Resistance to cell-mediated cytotoxicity is correlated with reduction of H-2K gene products in AKR leukemia

AKR leukemia cell lines differing in the amount of H-2K and H-2D antigens expressed on the cell surface were used to assess cell-mediated immune responses in syngeneic mice against Gross/AKR murine leukemia virus (MuLV)-induced tumors. Leukemic cells with reduced expression of H-2K^k antigens were inactive as inducers of Gross-MuLV/H-2^k-specific cytotoxic T lymphocytes (CTL) and resistant to lysis by CTL raised against H-2K^k positive AKR leukemia cells. H-2K^k positive leukemias induced cytotoxic effectors, which upon restimulation in vitro, lysed the stimulating and other H-2K^k positive leukemia cells. In antibody inhibition experiments, T-cell-mediated cytotoxicity to these leukemias could only be inhibited by antisera and monoclonal antibodies specific for the H-2K^k antigens. Due to this specific role of H-2K^k antigens in T-cell cytotoxicity to Gross/AKR MuLV-induced tumors, reduced expression of H-2K^k antigens on spontaneous AKR leukemic cells could have important implications for surveillance of these neoplastic cells.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MuLV murine leukemia virus     

Variation of expression of histocompatibility antigens on tumor cells: absence of H-2Kk-gene products from a gross-virus-induced leukemia in BALB.K

The antigenic profile of the K-GV tumor of BALB.K origin, induced by Gross virus and maintained in vitro and in vivo, was investigated by serological and immunochemical methods and techniques of cell-mediated immunity. The H-2K^k-gene products were absent by several criteria: (1) monoclonal antibody and conventional alloantisera directed against the H-2K^k antigenic specificities were nonreactive by direct testing and by absorptions. (2) H-2K^k products could not be precipitated from glycoprotein or protein extracts of the radiolabeled K-GV tumor. (3) Cytotoxic effectors against H-2K^k produced by sensitization in vitro and in vivo failed to kill K-GV target cells. (4) The tumor could neither stimulate BALB.B congenic mice to produce cytotoxic effectors nor specific cytotoxic antibody against H-2K^k-gene products. In contrast, the H-2D^k antigen was readily detectable by all these criteria. These findings therefore describe a tumor which has selectively lost the H-2K-gene products. The K-GV tumor was able to generate Gross-virus-specific CTL, but had greatly reduced susceptibility to lysis by Gross-virus-specific CTL generated by H-2K expressing AKR (H-2 ^k) tumors. These findings have important implications for the associative recognition of tumor antigens and the immune surveillance of virally induced tumors.Abbreviations used in this paper MHS major histocompatibility system - LcH Lens culinaris hemagglutinin - SDS sodium dodecyl sulfate - CTL cytotoxic T lymphocytes - GCSA Gross-virus-induced cell-surface antigen - MuLV murine leukemia virus     

Dual parameter, quantitative cytofluorometric analysis of endogenous H-2K and foreign HLA class I molecules expressed at the surface of murine transformed L cells

By using a calibrated dual laser cell sorter and monoclonal antibodies directly conjugated to fluorescein and rhodamine and specific for H-2K^k and HLA class I antigens, quantitative cytofluorometric analysis was performed on individual HLA-A3 or -CW3 transformed mouse L cells (H-2^k). More than 80% of these cells expressed both HLA class I and H-2K^k molecules. Their respective levels of expression were calculated: a mean of 4 × 10⁵ HLA class I and 2.3 × 10⁵ H-2K^k molecules per single cell. Quantitative comparison with control untransformed L cells and double fluorescence contour maps showed a positive correlation between the levels of expression of HLA class I and H-2K^k molecules suggesting that expression of foreign class I molecules did not occur at the expense of the endogenous H-2^k product.     

Quantitative variation in H-2-antigen expression

Minor differences in the expression of individual H-2K and H-2D antigens were detected on mouse spleen cells. The method involved the use of an¹²⁵I-protein A radioimmunoassay using highly specific anti-H-2 sera to make estimates of the number of cell-bound antibody molecules. The maximum number of antibody binding sites varied for each H-2 antigen reflecting differences of between 10 and 70 percent in the expression of any two antigens. The order of magnitude of expression was D^b>(K^d)=K^k=K^b=D^q>D^d>K^q>D^k. Minor background differences were detectable, but antigen expression was allele-specific and independent of the expression of other K, D or I antigens expressed on the same cell.     

