YAC-1 Lymphoma Research Articles

Eradication of spontaneous and experimental adenocarcinoma metastases with chronic indomethacin and intermittent IL-2 therapy.

We had earlier shown that tumor-bearing results in an inactivation of IL-2-dependent effector cells by host macrophage-derived PGE2, and that chronic indomethacin therapy (CIT) aimed at blocking prostaglandin synthesis, combined with multiple rounds of IL-2, can cure experimental metastases of a variety of tumors in mice. We have now tested the efficacy of this therapy on spontaneous as well as experimental metastasis of C3-L5 mammary adenocarcinoma in C3H/HeJ mice. Mice transplanted s.c. with C3-L5 cells (and showing visible spontaneous lung metastases between days 7 and 10) were given CIT starting on day 15, plus 2 5-day rounds of IL-2 or IL-2 alone. Mice injected i.v. with 10(4) C3-L5 cells (and showing lung micrometastases on day 5) were placed on CIT on day 5 and given 3 5-day rounds of IL-2 or treated with IL-2 alone. Control mice received vehicles alone. Results revealed that combined CIT + IL-2 therapy in the spontaneous metastasis model caused a regression of primary tumors, a marked reduction in lung metastases scored on days 25-35 and a marked prolongation of host survival (79% cured). Survivors rechallenged with 10(4) tumor cells i.v. on day 210 resisted tumor growth. In the experimental metastasis model, this therapy also markedly reduced lung metastases and prolonged animal survival (50% cured). In both models, the combination therapy led to the presence of highly active tumoricidal (for C3-L5 and YAC-1 lymphoma targets) lymphocytes with AGM-1+, Lyt-2- and Thy-1 +/- phenotype and macrophages in the spleen and the lungs, and ADCC-promoting activity in the serum. CIT + IL-2 therapy can thus effectively eradicate spontaneous and experimental mammary adenocarcinoma metastasis in mice. It activates natural effector cells in situ, generates ADCC-promoting activity in the serum and results in resistance to tumor take in this moderately immunogenic tumor model.

Effects of histamine type-2 receptor antagonists on indomethacin and IL-2 immunotherapy of metastasis

Histamine type-2 receptor antagonists (H-2RA) have been used chronically to prevent dyspepsia in cancer patients subjected to immunotherapy with chronic indomethacin (Indo) and intermittent IL-2 in our cancer centre. We tested the effects of these agents during immunotherapy of C3H/HeJ mice transplanted s.c. with 5 x 10(5) C3L5 mammary adenocarcinoma cells. Tumor-transplanted mice were divided into groups receiving: (1) Indo (14 micrograms/ml); (2) H-2RA, i.e. (a) ranitidine at 28.6 micrograms/ml (Ran-lo) or 143 micrograms/ml (Ran-hi), or (b) famotidine (Fam) at 4.3 micrograms/ml, or (c) cimetidine (Cim) at 107 micrograms/ml, all in the drinking water on days 5-24; (3) IL-2 (1.5 x 10(3) Cetus U i.p. every 8 h on days 10-14 and 20-24); (4) combinations of H-2RA + Indo; or (5) combinations of H-2RA + Indo + IL-2. Animals were killed on day 24 for examination of primary s.c. tumor growth, secondary lung metastasis and splenocyte cytotoxicity against YAC-1 lymphoma cells (51Cr release assay). Results revealed: (1) primary tumor growth was reduced in mice treated with Fam + Indo, Indo + IL-2 and any of the H-2RA + Indo + IL-2 (no difference observed within the last two groups); (2) lung metastases decreased in mice treated with IL-2 alone, Indo + IL-2, and Indo + IL-2 + Ran-hi; (3) splenic cytotoxicity was suppressed in tumor-bearing controls, with partial restoration seen in Ran (both doses), Ran-lo + Indo, Ran-lo + Indo + IL-2, and Cim + Indo + IL-2 treated groups. Nearly complete restoration was seen in Cim, Cim + Indo, Indo + IL-2, Ran-hi + Indo + IL-2, and Fam + Indo + IL-2 groups. Thus, addition of H-2RA did not alter the overall therapeutic efficacy of the standard Indo + IL-2 tumor immunotherapy.

