YAC-1 Lymphoma Research Articles

In vivo administration of Picibanil (OK‐432) prior to tumor harvest leads to an enhancement of tumor‐infiltrating lymphocyte (TIL) cytotoxicity

TILs can be isolated and expanded in vitro in the presence of RIL-2. The resulting cells are highly cytotoxic in vitro and in vivo against a variety of tumor targets. Although most of the TILs bear T cell antigens on their cell surface, recent evidence suggests that natural killer (NK) cells may be part of the overall population of infiltrating cells. Based upon this evidence, we have evaluated the effects of Picibanil (OK-432), a well-known inducer of NK cell and T cell cytotoxicity, on TILs. OK-432 was administered intravenously at a dose of 100 micrograms, previously determined to be optimal for NK stimulation, to tumor-bearing animals. Two days later, control and experimental animals had their tumors harvested and processed in order to grow their TILs in vitro in complete medium containing RIL-2 at a final concentration of 1,000 U/ml. The following observations were made: 1) a greater than 300% increase in overall TIL number compared to controls on day 10 of culture returning to normal by day 30; 2) a marked increase in the percent of cells expressing cytotoxic and differentiation antigens in the experimental group compared to controls, such increase seen mostly from day 7 to day 10; 3) a marked increase in the cytotoxic activity of the experimental TILs against an NK-sensitive tumor target, the YAC-1 lymphoma, throughout the period of growth of the TILs (3-4 times controls) and to a lesser extent against an NK-resistant tumor target. These findings may have potential application, in immunotherapeutic trials against human tumors and may help to understand the reasons for the effectiveness of OK-432 in vivo against selected murine tumors.

Cure of human melanoma lung metastases in nude mice with chronic indomethacin therapy combined with multiple rounds of IL-2: characteristics of killer cells generated in situ

We have shown that macrophage-derived prostaglandin (PG)E2 inactivates all interleukin 2 (IL-2) dependent killer cell lineages in the tumor-bearing host, so that chronic indomethacin therapy (CIT) combined with multiple rounds of IL-2 can cure experimental and spontaneous metastases of a variety of murine tumors. We tested the efficacy of this therapy on experimental human melanoma metastasis in nude mice and characterized the killer cells generated in situ. BALB/c nude mice were injected i.v. with 2 x 10(6) human lung-metastasizing line P52, MeWo melanoma cells. After 5 weeks, when lung nodules were well established, mice received vehicles alone (control) or were given (a) CIT (14 micrograms/ml in drinking water); (b) three rounds of IL-2, 25,000 Cetus U, 8 hourly i.p. (days 40-44, 50-54, 60-64); (c) CIT + three rounds of IL-2; (d) CIT + four rounds of IL-2 (round 4 on days 70-74); and (e) CIT + five rounds of IL-2 (round 5 on days 80-84). Control and experimental mice were killed on day 71 to score lung colonies and evaluate killer activity in splenic and lung lymphocytes and macrophages against murine YAC-1 lymphoma and B16F10 melanoma, human P52 melanoma, K562 erythroleukemia, and Raji lymphoma targets. Killer cells for P52 were phenotyped for Thy-1, Lyt-2, and asialo-GM-1 markers by ab + C'-mediated deletion of killer function. Mice in all groups were also kept for survival. CIT alone improved splenic NK activity but marginally reduced the lung colony counts or prolonged the survival time. Three rounds of IL-2 alone reduced the median colony counts by 50% and prolonged the survival by 2 weeks, but resulted in no long-term, disease-free survival, in spite of significant activation of LAK cells with Thy-1-, Lyt-2-, AGM-1+ phenotype in the spleen. CIT + 3 rounds of IL-2 reduced the median colony counts from 40 to 0 and improved the survival from a median of 66 (control) to 120 days (40% surviving 260 + days). CIT + four or five rounds of IL-2 caused long-term (260 + days) survival of 80% mice, most surviving 400 + days. The combination therapy activated killer lymphocytes (Thy-1-, Lyt-2-, AGM-1+) and, to a smaller extent, macrophages (AGM-1 +/-) in the spleen and the lungs, showing a high cytocidal ability for all the targets.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of ozone, hexachlorobenzene, and bis(tri- n-butyltin)oxide on natural killer activity in the rat lung