Lateral diffusion and patch formation of H-2Kk antigens on mouse spleen lymphocytes

We have studied the diffusion and aggregation of H-2K^k antigens labeled with a fluorescent anti-H-2K^k monoclonal antibody (IgG) on mouse splenic lymphocytes, employing fluorescence photobleaching recovery and fluorescence microscopy. The H-2K^k antigens were initially distributed homogeneously on all lymphocytes. Upon antibody binding, sub-micron patches were formed on 50–60% of the cells. A lateral diffusion coefficient, D, of 7.1·10^?10 cm²/s and a mobile fraction of 0.73 were found for H-2K^k antigens on diffusely-labeled cells, while these antigens were immobile (D?5·10^?12 cm²/s) on patched cells. The patched and nonpatched sub-populations did not correspond to B- and T-lymphocytes. Subjection to low temperature or treatment with NaN₃ or cytoskeleton-disrupting drugs did not affect the diffusion or patching of H-2K^k, indicating no involvement of metabolic energy or drug-sensitive cytoskeletal components. These findings could be related to the interactions of H-2 antigens on the cell surface, and to the different susceptibilities of various cells to lysis by cytotoxic T-cells.     

Spleen cells from animals neonatally tolerant to H-2Kk antigens recognize trinitrophenyl-modified H-2Kk spleen cells

A.TL mice injected with (A.AL × A.TL)F₁ cells within 24 hours after birth were rendered tolerant to H-2K^k antigens, as evidenced by acceptance of A.TL skin grafts. When spleen cells from these tolerant animals were cocultured with A.AL stimulator cells, no cytotoxic effector cells were generated in a cell-mediated lympholysis assay. However, when the A.AL stimulator cells were derivatized with trinitrophenol, effector cells that displayed a cytotoxic effect against trinitrophenyl-modified H-2K^k target cells were generated. These data indicate that animals tolerant to H-2 determinants but chimeric to only a minor extent possess cytotoxic precursor cells in sufficient frequency to mount a primary in vitro response against trinitrophenol in the context of an allogeneicH-2K region.     

Differential phosphorylation of murine class I major histocompatibility antigens

Recent studies have shown that the H-2K and H-2D transplantation antigens are expressed differentially in different tissues of mouse. Our previous investigations also established that in thioglycolate-stimulated peritoneal macrophages the H-2D^k antigen exists in distinct cell surface and intracellular forms. These two forms are glycosylated differently. In this report, we have found that (1) H-2D^k antigen is phosphorylated whereas H-2K^k antigen is not, and (2) only the cell surface form of H-2D^k antigen is phosphorylated in thioglycolate-stimulated macrophages derived from C3H/Heha mice. This differential phosphorylation of H-2 antigens will provide a model system for further studies on the molecular mechanism and function of phosphrrylation of H-2 antigens.     

H-2 class I antigen expression on mouse teratocarcinoma cell lines

Immunity against PCC3 teratocarcinoma cells (129, H-2 ^b) was induced in allogeneic (C3H, H-2 ^k) mice by preimmunization with L cells (C3H, H-2 ^k) expressing cosmid-introduced K ^b or D ^b genes, but not with nontransfected L cells. In addition, the growth of PCC3 cells in sublethally irradiated (C3H × B6-H-2 ^bm1)F₁ and (C3H × B6-H-2 ^bm13 )F₁ mice bearing the K ^bm1 and D ^bm13 mutations, respectively, was either prevented, stopped, or delayed in comparison with the (C3H × B6)F₁ (k × b) mice, which failed to reject the PCC3 cells. The teratocarcinoma line OC15S was exceptional because it reacted specifically with K^b- and D^b-specific (but not I^b-specific) alloantisera, and because K^b- and D^b-specific antibodies could be absorbed by OC15S cells. The subpopulation of OC15S cells bearing the ECMA-7 antigen characteristic for embryonic carcinoma (EC) cells was isolated by the fluorescence-activated cell sorter and was shown to react specifically with K^b- and D^b-specific antisera. These experiments show that teratocarcinoma cells express antigens similar or identical to the K-and D-region products of differentiated cells. The lack of expression of class I antigens is thus neither a condition nor a consequence of the pluripotentiality of the EC cells. The exact nature of the major histocompatibility complex antigens on EC cells has yet to be established using the methods of molecular biology and biochemistry.     

