Donald L. Evans

H-2 antigens incorporated into phospholipid vesicles interact specifically with allogeneic cytotoxic T lymphocytes

Abstract Subcellular fractions containing different H-2 antigens were tested for their ability to inhibit specific T cell-target cell conjugate formation. H-2-containing membrane vesicles, lentil-lectin-purified H-2 antigens solubilized with detergent (referred to in the text as high-density fraction) or incorporated into lipid vesicles, inhibited T cell-target cell conjugate formation effectively and specifically. However, two- to threefold more protein was required to inhibit T cell-target cell conjugate formation when detergent-solubilized lentil-lectin-purified H-2 antigens were tested. This suggests that a lipid matrix is advantageous for interaction with anti-H-2 T-cell receptors. Experiments were also undertaken to demonstrate specific binding of liposomes containing 125I-labeled H-2 antigen to anti-H-2-specific cytotoxic T lymphocytes (CTLs). The binding of the 125I-labeled H-2-containing liposomes was saturable and was specifically inhibited by unlabeled H-2 antigens. Monospecific anti-H-2 sera specifically inhibited the binding of liposomes containing H-2 antigen to the CTLs. The results suggest that a specific interaction can occur between serologically defined H-2 antigens and the receptor of anti-H-2 CTLs.

Effect of prostaglandins on elicitation of anti-viral cytolytic activity

We have studied the modulating effect of prostaglandins (PG) on the elicitation of secondary anti-vesicular stomatitis virus (VSV) cytotoxic T-lymphocytes (CTL) with viral antigens. The results indicate that PGE1 and PGE2 both inhibit the induction of anti-VSV CTLs by ultraviolet-inactivated VSV. Consistent with the result was the augmentation of anti-VSV CTL activity when VSV-primed spleen cells were cultured in the presence of indomethacin. These results suggest that PGs may modulate anti-viral CTL responses.

Age-related increases in mitogenic responses and natural immunity to a syngeneic fibrosarcoma in rats

An increase in natural (innate) immunity with age (through 65 weeks) of NBR rats to a syngeneic methylcholanthrene-induced fibrosarcoma (MCA) is described. This increase is characterized by the appearance of spontaneous tumor regressor animals and an increase in the incidence of tumor non-takes by rats 45 weeks of age and older when compared to younger animals. Age-related changes were observed in the in vitro proliferative responses of normal unfractionated and nylon wool fractionated spleen cells to Con A, PHA and irradiated MCA tumor cells. Certain of these different proliferative responses either increased and/or decreases independently at different ages. Relative to normal spleen cell proliferative responses, tumor progressors have decreased proliferative responses and tumor regressors display increased proliferative responses. The significance of this in light of current aging research is discussed.

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

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

Suppression of mitogen- and tumor-cell-induced lymphocyte stimulation by tumor-associated fetal antigens

Abstract The ability of tumor-associated fetal antigens (TAFA) to suppress mitogen and tumor-cell-induced blastogenesis was investigated. Three different TAFA (I, III, and IV) were tested in PHA and Con A lymphocyte proliferation assays. Spleen cells from New Zealand black rats (NBR) were fractionated over nylon wool; and nonadherent (NA) and adherent (AD) cells were compared with unfractionated (UF) cell responses. Preincubation of NA cells with TAFA-I was followed by addition of phytohemagglutinin (PHA) elicited suppression in a 3-to 4-day assay. UF cells were unresponsive to TAFA and/or PHA at all concentrations tested. TAFA dose—response titration curves in Con A proliferation assays revealed that all TAFA tested (TAFA I, -III, and -IV from fibrosarcomas; TAFA-I and -III from osteosarcomas) were suppressive. For some TAFA, nanogram quantities produced significant suppression. In mixed leukocyte tumor cell assays (MLTC) both UF and NA normal rat spleen cells were tested for proliferative responses to syngeneic tumor cells. Four distinct TAFA, purified by high-pressure liquid chromatography, suppressed lymphocyte proliferation when added to MLTC cultures in 5-day assays. Specificity experiments demonstrated that trinitrophenol-bovine serum albumin did not produce similar immunosuppression. TAFA did not block recognition of tumor antigen nor produce nonspecific cytotoxicity of the spleen cells. Significant suppression of DNA synthesis was produced by TAFA-1 following cocultivation with spleen and tumor cells for 1, 2, and 3 days, compared to no suppression when spleen and tumor cells were washed free of TAFA-I prior to tumor cell addition at Day 0. Similar experiments using rat embryo fibroblasts (REF) as stimulators demonstrated that pre-REF cocultivation treatment of lymphocytes with TAFA-I significantly reduced subsequent blastogenic responses. This effect was not reversible; however, if TAFA-I was added to responders previously stimulated by REF, a suppressive response was not seen. Experiments were also carried out to determine the reversibility of TAFA-I effects. Cells were treated with TAFA-I from 1 to 5 days, followed by determination of lymphocyte blastogenesis. TAFA-I effects are reversible and antigen presence is required to completely suppress (or inhibit) stimulation by tumor cells.