The respiratory tract is a major route of exposure to noxious agents as well as pathogens such as viruses. Natural killer (NK) activity is an important first line of defense to virally infected cells as well as certain neoplasms; therefore, testing the effects of exposure to toxic compounds on this activity is important in understanding the immunotoxic potential of the compound. Lymphoid cell suspensions, obtained after enzymatic dispersion of rat lungs and purification over nylon wool columns, showed in vitro natural killer activity toward YAC lymphoma cells. Validation of the test with well-known NK activity stimulators such as Bacillus Calmette-Guérin (BCG), interleukin-2 (IL-2), interferon (IFN), and inhibitors like anti-asialo-GM1 (ganglio- n-tetrasylceramide) antibody confirmed the reliability of the test as an assay for detecting NK activity in rat lungs. Using this assay, we studied the effects of exposure to ozone (O 3), hexachlorobenzene (HCB), and bis(tri- n-butyltin)oxide (TBTO) on NK activity in rat lung. Inhalation exposure to O 3 for 7 days at 0.4 and 0.8 mg/m 3 resulted in stimulation, and exposure at 1.6 mg O 3/m 3 resulted in suppression of NK activity. Oral exposure to HCB in concentrations of 150 and 450 mg/kg food for 6 weeks suppressed NK activity in rat lungs in a dose-related manner. This was also true for 6 weeks of oral exposure of rats to 20 and 80 mg TBTO/kg food, but to a lesser extent. In summary, we have developed and validated a method to measure the effects of (toxic) substances on NK activity in rat lung.

Mouse tumors are heterogeneous in their susceptibility to syngeneic lymphokine-activated killer cells and delineate functional subsets in such effectors.

We have analyzed whether lymphokine-activated killer (LAK) cells, generated from C57BL/6J (B6) spleen cells at different times after recombinant interleukin-2 (rIL-2) culture, could be heterogeneous in their ability to lyse a variety of tumor targets. When tested 3 days after exposure to 250 U/ml rIL-2 (day-3 LAK cells) a significant lysis was detected with the natural-killer(NK)-sensitive YAC lymphoma, the NK-resistant P815 mastocytoma, three different syngeneic melanomas and a syngeneic fibrosarcoma (group 1 targets), whereas no lysis was observed with a reticulum cell sarcoma, two different lymphomas or concanavalin A blasts, all of B6 origin (group 2 targets). LAK cells cultured for 5 days, however, lysed group 2 targets and showed a parallel increase of cytotoxic activity against group 1 targets. At day 7, LAK activity declined on all targets examined. In cold-target inhibition studies, the lysis of group 1 tumor targets by day-3 or day-5 LAK cells could be inhibited only by group 1 and not by group 2 unlabelled tumor cells. All group 1 tumors could effectively compete each other. Conversely, the lysis of group 2 tumor targets by day-5 LAK cells was inhibited by both group 1 and group 2 targets. These data indicate the presence of separate LAK effectors that appear to arise with different time kinetics and have different recognition structures. In vitro antibody depletion at the effector level showed that day-3 LAK cells with cytotoxic activity against group 1 tumors were ASGM1+. Day-5 LAK cells included both ASGM1+ and Lyt2+ effectors and both populations, although to a different extent, contributed to the lysis of all targets. Our results indicate that LAK cells are functionally heterogeneous. This heterogeneity is defined by their susceptible target cells and cannot be ascribed to different (Lyt2+ versus ASGM1+) lineages.