Effects of lipopolysaccharide on interleukin-2-induced cytotoxic activity of murine splenocyte cultures: role of prostaglandin E2 and interferons.

Splenocytes cultured for 24 h in the presence of interleukin-2 (IL-2), lipopolysaccharide (LPS) or both together expressed a cytotoxic activity against the YAC-1 lymphoma cell line and to a lesser extent against P815 mastocytoma cells. The association of IL-2 and LPS had an additive effect on induction of cytotoxicity. The IL-2-induced cytotoxic activity lasted for the whole of the culture; however, the addition of LPS at the initiation of the culture increased the cytotoxic activity during its the early phase, the increment being followed by a fall of lytic activity after 72 h of culture. Assessment of interferon (IFN) in the culture supernatants showed (a) a production of IFN gamma by IL-2-supplemented cultures, (b) a more potent IFN production by cultures treated with IL-2 plus LPS (including 20% IFN alpha/beta, (c) and that indomethacin (1 microM) potentiated the effect of either IL-2 or LPS used alone but did not significantly increase the cytotoxic activity of cultures treated with IL-2 plus LPS (the one that produced a high level of IFN). When cultures were treated by an anti-IFN gamma antibody we observed no change in the cytotoxic activity; however, in the presence of anti-IFN alpha/beta serum the cytotoxic activity of cultures treated with IL-2 plus LPS was inhibited after 24 h but stimulated after 72 h. When cultures treated with IL-2 plus LPS were supplemented with both indomethacin and anti-IFN alpha/beta the cytotoxic activity assessed after 72 h of culture was maintained at the same level as that of IL-2-treated cultures, hence the fall after 72 h of the cytotoxicity of cultures initiated in the presence of LPS alone was affected by both the immune serum and the cyclooxygenase inhibitor. Altogether these data show that when splenocytes are cultured for more than 72 h in the presence of IL-2 and LPS their cytotoxic activity decreases, and it is likely that this diminution is linked to the endogenous production of prostaglandin E2 and INF alpha/beta.

Ganglioside alterations in YAC-1 cells cultivated in serum-supplemented and serum-free growth medium.

Gangliosides of the 'GM1b-pathway' (GM1b and GalNAc-GM1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas GM2 and GM1 ('GM1a-pathway') occurred only in low amounts [Müthing, J., Peter-Katalinić, J., Hanisch, F.-G., Neumann, U. (1991) Glycoconjugate J 8:414-23]. Considerable differences in the ganglioside composition of YAC-1 cells grown in serum-supplemented and in well defined serum-free medium were observed. After transfer of the cells from serum-supplemented medium (RPMI 1640 with 10% fetal calf serum) to serum-free medium (RPMI 1640 with well defined supplements), GM1b and GalNAc-GM1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells. The expression of GM1a was also diminished but not as strongly as that of GM1b and GalNAc-GM1b. These growth medium mediated ganglioside alterations were reversible, and the original ganglioside expression was achieved by readaptation of serum-free growing cells to the initial serum-supplemented medium. On the other hand, a 'new' ganglioside, supposed to represent GalNAc-GD1a and not expressed by serum-supplemented growing cells, was induced during serum-free cultivation, and increased strongly after readaptation. These observations reveal that the ganglioside composition of in vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium.

The Na+/Ca2+ exchanger regulates cytolysin/perforin-induced increases in intracellular Ca2+ and susceptibility to cytolysis.