Reduced humoral and cellular cytotoxic sensitivity in major histocompatibility variants of the YAC (Moloney) lymphoma

Previously we have reported that two sublines of the YAC lymphoma selected for reduced expression of H-2^a and Moloney-virus determined cell-surface (MCSA) antigens are, in contrast to YAC, allotransplantable in H-2-incompatible recipients, and resistant to rejection by preimmunized semisyngeneic hosts. A third YAC variant with reduced MCSA but unchanged H-2-antigen expression, was not allotransplantable and showed only a slight decrease in its immunosensitivity in preimmunized semisyngeneic hosts in vivo. This suggested that H-2-antigen expression may be more important than MCSA expression for recognition and rejection by semisyngeneic mice. We have now tested the sublines expressing low H-2^a for their in vitro sensitivity to humoral and cell-mediated lysis. — The variants were more resistant than YAC to complement lysis by anti-H-2^a, anti-MCSA, anti-Thy 1.2 and antispecies sera. Absorption tests with antispecies serum indicated that the decreased cytolytic sensitivity of the variants was not related to the concentration of the relevant antigens, which was similar to that of the original YAC tumor. As expected from the low amount of H-2^a the variants showed a decreased sensitivity to the killing effect of allogeneic cytotoxic T lymphocytes (CTL). They were also lysed to a lesser extent than YAC by semisyngeneic CTL, probably directed against virally determined antigens. However, they were also less sensitive to lysis by natural killer (NK) cells, although NK lysis is probably unrelated to MHC expression. In conclusion, our selection for reduced H-2-expression appears to have resulted in the isolation of variants with a generally increased resistance to various humoral and cell-mediated lytic functions.On leave from Instituto Venezolano de Investigaciones Cientificas (IVIC), Caracas, Venezuela.     

Aberrancy in immunogenicity and cell-surface expression of H-2 antigens on erythrocytes

Immunogenicity for T cell-independent B-cell response assessed by splenic plaque-forming cell (PFC) response and cell-surface expression measured by laser flow cytometry of various class I H-2 antigens on mouse red blood cells (RBC) were compared. It was found that the order of magnitude of both immunogenicity and cell-surface expression on RBC is H-2D^d H-2D^b > H-2K^d, H-2K^b. Furthermore, H-2^d public antigens and H-2L^d antigens were neither immunogenic nor easily demonstrable on RBC. These findings contrasted with poor immunogenicity for PFC response (Nakashima et al. 1982, 1983) and proportionally strong expression of H-2 antigens on lymphoid cells. Immunogenicity and cell-surface expression of H-2D^d antigen on RBC were not shown to be controlled by the action of genes outside H-2D. It was therefore suggested that a number of H-2 antigens, including H-2K^d private, H-2K^b private, and H-2^d public specificities are at least functionally defective on RBC. This is possibly due to the structural characteristics of the antigens. Since immunogenicity and cell-surface expression were in parallel, the expression of H-2 antigens on RBC must be dictated by a subset of B cells whose activity was assessed by PFC response. This finding supports the view that the H-2 molecules display a new category of activity which is different from their ability to activate T cells and depends on their expression on RBC.     

H-2 class I loci determine sensitivity to MCMV in macrophages and fibroblasts

Peritoneal (PM) and bone marrow-derived (BMM) macrophages and lung fibroblasts (LF) from inbred, intra-H-2 recombinant, H-2 mutant, and hybrid mice were infected with murine cytomegalovirus (MCMV) under centrifugal enhancement. At the concentration of virus employed, peritoneal macrophages from strains carrying Kd, Kb, D^d, KS and/or D^s, K4 and/or D4 alleles could be infected to a level of 80%–100%, as assessed by viral antigen expression or loss of Fc receptors. Cells lacking these haplotypes and carrying K^k, K^f, D^k, Df, or Db were resistant, yielding levels of infection below 20% . The background (non-H-2) and class II genotype and the S allele did not influence the proportions of cells infected. Furthermore, sensitivity was dominant in the F, progeny of H-2 b x H-2 k and H-2d x H-2 k crosses, and was not compromised by thebm1, bm3, bm10, or bm14 mutations in the al or2 regions of Kb orD ^b. The proportions of cells able to release infectious virus were low, but paralleled the frequencies of viral antigen expression. The class I genotype also determined susceptibility to MCMV infection in BMM and LF, although up to 35% of H-2 k BMM and 46% of H-2 k LF could be infected. The findings are consistent with an association between K and D antigens and a cellular receptor for MCMV on all three cell types.     