Effect of IFN‐γ treatment and in vivo passage of murine tumor cell lines on their sensitivity to lymphokine‐activated killef (LAK) cell lysis in vitro; association with H‐2 expression on the target cells

Interferon-gamma (IFN-gamma) treatment or in vivo passage of the murine YAC-1 lymphoma resulted in reduced sensitivity to in vitro lysis by syngeneic murine spleen cells cultured in rIL-2 (LAK-cells). IFN-gamma treatment also rendered the murine B16 melanoma less sensitive to lysis by syngeneic LAK cells, whereas in vivo passage did not alter LAK sensitivity. The reduction in sensitivity to lysis correlated with enhanced expression of cell surface H-2 on the target cells. The possible role of H-2 was studied with a beta 2-microglobulin-deficient, and thus H-2-deficient, variant of the YAC-1 lymphoma. This variant line remained H-2 negative even after IFN-gamma treatment or in vivo passage, and was highly sensitive to LAK-cell-mediated lysis, even after IFN-gamma treatment or in vivo passage. The present results are discussed in relation to IFN-gamma and in vivo induced modulation of MHC class-1 molecules on target cells and the possible consequences for interaction with activated as well as "natural" effector cells.

Failure of dietary restriction to influence natural killer activity in old rats

Natural killer activity of Sprague-Dawley rats maintained on an ad libitum versus restricted diet was compared using an 18 hour 51Cr-release assay, against the K562 erythroleukemic line, Yac-1 lymphoma cells and SV40-3T3 cells. The results indicated that no enhancement of natural killer function was induced by dietary restriction of 10.5-month-old rats from weaning. Prolongation of the restricted diet into late life (24 months) similarly did not enhance basal natural killer activity over levels observed in the ad libitum controls. This suggests that the improved resistance to some tumours seen after prolonged dietary restriction depends on another defensive mechanism, reduced metabolic activity and/or a reduction of available nutrients at cancerous foci.

Lack of an Influence of Dietary Fat on Murine Natural Killer Cell Activity

The influence of the degree of saturation and the concentration of dietary fat on murine natural killer (NK) cell activity was investigated using C3H/HeN and C57BL/6N mice. Mice were fed purified diets containing either 0% fat (fat free), 5 or 20% safflower oil or 5 or 20% palm oil. Safflower oil and palm oil were used because they are comparable in carbon chain length but vary in the amount of linoleic acid [18:2(n-6)]. Cytotoxicity of splenic NK cells from mice stimulated or unstimulated by the interferon inducer poly I:C was measured against either the YAC-1 lymphoma or line 168 mammary tumor targets. The number of asialo GM1+ cells was not influenced by concentration or degree of saturation of dietary fat. Generally, dietary fat had no consistent influence on NK cell cytotoxicity of spleen cells from either the high responder C3H/HeN mice or the moderate responder C57BL/6N mice against either tumor target. The level of 18:2(n-6) in NK cell-enriched splenic lymphocytes increased with greater levels of dietary safflower oil. Nevertheless, there appeared to be no correlation between lymphocyte fatty acid composition and NK cell cytotoxic capabilities. Therefore, the concentration of dietary safflower or palm oil, and thus 18:2 (n-6), did not appear to effect the number or activity of murine NK cells.

Enhanced H-2 expression and T-cell-dependent rejection after intracerebral transplantation of the murine lymphoma YAC-1

The relationship between MHC class I (H-2) expression and tumorigenicity was investigated after intracerebral inoculation of the murine lymphoma YAC-1 and its H-2 negative variant, A.H-2 −, YAC-1 was less tumorigenic than A.H-2 − in normal as well as NK-depleted syngeneic A/Sn mice. However, in T-cell-depleted syngeneic mice YAC-1 was as tumorigenic as A.H-2 −. Following intracerebral growth, the H-2 expression of YAC-1 was markedly enhanced in a similar fashion as after intraperitoneal passage. The A.H-2 − variant remained H-2 negative after intracranial passage. The H-2 negative variant cells were not rejected from the brain even when intermixed with wild-type YAC-1 cells prior to intracerebral inoculation, excluding an “innocent bystander” effect. In vitro, the intracerebrally passaged YAC-1 line showed enhanced sensitivity to lysis by H-2 K k D d (H-2 a) specific CTLs but decreased sensitivity to NK cells. The A.H-2 − line was unchanged. Our data suggest that the lack of H-2 molecules may facilitate the growth of antigenic tumor cells in the brain due to escape from T-cell-mediated immunosurveillance. Our data also suggest, in line with other recent findings, that intracerebrally growing tumor cells are sheltered from NK cell-mediated rejection.