The Ca2+ dependency of NK cell-mediated and cytolysin-mediated cytolysis may be related to increases in target cell intracellular Ca2+. In a previous study we hypothesized that the Na+/Ca2+ exchanger can act as a counter-lytic mechanism by regulating the damaging increases in intracellular free calcium ([Ca2+]i) produced by cytolysin. We found that conditions said to inhibit Ca2+ extrusion by Na+/Ca2+ exchange, namely low extracellular Na+ or the presence of certain amiloride analogs which block Na+/Ca2+ exchange, enhanced the cytolysin-mediated cytolysis of YAC-1 lymphoma cells. In the present work we have confirmed the above hypothesis by measuring the [Ca2+]i of fura-2- or aequorin-labeled YAC-1 cells treated with cytolysin and low Na+ medium or amiloride analogs. YAC-1 cells appear to have a Na+/Ca2+ exchange system: low Na+ medium caused gradual increases in [Ca2+]i, and this effect was reversed in Na(+)-replete medium. Cytolysin purified from NK cell granules caused rapid dose-dependent increases in [Ca2+]i, and low Na+ medium enhanced these cytolysin-mediated increases. The Na+/Ca2+ exchange system appeared to be more active in cytolysin-challenged cells: amiloride analogs, which inhibit Na+/Ca2+ exchange in other systems, acted synergistically with cytolysin to cause large increases in [Ca2+]i, but had little effect, if any, on their own. 5-(N-4-Chlorobenzyl)-2',4'-dimethylbenzamil, the amiloride analog which has the greatest specificity for the Na+/Ca2+ exchanger and which previously was found to be the most potent enhancer of cytolysin-mediated cytolysis, was the most potent enhancer of cytolysin-mediated increases in [Ca2+]i. The above results suggest that Na+/Ca2+ exchange may be one of the target cell mechanisms of resistance to cytolysin and NK cell-mediated cytolysis.

Effects of prodigiosin 25-C on cultured cell lines: its similarity to monovalent polyether ionophores and vacuolar type H(+)-ATPase inhibitors.

Prodigiosin 25-C inhibited the proliferation of various cultured cell lines more strongly when concanavalin A (Con A) was added to the cultures. The increase in sensitivity was most evident in T lymphoma YAC-1 cells. The combination of prodigiosin 25-C and Con A induced characteristic morphological changes in these cells. In the presence of Con A, monovalent polyether ionophores and vacuolar type H(+)-ATPase inhibitors induced effects similar to those of prodigiosin 25-C on YAC-1 cells. Prodigiosin 25-C had neither K+ionophore activity nor inhibitory effect on vacuolar type H(+)-ATPase. A Golgi mannosidase II inhibitor, swainsonine, inhibited the proliferation of YAC-1 cells only when Con A was added. Prodigiosin 25-C and swainsonine increased Con A binding receptors on the surface of YAC-1 cells. These results suggest that prodigiosin 25-C affects the intracellular transport and/or processing of glycoproteins.

Recognition of beta 2-microglobulin-negative (beta 2m-) T-cell blasts by natural killer cells from normal but not from beta 2m- mice: nonresponsiveness controlled by beta 2m- bone marrow in chimeric mice.

The role of major histocompatibility complex (MHC) class I expression in control of the sensitivity of normal cells to natural killer (NK) cells was studied by the use of mutant mice made deficient for expression of beta 2-microglobulin (beta 2m) through homologous recombination in embryonal stem cells. T-cell blasts from beta 2m-deficient (beta 2m -/-) mice were killed by NK cells from normal mice in vitro, while beta 2m +/- blasts were resistant. The beta 2m defect also affected the NK effector cell repertoire: NK cells from beta 2m -/- mice failed to kill beta 2m -/- blasts, while they retained the ability to kill the prototype NK cell target lymphoma YAC-1, although at reduced levels. The inability to recognize beta 2m -/- blasts could be transferred with beta 2m -/- bone marrow to irradiated beta 2m-expressing mice. In contrast, the development of CD8+ T cells (deficient in beta 2m -/- mice) was restored in such chimera. These results indicate that loss of MHC class I/beta 2m expression is sufficient to render normal cells sensitive to NK cells, and that the same defect in the hemopoietic system of a mouse renders its NK cells tolerant to beta 2m-deficient but otherwise normal cells. In the beta 2m -/- mice, NK cells may be selected or educated by other bone marrow cells to tolerate the MHC class I deficiency. Alternatively, the specificity may be controlled directly by the class I molecules on the NK cells themselves.

