Wilhelm Schmidt

Variation in mhc antigenic profiles of tumor cells and its biological effects.

It is now well established that the major histocompatibility system (MHS), Ly, and other alloantigenic systems play important roles in cell-cell interactions, which are particularly relevant for immune responses. For example, 1-region gene products are involved in the presentation of antigen by macrophages and may be part of antigen-specific and nonspecific factors that are released by helper and suppressor cells, respectively. In contrast, T-cell in vitro cytotoxicity appears to depend on the sharing of Class I ( H 2 K / D / L ) gene products between target and effector cells in specific instances. This is true for virally infected and chemically modified cells and for effector cells sensitized against the H-Y antigen and a variety of non-H-2 antigens including certain tumor-associated antigens (for references see Snell 1979, Zinkernagel & Doherty 1980). Any modification of the antigenic profile of effector or target cells, or both, is thus likely to substantially influence the outcome of immune reactivity (Festenstein & Demant 1978).

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-2Kk antigens were inactive as inducers of Gross-MuLV/H-2k-specific cytotoxic T lymphocytes (CTL) and resistant to lysis by CTL raised against H-2Kk positive AKR leukemia cells. H-2Kk positive leukemias induced cytotoxic effectors, which upon restimulation in vitro, lysed the stimulating and other H-2Kk 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-2Kk antigens. Due to this specific role of H-2Kk antigens in T-cell cytotoxicity to Gross/AKR MuLV-induced tumors, reduced expression of H-2Kk antigens on spontaneous AKR leukemic cells could have important implications for surveillance of these neoplastic cells.

Interspecies exchange of β2-microglobulin and associated MHC and differentiation antigens

Radiolabeled human β2-microglobulin (β2m) can bind to mouse histocompatibility (H-2) antigens on the cell surface or to partially purified H-2 antigens in solution. The complexes containing human β2m and H-2 antigens from C3H (H-2k) mice could be immunoprecipitated specifically with alloantisera, rabbit anti-H-2 xenoantisera, and with monoclonal H-2-specific antibodies. Specific association with H-2 antigens was also observed with other haplotypes. The only exception was B10.D2 (H-2d) from which complexes containing human β2M could only be precipitated with anti-H-2 xenosera. Thus radiolabeled human β2M can be used as a specific label for mouse H-2 antigens in precipitation and radioimmunoassays. The application of this finding extends to major histocompatibility complex antigens of other species, and to differentiation antigens with primary association with β2m.

Loss of H-2Kk gene product(s) from an AKR spontaneous leukaemia

Serological absorption analyses and immunochemical studies of the H-2 antigenic specificities on the AKR (H-2k) spontaneous leukaemia K36 have been performed. The results confirm the absence of the H-2Kk gene product from the cell surface and from detergent solubilised tumour cells. A genetic mechanism is considered for this phenotypic alteration.

β-endorphin: Interaction with specific nonopioid binding sites on EL4 thymoma cells

Binding of 125I-labeled camel beta-endorphin (125I-beta C-endorphin) to cells of several mouse thymoma cell lines was examined and was highest to EL4 cells. 125I-beta C-endorphin binding to EL4 cells was temperature-dependent; it was further characterized at 4 degrees C and exhibited saturability, complete reversibility, structural specificity and pH-dependence. 125I-beta C-endorphin binding was not inhibited by the opioid pentapeptides [Leu] enkephalin or [Met] enkephalin (which share common sequences with the N-terminus of beta C-endorphin) or by the N-terminal beta C-endorphin fragments beta C-endorphin (1-16) or beta C-endorphin (1-27). In contrast, binding was inhibited by beta C-endorphin (1-31), indicating that beta C-endorphin binding to EL4 cells was with a C-terminal beta C-endorphin segment. We suggest that binding of beta-endorphin to such nonopioid binding sites may precede its apparent effects on the proliferation of T-lymphocytes (5,6).

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-2Kk-gene products were absent by several criteria: (1) monoclonal antibody and conventional alloantisera directed against the H-2Kk antigenic specificities were nonreactive by direct testing and by absorptions. (2) H-2Kk products could not be precipitated from glycoprotein or protein extracts of the radiolabeled K-GV tumor. (3) Cytotoxic effectors against H-2Kk 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-2Kk-gene products. In contrast, the H-2Dk 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-2k) tumors. These findings have important implications for the associative recognition of tumor antigens and the immune surveillance of virally induced tumors.

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-2Kk-restricted cytotoxic T lymphocytes, we consider the possibility that antibodies detecting conformational alterations induced in H-2Kk molecules by viral association may be present in syngeneic AKR antileukemia sera.

Control of synthesis and expression of H-2 heavy chain and beta-2 microglobulin in AKR leukemias

In a series of newly isolated AKR leukemias, some tumors expressed large amounts of both H-2K and H-2D molecules, while others had reduced levels of both antigens. The number of H-2 antigens expressed in steady state showed a consistent correlation with the rate of synthesis of the H-2 class I heavy chain and the beta-2 microglobulin (β2m) light chain, and with the amount of H-2-and β2m-specific mRNA present in the tumors. Stimulation of leukemic cells with interferon induced an increased transcription of both H-2 and β2m mRNA. These results suggest that there is a mechanism that regulates, at the transcriptional level, the coordinate expression of H-2K and H-2D heavy chains, and the β2m light chains encoded by genes on chromosomes 17 and 2, respectively.

Identification of murine H-2Db histocompatibility antigens in cells transfected with cloned H-2 genes

Clones of mouse L-cells transformed with 21 cosmids containing 15 major histocompatibility complex class I genes of C57BL10 (H-2b) sperm cell DNA were analyzed for the expression of their transfected H-2 and Qa/Tla genes. Three cosmids contained a single gene, mapping to the H-2D region. This gene encodes the H-2Db alloantigen: mouse L-cells transfected with cosmids containing this gene reacted with monoclonal antibodies and alloantisera specific for the H-2Db antigen and expressed a 46-kd H-2 heavy chain associated with beta 2-microglobulin in their cell membranes. Furthermore, these transfected cells were stimulators of, and targets for, anti-H-2Db cytotoxic T-lymphocytes. Eighteen cosmids contained 14 different genes mapping to the Qa and Tla regions. L-cells transfected with these genes did not express class I genes reacting with alloantisera or monoclonal antibodies against Qa2, Qa4 or TL differentiation antigens. In particular, the Qa2,3 gene of C57BL10 was not identified.

Exchange OF β2-Microglobulin in Histocompatibility Antigens by its Human Analogue

Abstract Human β2-microglobulin (β2m) can be exchanged in mouse histocompatibility (H-2) antigens for native β2m The exchange can be achieved at the cell surface by incubating mouse cell suspensions in medium containing free β2m, or in cell free suspensions using purified H-2 antigens. The complexes containing radiolabelled human β2m and H-2 antigens can be immunoprecipitated specifically with alloantisera and with monoclonal anti H-2 antibodies. Specific association of human β2m with H-2 antigens was observed with several haplotypes including H-2. Radiolabelled H-2 antigens associated with unlabelled human β2m can be precipitated with monoclonal antibody against human β2m. Thus radiolabelled human β2m can be used as a specific label for mouse H-2 antigens in precipitation and radioimmunoassays, and monoclonal anti-human β2m antibody can be used for affinity purification of H-2 antigens. The application of this method of exchange can be extended to major histocompatibility complex (MHC) antigens of other species, and to MHC linked differentiation antigens having a natural association with β2m.