Molecular analysis of H-2-deficient lymphoma lines. Distinct defects in biosynthesis and association of MHC class I heavy chains and beta 2-microglobulin observed in cells with increased sensitivity to NK cell lysis.

It has previously been reported that MHC class I-deficient RBL-5 and YAC-1 lymphoma sublines show an enhanced NK sensitivity in vitro. In the present study such lymphoma variants were found to have different defects in H-2 biosynthesis such as 1) reduced beta 2-microglobulin and H-2 H chain transcription, 2) block in beta 2-microglobulin translation, and 3) impaired association between beta 2-microglobulin and H-2 H chains. For lines with the latter two defects, the results suggested that a major part of the H chains were arrested before the middle Golgi complex as indicated by their failure to undergo carbohydrate side chain trimming. The data suggest that NK sensitivity can be directly influenced by the membrane expression of MHC class I gene products of tumor targets because three independent molecular defects, all interfering with the cell surface H-2 expression, gave rise to NK-sensitive phenotypes. These variant lines will also be useful tools for studies of H-2 glycoprotein assembly and transport and for Ag presentation to CTL.

Characteristics of yorkshire swine natural killer cells

The present study examined the properties of NK activity in Yorkshire swine. The results support other porcine studies which indicate the swine NK system has both similarities and differences to this system in other species. Profiles of NK activity indicated swine NK cells are highly reactive against the YAC-1 lymphoma, the K-562 myeloid leukemia, the P-815 mastocytoma, and the TU-5 virally transformed fibroblast. In contrast, the MOLT-4 and SB leukemias are NK resistant. Kinetic studies indicated that in Yorkshire swine, NK lysis begins 6 h after mixing effectors and targets. The kinetics of the lytic reaction differ both from other breeds of swine and from other species, where cytotoxicity is readily measured in 4-h assays. The delayed lysis was not due to delayed target cell recognition, because Yorkshire swine NK cells are rapidly bound to tumor targets. The delayed lysis seems to be due to a refractoriness in the NK lytic mechanism. This delay may relate to the morphologic finding that the target-binding cell in Yorkshire swine appeared quite different from the large granular lymphocyte (LGL) reported as the NK effector in humans and rodents. Indeed, in light microscopic studies the typical tumor-binding cell in Yorkshire swine is a small, apparently nongranular, lymphocyte. Analysis of NK activity at the single cell level was performed with single effector-tumor conjugates immobilized in agarose. Generally, lysis by target binders paralleled sensitivity to lysis in 51Cr release tests, indicating lysis in agarose may be used as an NK index in swine. Like other species, swine NK cells were found to be non-adherent lymphocytes with a characteristic tissue distribution. Peripheral blood and spleen had the highest levels of NK activity. Lymph node cells displayed a small amount of NK activity which was limited to the YAC-1 target, while thymocytes showed no appreciable NK activity against any of the cell lines tested.

Interleukin 1-induced, T cell-mediated regression of immunogenic murine tumors. Requirement for an adequate level of already acquired host concomitant immunity.

Intraperitoneal injection of human rIL-1 in a dose of 0.5 microgram daily for 5 d, or 1 microgram daily for 3 d, was capable of causing complete regression of immunogenic SA1 sarcoma growing subcutaneously in syngeneic or semisyngeneic mice. Higher doses of IL-1 were not more therapeutic against the SA1 sarcoma, but needed to be given to cause complete regression of the immunogenic L5178Y lymphoma. On the other hand, the P815 mastocytoma was much less responsive to IL-1 therapy, in that it failed to undergo complete regression in response to doses of IL-1 capable of causing regression of the L5178Y lymphoma. IL-1 caused regression of the SA1 sarcoma when given on days 6-8 of tumor growth, but not when given on days 1-3. This refractoriness of a small tumor to IL-1 therapy suggests that the antitumor action of IL-1 is based on an underlying host-immune response that is not generated until after day 3 of tumor growth. Direct evidence for the participation of host immunity in IL-1-induced tumor regression was supplied by results showing that IL-1 was not therapeutic against the SA1 sarcoma growing in T cell-deficient (TXB) mice, unless these mice were first infused with Ly-2+ and L3T4+ T cells from donor mice bearing an established SA1 sarcoma. In contrast, normal T cells, or T cells from donor mice bearing a YAC-1 lymphoma, failed to provide TXB recipients with the ability to cause regression of their SA-1 sarcoma in response to IL-1 treatment. The results are in keeping with the interpretation that exogenous IL-1, by augmenting the production of tumor-sensitized T cells, converts a subtherapeutic level of host immunity to a therapeutic level. The results suggest, in addition, that IL-1 only stimulates the replication of T cells that are already engaged in the antitumor immune response.