Inflammatory response induced with an isolated syngeneic activation antigen shared by normal lymphoblasts and YAC lymphoma cells

An immunogenic protein with an identical M r (64 kDa) was isolated from syngeneic concanavalin A-induced lymphoblasts (syn-Con A-blasts) and YAC lymphoma cells, both derived from A mice. The 64-kDa protein was purified by a sequence of biochemical steps: Sephadex G-100 gel filtration, ion-exchange chromatography in a fast protein luquid chromatography system, Con A-Sepharose affinity chromatography, and preparative gel electrophoresis. The immunogenic fraction isolated in each step was moved to the next one, and so on. The immunogenicity of the separated fractions was measured by a lymph node proliferation (LNP) assay, which is indicative of a delayed-type hypersensitivity response. For instance, the final 64-kDa isolated protein of the syn-Con A-blasts induced an efficient LNP response in A mice which was detected after challenge with the final 64-kDa isolated protein of YAC cells. In addition to their identical molecular weight, both proteins were eluted at the same ionic strength and both expressed affinity to Con A-Sepharose beads, suggesting that they were glycosilated. Similar 64-kDa proteins were isolated by a different purification procedure, which was performed in the presence of protease inhibitors, excluding the possibility that the final antigen was an autodigested product. As the 64-kDa protein is immunogenic in the syngeneic host, it may be employed as a immunotherapeutic reagent against the original tumor and perhaps against other tumors expressing the same antigen.

Effect of culture media on lymphokine-activated killer effector phenotype and lytic capacity.

We compared the ability of murine lymphokine-activated killer (LAK) cells grown in either a serum-supplemented "standard" medium (alpha MEM plus fetal calf serum) or a serum-free medium (AIM-V) to lyse a range of tumour targets. LAK cells grown in either of the media killed a cultured murine tumour line (YAC-1 lymphoma) well and spared syngeneic "self" cells (concanavalin-A-stimulated splenocytes). However, a striking difference was noted in the ability of LAK cells grown in alpha MEM plus fetal calf serum (as opposed to AIM-V) to kill "modified self" cells (trinitrophenol-modified concanavalin A blasts); LAK cells grown in the former always killed modified self cells better than those grown in the latter. This pattern held under a broad range of experimental manipulations and was found to be related to a relative increase in CD3-bearing LAK cells grown in the standard medium. These data suggest that the two media cannot be used interchangeably. This conclusion may have clinical implications for the use of LAK cells, as animal studies have been done using LAK cells generated in serum-containing medium and clinical studies have used LAK cells generated in serum-free medium.

Effect of repeated oral administration of tobacco snuff on natural killer-cell activity in the rat

The possible immune effect of oral snuff was investigated. In male adult rats, oral snuff given for 15 weeks significantly decreased NK-cell activity in peripheral blood against murine NK cell-sensitive target cells (YAC-1 lymphoma).

The mechanism of action of a synthetic immunomodulator, 3,6-Bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246, 738), in natural killer cell activation in animals

CL 246,738 is a low molecular weight, synthetic immunomodulator. The present study was done to determine the interaction among interferon (IFN), macrophages, and natural killer (NK) cells in mice following oral administration of CL 246,738. Splenic NK activity as evidenced by lysis of YAC-1 lymphoma cells in vitro was found to be augmented by the compound not only in normal mice, but also in immunodeficient beige and nude mice. Lytic activity remained elevated from one to seven days after a single treatment and the peak activation varied depending on the source of NK cells. NK cell activity associated with the peritoneal exudate cell population peaked at day 1 and returned to normal by day 2, whereas NK cell activity of peripheral blood lymphocytes peaked at day 3 and remained significantly elevated until day 7. Liver associated NK activity peaked at day 4 and remained significantly elevated at day 7 after treatment with CL 246, 738. Lung associated NK activity was elevated by day 1 after treatment, peaked at day 4 and returned to normal by day 7 after drug administration. The drug was also effective in inducing IFN in all mouse strains tested. When these drug-treated mice were given antibody to IFN-(α + β) but not to IFN-(β), both IFN levels and NK cell activity decreased, suggesting the importance of IFN-(α) in this system. Furthermore, mice that had received carrageenan prior to, but not after CL 246,738 administration showed reduced serum IFN titers as well as decreased NK cell activity, indicating that macrophages played an intermediate role in immune enhancement by the drug. Taken together, these studies indicate that the mechanism of action of CL 246,738 in vivo is to induce macrophages to release IFN-(α) which subsequently activates NK cells.

Role of histamine in natural killer cell-mediated resistance against tumor cells.