New evidence fortrans-species evolution of theH-2 class I polymorphism

A serological survey using alloantisera specific for the H-2 class I antigens in Japanese wild mice,Mus musculus molossinus, revealed a high frequency of the H-2K^f antigen. This antigen has also been found in European wild mice,M. m. domesticus andM. m. musculus. In this survey, the H-2K^f antigen was characterized through the use of ten newly isolated monoclonal antibodies raised against cells of a Japanese wild mouse, and by Southern blot analysis using anH-2K locus-specific probe which hybridizes with the 3′ end of the gene. The serologically identified H-2K^f antigens revealed several minor variations in reactivities to the monoclonal antibodies. However, all the antigens examined could be clearly separated into two types with respect to the restriction fragment length polymorphism (RFLP) pattern. The first type, found together with a single, characteristic RFLP pattern, was always associated with the presence of reactivity to one particular monoclonal antibody, MS54. The second type, found to represent different RFLP patterns, is associated with the absence of reactivity to MS54. This concordance between the presence of an antigenic determinant and a particular RFLP was observed not only withinMus musculus subspecies but also in a different species:M. spretus, carrying the same antigenic determinant, gave an identical RFLP to that of the other MS54-positiveMus musculus subspecies. The data suggest that the antigenic determinant specific for MS54 is an ancient polymorphic structure which has survived the long period of diversification ofMus species (approximately 2–3 million years) without alteration, and is associated with a stable DNA structure at the 3′ end of theH-2K gene.     

The spatial relationship of the viral and H-2 antigens recognized by anti-viral CTLs

With the use of monospecific rabbit anti-G protein and mouse monoclonal anti-H-2K^k, we have analyzed the spatial relationship of the serologically defined H-2K^k antigens and the major surface glycoprotein (G protein) of vesicular stomatitis virus (VSV) to those antigens recognized by B10.A (k, d) anti-VSV cytotoxic T lymphocytes (CTLs). The ability of monoclonal anti-H-2K^k or rabbit anti-G protein to inhibit specifically the cytolytic activity of B10.A anti-VSV CTLs indicates that the G protein and the H-2K^k molecules are in close proximity to the viral and H-2K^k antigens recognized by the anti-VSV (CTLs. By the method of sequential immunoprecipitation, we also demonstrated that only 10–30 percent of the serologically defined G and H-2K^k molecules are in theG-H-2K ^k complexes.Abbreviations used in this paper Con A Concanavalin A - cpm counts per minure - CTLs cytotoxic T lymphocytes - E: T ratio effector: target ratio - G major surface glycoprotein of VSV - MHC major histocompatibility complex - MOI multiplicity of infection - NP40 Nonidet-P40 - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - SaCI Staphylococcus aureus, Cowan I strain - SDS sodium dodecyl sulfate - UV ultraviolet light     

H-2-specific antibodies induced by injection of syngeneic leukemia cells

AKR/J mice immunized with several syngeneic leukemia cells contained antibodies in their sera which reacted with certain AKR leukemia cell lines, depending on their H-2 expression, and precipitated H-2K antigens from lysates of leukemia cells. Precipitation of H-2K was not due to virus-specific antibodies: it could not be blocked by prior absorption with H-2-negative leukemias, but was blocked by certain allogeneic lymphocytes. Tumor-specific H-2K antibodies did not react with H-2K from normal AKR lymphocytes either on the cell surface or after detergent solubilization; however, they did react with H-2K from mitogen-activated AKR and BALB.K lymphoblasts. Since both these latter cells were also lysed by AKR-Gross/MuLV-specific and H-2K^k-restricted cytotoxic T lymphocytes, we consider the possibility that antibodies detecting conformational alterations induced in H-2K^k molecules by viral association may be present in syngeneic AKR antileukemia sera.Abbreviations used in this paper GCSA Gross-virus-induced cell-surface antigen - MCF mink cell focus-forming virus - MuLV murine leukemia virus - Th T helper     