Cell Requirements for Optimum Proliferative Responses of Rat Spleen Cells

Abstract Spleen cells from New Zealand black rats (NBR) were separated by nylon wool (NW) and by density gradient centrifugation (Percoll) to identify certain physical, chemical, morphological and functional attributes of mononuclear cells. Density distribution profiles of nylon wool nonadherent (NA) and adherent (ADH) cells indicated that at least two different populations of spleen cells existed (NA = 1.070–1.075 g/ml; ADH = 1.064 g/ml). 38% of all NA cells and 13 % of all ADH cells are T-cells (mean density = 1.080 g/ml). Nonspecific esterase staining indicated gradient enrichment of ADH cells at densities of 1.064 g/ml (mononuclears) and 1.080 g/ml (polymorphonuclears). NA cells contained 3 % esterase positive cells. Plastic adherent cells had a mean density distribution of 1.064 g/ml. Mitogen binding cells were identified by function and density. ADH cells (1.064 g/ml) demonstrate higher specific binding of PHA-I 125 compared to NA cells (1.072 g/ml). Unlike the PHA binding cells, Con-A binding ADH and NA cells share the same mean densities (1.055–1.064 g/ml). As with the PHA responses, ADH cells have greater specific binding activities for Con-A than NA cells. Mitogen binding cells have lower mean densities than cells which incorporate tritiated thymidine at 5 days post-cultivation with PHA or Con-A. Adherent cells enriched for T cells and polymorphonuclears produced the highest PHA responses. PHA responsive NA cells were not enriched by gradient separation. NA cells produced a suppressive effect when mixed with cells which produced optimum PHA responses. ADH cells (density 1.073 g/ml) produced a significant PHA response helper effect when mixed with gradient separated cells. A helper effect was not produced by this fraction in the presence of Con-A responsive cells. Unlike PHA responses (where optimum binding and proliferation occurred by ADH cells), enrichment for Con-A responders occurred in NA gradient fractions (1.073–1.080 g/ml).

Interactions of tumor-associated fetal antigens with rat histocompatibility antigens☆

Abstract The physical interactions of fetal antigens (tumor-associated fetal antigens; TAFA-I, TAFA-II, and TAFA-III) with rat histocompatibility antigens were studied. TAFA-I and TAFA-III are present on syngeneic (NBR) and allogeneic (Fisher F344, Wistar Furth, and White Buffalo) rat embryo fibroblasts and on tumor cells. TAFA-II was found only on NBR (syngeneic) rat embryo fibroblasts and on NBR tumor cells. Antibody-blocking experiments were used to examine the fetal and histocompatibility antigen topography on cell membranes of tumor cells transformed by chemical and viral carcinogens. Precoating the tumor cells with alloantisera inhibited the subsequent adsorption of anti-NBR embryo, anti-TAFA-I, and anti-TAFA-III sera, but not anti-TAFA-II serum. Immunofluorescent cocapping experiments indicated that TAFA-I and TAFA-III, as well as other fetal antigens found on cells from 14-day gestation NBR embryos cocap with histocompatibility antigens when tested on syngeneic embryo fibroblasts and on sarcoma cells. TAFA-I cocapped with White Buffalo (Buf) strain rat histocompatibility antigens on herpes simplex Type II virus-transformed cells. The specificity of the TAFA-histocompatibility interactions was confirmed by demonstrating that the different anti-TAFA sera did not have contaminating antiviral antigen specificity; and also that these interactions did not occur on normal adult fibroblasts or spleen cells.