The formation of lung metastases by i.v.-injected B16 melanoma (F1 and F10 strain) cells in Swiss albino, C57BL/6, and BALB/c mice was reduced by a single dose of histamine given 24 h before tumor cell inoculation. The antimetastatic effect of histamine was specifically mediated by histamine H2-receptors (H2R): it was blocked by the H2R antagonist ranitidine and mimicked by dimaprit, a specific H2R agonist but not by an H2R-inactive structural analog of this compound, nor-dimaprit, or the H1R agonist 2-thiazolyl-ethylamide. A single dose of any of the H2R antagonists ranitidine, tiotidine, famotidine, or cimetidine drastically augmented metastasis. Effects of H2R-interactive compounds on B16 metastasis required intact NK cells, as judged by the inability of histamine or ranitidine to affect B16 metastasis after NK cell depletion in vivo using antibodies to asialo-GM1. NK-cell-mediated lysis of YAC-1 lymphoma cells in vivo was enhanced by histamine and reduced by ranitidine within 4 h after inoculation of tumor cells. The antimetastatic effect of IL-2 was potentiated by histamine; in some experiments, combined treatment with a low dose of IL-2 (6000 U/kg) and histamine completely eliminated metastasis, whereas concomitant treatment with ranitidine abrogated antimetastatic effects of IL-2; animals treated with ranitidine and IL-2 displayed the same level of enhanced metastasis as those treated with ranitidine alone. The presented data are suggestive of an earlier unrecognized role for histamine in NK cell-mediated resistance against metastatic tumor cells.

Aldicarb immunotoxicity: Functional analysis of cell-mediated immunity and quantitation of lymphocyte subpopulations

Adult female B6C3F1 mice received distilled water only or water containing 1.0, 10, or 100 ppb of aldicarb daily for 34 days. The target concentration of aldicarb present in the 100 ppb dosing solution was analytically verified. To further develop an immune profile of this compound, following aldicarb exposure, the ability of splenic natural killer cells and specifically sensitized cytotoxic T-lymphocytes to lyse YAC-1 lymphoma and P815 tumor cells, respectively, was evaluated. To supplement the functional assays, the impact of aldicarb exposure on the percentages and absolute numbers of total T-cells, T-suppressor, T-helper, and B-cells was evaluated. The absence of statistically significant effects on any of these parameters supports earlier reports that aldicarb does not result in adverse effects on the immune system of mice.

Transfection of beta 2-microglobulin restores IFN-mediated protection from natural killer cell lysis in YAC-1 lymphoma variants.

A beta 2-microglobulin (beta 2m)-deficient variant of YAC-1, A.H-2-, was transfected with a genomic beta 2m clone. Transfected cells were used to investigate the role of beta 2m in IFN-induced protection from NK cell lysis. IFN-gamma treatment of the NK-sensitive murine YAC-1 lymphoma results in reduced sensitivity to NK cell-mediated lysis in parallel with increased expression of its constitutively low MHC class I expression. It was previously shown that the A.H-2- variant had lost both these capacities, although it retained other responses to IFN-gamma. Here beta 2m transfection restored the YAC-1 phenotype with respect to an inducible expression of MHC class I molecules and a concomitant protection from NK cell lysis after treatment with IFN-gamma. In the absence of IFN-gamma the NK sensitivity of the transfectants did not differ significantly from A.H-2-. A similar protection from NK cell lysis, in parallel with enhanced MHC class I expression, was observed for in vivo-passaged beta 2m transfectants whereas no protection was found for in vivo-passaged A.H-2- cells. The present study provides evidence that the IFN-gamma-mediated protection from NK cell lysis is dependent on beta 2m expression in the YAC-1 lymphoma. Restoration of MHC class I assembly, transport, and concomitantly an IFN-gamma augmentable cell surface expression of MHC class I molecules is a possible explanation for the effect of beta 2m.