Analysis of intrathymic differentiation patterns during the course of AKR leukemogenesis

Thymocytes from AKR mice in different stages of leukemia development were analyzed with the fluorescence-activated cell sorter (FACS) using monoclonal antisera to Lyt-1, Lyt-2, Thy-1.1, H-2K^k, and Ia^k. In addition, the number of cells bearing receptors for peanut agglutinin (PNA) was assessed. The results were correlated with the expression of murine leukemia virus (MuLV) antigen. Thymocytes from late preleukemic and leukemic stages were found to have a phenotype characteristic of a more mature cell population in that there was an increase in the expression of determinants encoded within the K end of H-2^k and Ia^k. This was associated with a decrease in the number of thymocytes bearing receptors for PNA during the leukemic stage. Simultaneously, a shift from a Lyt-1⁺ 2⁺ thymocyte population to cells with varying expressions of Ly antigens was observed. Analysis of Lyt determinants on thymomas indicated that they could arise from cells bearing any of the different possible combinations of Ly phenotypes. The cell surface antigen changes occurred in temporal correlation with an increased expression of MuLV antigens.     

Isolation of twelve monoclonal antibodies against Ia and H-2 antigens. Serological characterization and reactivity with B and T lymphocytes

The cell hybridization technique was used for the production of 12 monoclonal antibodies against H-2K^k, H-2D^b, I-A^k and I-E^k antigens. The strain distribution pattern indicated that three antibodies reacted with new H-2 and Ia determinants, respectively, while the majority of determinants defined by the monoclonal antibodies showed good correlation with H-2 and Ia determinants described by conventional alloantisera.Monoclonal Ia antibodies showed strong reactivity with about 90% of surface IgM positive B cells, but not with T cells. In double fluorescence studies, both I-A and I-E determinants were always found to be coexpressed on the same B cells. When the high sensitivity of the fluorescence activated cell sorter was utilized, about 30 to 40% of purified lymph node T cells were found to carry both I-A and I-E antigens, although in a much lower density than B cells. In conclusion, monoclonal Ia antibodies appear to display the same serological and cellular reactivity pattern as do conventional antisera.     

A study of the ability of H-2Kk-Iak containing subcellular fractions to elicit primary anti-H-2 cytotoxic T lymphocytes

In order to identify an antigen required for elicitation of anti-H-2 cytotoxic T lymphocytes (CTLs), we have purified the H-2-K^k glycoprotein, incorporated it into a lipid vesicle, and tested it for its ability to elicit anti-H-2K^k CTLs. The results indicate that a lipid vesicle containing purified H-2K^k can elicit specific secondary anti-H-2K^k CTLs. In addition it was shown that in association with a partially purified Ia^k fraction the primary anti-H-2K^k CTL response was enhanced. It was also shown that Ia^k antigens alone could elicit an anti-H-2^k CTL response. Although a low primary response was found with purified H-2K^k, it was observed that lipid vesicles containing both H-2K^k and Ia^k glycoproteins could elicit a significantly enhanced primary anti-H-2K^k CTL response. Lipid vesicles containing H-2K^k-Ia^k were tested for their enhanced capacity to elicit anti-H-2 CTLs as well as for their ability to elicit anti-H-2K^k CTLs in the presence of supernatants from concanavalin A-stimulated spleen cells.     