Histocompatibility requirements of rat lymphocytes for fetal antigen recognition

Abstract The primed lymphocyte test (secondary mixed lymphocyte-fibroblast reaction) and chromium-51 release cytotoxicity assay were used to study the involvement and/or requirement of major histocompatibility antigens in the interaction of anti-fetal effector lymphocytes with rat embryo fibroblasts (REFs) and tumor cells. Rat spleen cells previously sensitized in vitro against 14-day gestation REFs produced significant secondary blastogenic reactions when restimulated with REFs or tumor cells possessing the same major histocompatibility complex haplotype as the primary REF stimulators. The fetal specificity of primed lymphocytes was demonstrated by the inability of either syngeneic or allogeneic adult normal fibroblasts to induce a secondary proliferative response. Analogous results were seen using the chromium-51 release cytotoxicity assay. Anti-F344 cytotoxic T lymphocytes (CTLs) from W/F × F344 F1 rats significantly kill F344 (Ag-B1) REFs and NBR (Ag-B1) REFs, but not W/F (Ag-B2) or Buf (Ag-B6) REF targets. Involvement of histocompatibility antigens in the recognition of fetal antigens by CTLs was further indicated by the ability of alloantiserum, as well as anti-fetal serum, to inhibit the lysis of REF target cells by the anti-fetal CTLs.

Surface analysis of early and late MOPC-315-EL tumor cells

Abstract The surface properties of a tumor line which undergoes a cyclic change in reactivity with cytotoxic T lymphocytes (CTLs) have been analyzed. The findings indicate that MOPC-315-EL when transplanted into the peritoneum of a BALB/c mouse for 4–5 days (early MOPC315-EL) becomes sensitive to lysis by anti-H-2 d CTL. After 9 to 10 days of growth in the peritoneal cavity these tumor cells (late MOPC-315-EL) become unreactive to CTLs. The amount of H-2 antigens on the surface of the cells is the same on the early and late tumor cells. By scanning electron microscopy (SEM) we have shown that the early population is a mixture of two cell types which are characterized by surface villi or surface blebs; the late cells are essentially one population which are characterized by surface villi. The net charge on the surface of the cell was evaluated by two methods: (i) rate of cell migration across an electric potential and (ii) binding by cationic ferritin to the glycocalyx. Both techniques indicated that the early cells have a higher net negative charge. The implication of this work is that surface charge may be involved in T cell-target cell interactions.

Antigenic differences between tumor-associated fetal antigens and rat histocompatibility antigens

Abstract Certain molecular properties of purified tumor-associated fetal antigens (TAFA) were analyzed by sequential immune precipitation (SIP), isoelectric focusing, and high-pressure liquid chromatography (HPLC). The antigenic relatedness of rat histocompatibility antigens and the various TAFA were determined by SIP. SIP of chloramine-T-labeled purified TAFA or lactoperoxidase-iodinated tumor cell membranes, in the presence of rat alloantisera and monospecific rabbit anti-TAFA sera demonstrated no antigenic cross-reactivities or similarities between H-antigens and TAFA. TAFA were also compared with histocompatibility antigens for isoelectric point optima and molecular weight. Rat H-antigens had isoelectric points in the 7.0–8.5 pH range, whereas all TAFA focused at pH 5.0–6.5 or above pH 8.0. Molecular weights were determined by HPLC. TAFA-I and TAFA-III had molecular weights of 16,000–17,500 daltons, whereas TAFA-II had a molecular weight of 12,000. The antigens were not coprecipitated by the rat alloantisera. Each TAFA was also isolated (via immune precipitation) from NP-40-solubilized tumor cell membranes. These TAFA were identical to the chloramine-T-labeled TAFA which had been previously extracted and purified from rat fibrosarcomas and osteosarcomas. These studies demonstrated that although TAFA and H-antigens cocap on embryonic and transformed cell membranes, these determinants are different molecules; they are not covalently linked on cell membranes; and TAFA are not cleavage products of normal NBR H-antigens.