Mast cell tumor destruction by deionized water

SUMMARY In a controlled study, malignant murine P815 mastocytoma cells exposed in vitro to distilled and deionized water died as a result of progressive swelling, degranulation, and membrane rupture. A 90% mean cell death occurred when cells obtained directly from culture were exposed to deionized water for 2 minutes. Of 6 cryopreserved malignant murine cell lines, which included Cloudman S91 melanoma, CMT-93 rectum carcinoma, MMT-06052 mammary carcinoma, and S-180 Sarcoma, only P815 mastocytoma and YAC-1 lymphoma were significantly (P < 0.05) affected by hypotonic shock; Cloudman S91 melanoma cells were the most resistant. Mastocytoma cells were selectively killed by hypotonic solution, and lymphoma cells were also killed by isotonic saline solution. Local mast cell tumor (mct) recurrence and percentage survival were evaluated in 12 cats (21 mct) and 54 dogs (85 mct) subjected to surgery alone or local infiltration of deionized water as an adjunct to surgery. Of all 16 incompletely excised mct in cats, there was no local recurrence following injection. Four mast cell tumors (2 cats) regressed after being injected in situ. In dogs with clinical stage-I mct, local recurrence was detected in 50% (5/10), but with injection after incomplete excision, local mct recurrence was significantly (P < 0.05) less (6.6%, 1/15). Percentage recurrence was significantly (P < 0.05) less and survival significantly greater when incompletely excised grade-II mct were injected. Mean follow-up period after surgery in cats and dogs was 35 and 23.4 months, respectively.

Activation of maternal killer cells in the pregnant uterus with chronic indomethacin therapy, IL-2 therapy, or a combination therapy is associated with embryonic demise

We have previously shown that NK lineage cells migrate to the murine decidua of pregnancy; but with advancing gestation, they are progressively inactivated in situ by prostaglandins of the E series (PGE 2) secreted by decidual cells and decidual macrophages. We have also shown that the same mechanism inactivates all killer lineage cells in the human decidua, and that this inactivation is at least in part due to a down-regulation of IL-2 receptors and an inhibition of IL-2 production in situ. We examined whether chronic indomethacin therapy (to block prostaglandin synthesis), or a systemic administration of a high dose of IL-2, or a combination of both agents administered to pregnant mice could activate killer cells in situ and interfere with the progress of pregnancy; and if so, whether there was a causal relationship between the two events. Pregnant CD1 mice (Day 5 of gestation) were subjected to chronic indomethacin therapy (14 μg/ml in drinking water up to Day 15, or 50 μg twice daily sc or ip up to Day 10), high dose IL-2 therapy (25000 Cetus U of human recombinant IL-2, ip every 8 or 12 hr for 3–5 days), or a combination of the two. These treatments led to pregnancy loss in 89–100% of mice, in contrast to 1% loss in control, vehicle-treated mice. Uterine mononuclear cells isolated from the embryo resorption sites exhibited high killer activity against YAC-1 lymphoma as well as murine trophoblast targets, with NK-like phenotype (Asialo GM-1 +, Thy-1 −) after indomethacin therapy and LAK-like phenotype (AGM-1 +, Thy-1 +) after IL-2 or indomethacin + IL-2 therapy. That AGM-1 + killer cells resulted in the pregnancy loss was suggested by the findings that in two of three separate experiments, iv injections of AGM-1 ab into pregnant indomethacin + IL-2-treated mice nearly completely prevented the fetoplacental demise (reducing it to 7.7% from 100%). These results reveal that PGE 2-mediated inactivation of killer lineage cells in the decidua in situ is conducive to the survival of the conceptus.

Cure of murine Ehrlich ascites tumors with chronic oral indomethacin therapy combined with intraperitoneal administration of LAK cells and IL-2

We have shown that prostaglandin E 2-(PGE 2) mediated inactivation of all killer lineage cells is a common event in the tumorbearing host, so that chronic indomethacin therapy (CIT) combined with multiple rounds of IL-2 cures spontaneous and experimental metastases of a variety of murine tumors by activating killer cells in situ. Ehrlich ascites tumor (EAT) is fatal for any mouse strain even with a small i.p. inoculum. We compared the therapeutic efficacy of chronic indomethacin therapy (CIT), CIT + interleukin-2 (IL-2), IL-2 + lymphokine activated killer (LAK) cells and CIT + IL-2 + LAK cells on EAT grown in CBA mice. CIT alone retarded tumor growth and stimulated cytotoxic activity in splenocytes as well as tumorinfiltrating lymphocytes against YAC-1 lymphoma and EAT targets but resulted in no cure. The therapeutic efficacy in prolonging the median survival improved in the following order: CIT < IL-2 + LAK cells < CIT + IL-2 < CIT + IL-2 + LAK cells. The last regimen cured 90% of mice and resulted in a short-lasting immunity against a second tumor challenge in some of the animals. Thus, this triple combination therapy may hold promise for eradicating carcinomatous ascites in the human.