Adjuvant Activity of Mycobacterial Fractions

A quantitative assay and characterization of oil-attached cell wall of Mycobacterium bovis BCG (BCG-CWS) which stimulates cell-mediated immunity of spleen cells to alloantigens in mice were carried out by an in vitro cell-mediated cytotoxicity test using ⁵¹Cr-labeled target cells. C57BL/6J mice (H-2^b) were immunized intraperitoneally with mastocytoma cells (H-2^d) with or without oil-attached BCG-CWS. The cytotoxicity, comparable to that of spleen cells from mice immunized with mastocytoma cells (3 × 10⁷), could be induced in spleens of mice immunized with a mixture of mastocytoma cells (10⁴) and oil-attached BCG-CWS. The enhancing effect persisted for 55 days or more after the alloantigenic immunization. Oil-attached BCG-CWS enhanced cell-mediated cytotoxicity of T cells in the spleen and the mesenteric lymph node, but not in the thymus. The cytotoxicity showed specificity toward the alloantigen used for immunization. In addition to BCG-CWS, the cell walls of Nocardia rubra and Corynebacterium diphtheriae PW8 and the peptidoglycolipids of Mycobacterium tuberculosis Aoyama B were found to be potent stimulants of cell-mediated cytotoxicity in mice. Oil-attached BCG-CWS did not enhance humoral response to mastocytoma cells but enhanced cell-mediated cytotoxicity when viable mastocytoma cells were used as antigen. The above result was supported by the fact that anti-hapten antibody response induced by viable trinitrophenyl (TNP)-mastocytoma cells (10⁴) plus oil-attached BCG-CWS did not increase to the maximum level as was observed in mice immunized with a larger number of mastocytoma cells (3 × 10⁷) alone, while cell-mediated cytotoxicity induced by the same treatment increased to the maximum level obtained by immunization with mastocytoma cells (3 × 10⁷) alone.     

Natural resistance of irradiated 129-strain mice to bone marrow allografts: Genetic control by theH-2K region

A major genetic determinant of natural resistance to bone marrow allografts, designated asHh-3, was mapped to theH-2K region. This gene may code for or regulate the expression of cell surface structures selectively expressed on donor hemopoietic cells and recognized by naturally occurring cytotoxic effectors. Resistance was observed as failure of donor cell growth in the spleen of irradiated 129-strain (H-2 ^bc ) recipients of H-2^k bone marrow cells. The mapping was accomplished by substituting donor cells bearingk alleles throughout theH-2 complex with cells of recombinant mouse lines bearingk alleles at definedH-2 regions. The host antigraft reaction underlying resistance was abrogated by pretreating 129-strain mice with either rabbit antimouse lymphocyte serum or the antimacrophage agent silica. Grafting of H-2K^k cells into mice ancestrally unrelated to 129 but sharing theH-2 ^bc or the similarH-2 ^b haplotype, and intoH-2 ^b/k ,H-2 ^k/bc , andH-2 ^k/d F₁ hybrids revealed that resistance was unique to 129 mice, since mice of the other strains, including F₁ hybrids, were susceptible to the grafts. Thus,Hh-3 incompatibility was a necessary but insufficient condition for the manifestation of allogeneic resistance; other genetic factors not associated withH-2 conferred responder status to 129-strain mice and nonresponder status to D1.LP, B10.129(6M), B10, B6, and possibly to F₁ hybrid mice. The possible relationships between allogeneic resistance to H-2^k marrow grafts, hybrid resistance to H-2^k lymphomas, and F₁ hybrid antiparental H-2^k cytotoxicity induced in vitro are discussed.     

Biochemical analysis of an MHC-linked hematopoietic cell surface antigen,Qa-2

The region of the murine 17th chromosome telomeric to H-2D encodes a group of serologically defined cell surface antigens termed Qa-1-5. These antigens are of interest because their expression is restricted to hematopoietic cells. In addition, the molecular weight and subunit structure (ie, association with β-2 microglobulin) of Qa-2 molecules are similar to H-2 and TL antigens. In the present studies, we have prepared isotopically labeled Qa-2 and H-2 molecules from mitogen-stimulated C57BL/6 spleen cells. Comparative peptide mapping of tryptic peptides from Qa-2 and H-2 molecules (K^b, D^bK^k, D^d) reveal that Qa-2 has a unique primary structure. However, considerable homology is indicated since 30–40% of the Qa-2 peptides cochromatograph with peptides derived from H-2K^b, H-2D^b, H-2K^k, and H-2D^d. Studies by other investigators have demonstrated that similar levels of structural homology are observed when H-2K, H-2D, and H-2L tryptic peptides are analyzed. We conclude from these studies that the Qa-2 alloantigen is structurally related to a class of cell surface molecules (ie, H-2) that play critical roles in immune recognition processes. These data further suggest that the genes encoding Qa-2 and H-2 molecules have arisen from a common primordial gene.