^k serologically defined private specificities. Absorption experiments of D-23^b and ASA-21 sera, using infected or uninfected L_s cells, also indicate that after virus infection, H-2K^k and H-2D^k private specificities are equally altered in their serological expression. We finally show that this alteration consists in a mere quantitative decrease of H-2 molecules, since the cytolytic capacity of a rabbit anti-H-2 serum, specifically reacting against the constant part of the heavy chains of H-2 molecules, was significantly more absorbed by uninfected cells than by infected cells. We conclude that no preferential decrease in the expression of H-2 K^k and H-2 D^k molecules is induced during cell infection by vaccinia virus.     

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.     

THE COMPONENT FRAGMENTS OBTAINED BY ACID DISSOCIATION OF PAPAIN-SOLUBILIZED H-2 MOLECULES*

H-2K^k and H-2D^d molecules were specifically purified from a radioiodinated H-2^a preparation obtained by papain digestion of spleen cell membranes of A/J strain mice. The molecules were isolated by binding to H-2 alloantisera of the corresponding private specificity followed by precipitation with rabbit anti-mouse IgG antiserum. The specifically precipitated radioiodinated H-2K^k and H-2D^d molecules were dissociated by acid treatment into large and small components of about 37,000 and 11,000 daltons respectively. These were separated by gel filtration at acid pH or by gel isoelectric focusing in the presence of 6 M urea. Each component separated by gel filtration of the acid-dissociated H-2 molecules showed a high degree of size homogeneity as determined by sodium dodecyl sulphate-acrylamide gel electrophoresis. Upon gel isoelectric focusing, however, the small components showed two peaks of radioactivity closely located together at pH 7–8, both of which had a restricted pH range, while the large components gave one peak of a relatively wide pH range of pH 5–6. The H-2K^k and H-2D^d molecules gave essentially the same pattern in terms of the numbers and the positions of the radioactivity bands. Under the iodination conditions used the large components of H-2K^k molecules contained more radioactivity than the small components, while the reverse was true in case of H-2D^d molecules. Such a difference was also found with H-2K^k and H-2D^d molecules isolated by use of alloantisera of the respective public specificity. The assay of binding of the isolated components with H-2 alloantisera of defined specificity revealed that the large components retain most of the allospecificities of the parental H-2 molecules. No H-2 allospecificities were found on the small components. The small components showed extensive binding with rabbit antiserum against mouse β₂-microglobin. The same antiserum did not show any binding with the large components. On the other hand, both of the components did bind with rabbit antiserum against papain-solubilized H-2 molecules.     

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.     

Presentation of viral antigens restricted by H-2Kb,Db or Kd in proteasome subunit LMP2-and LMP7-deficient cells

In the class II region of the major histocompatibility complex (MHC), four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP 1 and TAP 2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP 2 and LMP 7) code for subunits of the proteasome. While TAP 1 and TAP 2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP 2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP 1/2 and LMP 2/7 genes, it was recently shown that expression of TAP 1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP 2/7, we transfected T2 cells with TAP 1, TAP 2 and either of the H-2K^b, D^b or K^d genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP 1/2 gene products, presented all tested viral antigens restricted through either the H-2K^b, D^b or K^d class I molecules. We conclude that the proteasome subunits LMP 2/7 as well as other gene products in the MHC class II region, except from TAP 1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP 2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.     

Down‐regulated expression of monocyte/macrophage major histocompatibility complex receptors in human and mouse monocytes by expression of their ligands

Mouse monocyte/macrophage major histocompatibility complex (MHC) receptor 1 (MMR1; or MMR2) specific for H-2D^d (or H-2K^d) molecules is expressed on monocytes from non-H-2D^d (or non-H-2K^d), but not those from H-2D^d (or H-2K^d), inbred mice. The MMR1 and/or MMR2 is essential for the rejection of H-2D^d- and/or H-2K^d-transgenic mouse skin onto C57BL/6 (H-2D^bK^b) mice. Recently, we found that human leucocyte antigen (HLA)-B44 was the sole ligand of human MMR1 using microbeads that had been conjugated with 80 types of HLA class I molecules covering 94·2% (or 99·4%) and 92·4% (or 96·2%) of HLA-A and B molecules of Native Americans (or Japanese), respectively. In the present study, we also explored the ligand specificity of human MMR2 using microbeads. Microbeads coated with HLA-A32, HLA-B13 or HLA-B62 antigens bound specifically to human embryonic kidney (HEK)293T or EL-4 cells expressing human MMR2 and to the solubilized MMR2-green fluorescent protein (GFP) fusion protein; and MMR2⁺ monocytes from a volunteer bound HLA-B62 molecules with a K_d of 8·7 × 10⁻⁹ M, implying a three times down-regulation of MMR2 expression by the ligand expression. H-2K^d (or H-2D^d) transgene into C57BL/6 mice down-regulated not only MMR2 (or MMR1) but also MMR1 (or MMR2) expression, leading to further down-regulation of MMR expression. In fact, monocytes from two (i.e. MMR1⁺/MMR2⁺ and MMR1^–/MMR2^–) volunteers bound seven to nine types of microbeads among 80, indicating ≤ 10 types of MMR expression on monocytes.The physiological role of constitutive MMRs on monocytes possibly towards allogeneic (e.g. fetal) cells in the blood appears to be distinct from that of inducible MMRs on macrophages toward allografts in tissue.     

Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2^bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2K^b, H-2D^b, and H-2K^bm1 class I molecules, although the sequence lacks the usual structural K^b and D^b peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2^b) targets as well as the L cell transfectants, L + K^b, L + D^b, and L + K^bm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro⁴⁴ and Thr⁴⁷, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2K^b, H-2D^b, and mutant H-2K^bm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.     

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.     

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.     

EVIDENCE OF ONLY ONE H-2D/L - RELATED GLYCOPROTEIN IN H-2dm1 MUTANT CELLS

Analyses of the H-2D/L-related glycoproteins from dm1 mutant cell extracts by sequential immunoprecipitation, by SDS gel electrophoresis and by tryptic peptide mapping indicate that dm1 cells express only a single glycoprotein with H-2D/L-related determinants. In contrast to the four H-2D/L-related antigens identified for the parental d haplotype viz. H-2D^d, H-2M^d, H-2L^d and H-2R^d, separate and distinguishable “H-2D^dm1”, “H-2M^dm1”, “H-2L^dm1” and “H-2R^dm1” glycoprotein counterparts are apparently lacking in the dm1 mutant haplotype. Only a single H-2D/L-related glycoprotein is identified in dm1 extracts by standard serological methods and this glycoprotein is designated H-2D/L^dm1 because of its H-2D^d/H-2L^d hybrid characteristics, as recently shown by Burnside and colleagues (1984). Thus, the seemingly complex phenotype of the dm1 mutant appears to originate primarily from one molecule having properties of two (or more) molecules of the parental haplotype.     

H-2K RESTRICTION OF THE T CELL-MEDIATED LYSIS OF A CHEMICALLY-INDUCED BALB/c FIBROSARCOMA

Previous work has shown that a cytotoxic T lymphocyte (CTL) immune response of syngeneic mice immunized with a chemically-induced BALB/c (H-2^d) fibrosarcoma was directed against an individual tumour-associated antigen. To see whether this reaction was restricted by products of the major histocompatibility complex (MHC), anti-H-2 alloantisera to K or D antigens were used to interfere with the CTL-mediated immune response. Antisera to K^d but not to D^d antigens inhibited the lytic activity of CTL against fibrosarcoma cells. In addition, the study of the CTL response in F₁→ P antitumour immunized chimeric mice showed that antitumour cytotoxicity developed only when F₁ and parental host shared the K^d region. Both experiments strongly indicate that recognition of the individual tumour-associated antigen of the BALB/c fibrosarcoma is restricted by the products of H-2K^d genes.     

Ir GENE TESTING IN H-2 MUTANTS

Four different C57BL/6 mutants, each with a mutation presumably in the H-2K^b locus, were studied to determine whether the mutations had affected Ir gene function. When tested with the antigens, (TG)-A-L, ovalbumin (OA), and lactic dehydrogenase (LDH₈), there was no difference in the antibody response of the mutants when compared to the parental C57BL/6 strain and there was also no difference in the response to the histocompatibility H-Y antigen. The responses to these antigens have been mapped, wholly or partially in the K-IA, IA or IB subregions and therefore the identical response of parent and mutants may provisionally exclude H-2K^b as a site of Ir gene function for the four antigens tested. In addition, the BALB/c-dm² mutant, a mutation in the H-2L^d locus, gave identical responses to the parental BALB/cKh strain.     

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.     

The ability of H-2Dd molecule to affect natural resistance to hemopoietic allografts is an intrinsic property shared by Ddm1 but not Ld

F₁ hybrid resistance (HR) to parental bone marrow grafts is mediated by natural killer (NK) cells, and thought to be controlled by the non-class I hemopoietic histocompatibility (Hh) genes linked to the major histocompatibility complex (MHC). However, as in the in vitro NK cytotoxicity against hemopoietic targets, expression of certain class I MHC molecules does affect HR, although mechanisms underlying such an effect are not understood. In this study, we examine the relevance of the “self/non-self” property of class I molecules and the molecular domains responsible for this function. H-2^b/Hh-1^b lymphoma cells were transfected with class 1 H-2D^d or L^d gene, and its effect on the Hh-1 phenotype was examined by testing the transfectant's ability to competitively inhibit the in vivo rejection of parental H-2^b/Hh-1^b bone marrow grafts by irradiated F₁ hybrid hosts. Multiple independent clones of transfectants show that the genomic or cDNA of the D^d gene, but not of L^d, renders the Hh-1^b-positive cells incapable of inhibiting HR in F₁ mice, although both genes belong to the same region of the same haplotype. The same effect could be observed not only in H-2^b/d F₁ mice for which D^d and L^d are self, but also in H-2^b/k F₁ mice for which both D^d and L^d are non-self. Thus, this function of the D^d molecule is an intrinsic property, not necessarily related to its self/non-self characteristic relative to the effector cells. Furthermore, given the nature of the assay used in this study, the results favor a “target interference” model as the underlying mechanism of the D^d effect. To locate the relevant domain(s) of the D^d molecule, mutant D^dm1 gene was tested and found to have the same effect as the non-mutant D^d. D^dm1 is a hybrid molecule between D^d and L^d, sharing with D^d only the α1 domain and a portion of the α2 domain. The two N-terminal domains of D^dm1 differ from those of D^d by three amino acid substitutions, two of which affect the molecules' peptide-binding properties.