Prostaglandin-mediated inactivation of natural killer cells in the murine decidua

Previous studies from this laboratory have demonstrated a large influx of null lymphocytes into the murine decidua during pregnancy. We had also shown that trophoblast cells of the murine placenta bear target structures recognized by NK cells. Since NK lineage cells belong to the null category of lymphocytes, we examined whether cells of this lineage appear in the murine decidua, and if so, whether their activity is locally regulated by NK suppressor cells. We further investigated the identity of the suppressor cells as well as their suppressor products. NK lineage cells, irrespective of their activation status, were identified morphologically in radioautographic preparations as the non-T, non-B (null) lymphocytes capable of binding YAC-1 lymphoma targets. NK activity of nucleated cells was measured with a 4-hr 51Cr-release assay against labeled YAC-1 targets. Studies with outbred CD1 mice, and to a smaller extent, inbred CBA mice revealed that the incidence of NK lineage cells remained fairly constant within the decidua throughout pregnancy, but their activity decreased steadily to negligible levels by Day 12–14 of gestation. This was found to result from an inactivation caused by NK-suppressor cells in the decidua. A mixing of Ficoll-Paque-separated nucleated cells of the decidua with normal splenic effector cells (at 1:1 ratio) led to a suppression of their NK activity tested immediately or after a 20-hr coculture. This suppression was MHC unrestricted. Suppressor cells were identified both in plastic nonadherent fraction highly enriched for typical decidual cells as well as in the plastic adherent fraction containing decidual cells and macrophages. Addition of indomethacin (10 −5 M), an inhibitor of prostaglandin synthesis, or anti PGE 2 antibody, revived the NK activity in the mixed population, as well as in the decidua, suggesting a PGE 2-mediated suppression. High levels of PGE 2 were detectable in decidual cell supernatants with a sensitive radioimmunoassay. Addition of pure PGE 2 (10 −7–10 −6 M) but not PGF 2α (10 −6 M) during the NK assay or to the effector cells for a 20-hr period prior to the assay led to an inhibition of NK activity. These results reveal that NK cells appearing in the murine decidua are progressively inactivated by PGE 2 produced by decidual cells and decidual macrophages.

Class I (H‐2Kb) gene transfection reduces susceptibility of YAC‐1 lymphoma targets to natural killer cells

A "hybrid gene" (MTKb) comprised of the human metallothionein IIA promoter ligated to the genomic sequence of the major histocompatibility complex class I (H-2Kb) gene was subcloned into the expression vector pSV2neo and transfected into the natural killer (NK) cell-sensitive YAC-1 lymphoma. The Kb gene product was readily detectable on the cell surface of G418-resistant transfectants using both Kb-specific monoclonal antibodies and H-2b-specific cytolytic T cells. Unlike control pSV2neo transfectants, MTKb-pSV2neo transfectants were relatively resistant to lysis by NK cells from H-2a, H-2b, H-2k or H-2 (a x b)F1 haplotype mice. These data strongly suggest that the effects of MHC expression on susceptibility to NK cells can be mediated by a single and well-defined class I molecule, Kb.

Rapid detection of extended ganglio‐series gangliosides with terminal GalNAcβl‐4Gal sequence on high performance thin layer chromatography plates

The mouse monoclonal antibody 2D4, which recognizes the terminal GalNAc beta 1-4Gal-disaccharide of GgOse3Cer and GgOse5Cer, was used for the detection of ganglio-series gangliosides. The method involves separation of gangliosides on thin layer chromatography plates, followed by silica gel fixation, Arthrobacter ureafaciens neuraminidase treatment and final immunostaining of desialylated gangliosides with the monoclonal antibody 2D4. Both neuraminidase and the hybridoma 2D4 producing the specific monoclonal antibody are commercially available and therefore accessible to all researchers working in this field. Gangliosides from mouse T lymphocytes and the mouse T cell lymphoma YAC-1 have been used as examples. This technique may be used for fast screening of gangliosides with the GgOse5Cer core structure which have been described as T cell markers, antigens in human neuronal disease and receptors for certain pathogenic bacteria.