Properties and characterization of a rat spleen cell-derived factor that induces resistance to natural killer cell lysis in YAC lymphoma cells.

Supernatants of Con A-stimulated rat spleen cell cultures contain a factor that induces relative resistance to NK cell-mediated cytotoxicity in the YAC cell line, a line that is otherwise highly susceptible to murine NK cell-mediated lysis. This NK-lysis resistance-inducing factor (LRIF) has a Mr of 12,600 Da, as determined by gel filtration chromatography, and an isoelectric pH of 4.8. NK-LRIF is heat labile and is de-activated by treatment with proteolytic enzymes. Unlike immune-IFN (IFN-gamma), NK-LRIF is not inactivated by pH 2 treatment, and antibodies capable of neutralizing IFN-alpha and IFN-gamma do not abrogate the effect of NK-LRIF. Highly purified IL-2 preparations lack NK-LRIF activity. NK-LRIF does not induce a general resistance to lysis in YAC cells, because control and NK-LRIF-treated YAC cells were equally susceptible to alloimmune cytotoxic T cells. YAC cells treated with NK-LRIF showed a marked enhancement (5- to 10-fold) in the expression of class I MHC Ag. This observation supports the proposition that the NK susceptibility of target cells could be inversely related to the expression of class I MHC Ag.

Induction of lymphokine-activated killer cells from rat thymocytes using recombinant human interleukin-2.

Using a 4-h 51Cr release assay, we observed that thymocytes from Fischer strain rats incubated with recombinant human interleukin-2 (rhIL-2) developed cytotoxicity to YAC-1 lymphoma, 9L-glioma, and B-16 melanoma cells (effector/target ratio = 25/1). Induction of the lymphokine-activated killer (LAK) cells was as follows: (1) when 5 x 10(6)/ml thymocytes were cultured with various concentrations of rhIL-2 (50, 125, 250, 500, or 1,000 units/ml) for 4 days, no cell proliferation was observed at any concentration. However, the LAK cells showed significant cytotoxicity toward all tumor cells at more than 50 units/ml. (2) When 5 x 10(6)/ml thymocytes were cultured for 1 to 6 days with 250 units/ml of rhIL-2, the harvested cell count decreased markedly after the 2nd day. The cytotoxicity of all the tumor cells became significant after the 2nd day, with peak activity on the 4th day. In rat splenocytes, on the other hand, the LAK cells could not be identified because rat splenocytes developed nonspecific cytotoxicity in medium containing fetal calf serum without adding rhIL-2.

Fate of tumor cells injected into left ventricle of heart in BALB/c mice: role of natural killer cells.

The arrest, retention, and elimination (i.e., clearance) of radiolabeled YAC-1 lymphoma cells injected either iv or into the left ventricle (LV) of the heart were studied in male BALB/c mice, with special emphasis on the role of natural killer (NK) cells. After iv injection YAC-1 cells were arrested and, to a large extent, destroyed in the lungs, which contain the first capillary bed that iv injected tumor cells meet. After LV injection the initial distribution of the tumor cells, which depends on the distribution of cardiac output at the time of injection, was estimated by use of radiolabeled microspheres. Using this technique, we have shown that LV-injected tumor cells, in contrast to iv injected tumor cells, were not arrested in the first capillary bed that they encountered but passed viably through the microvasculature of the brain, heart, kidneys, intestinal tract, and to some extent, the bone, skin, and muscle. The only organs that could arrest the LV-injected tumor cells were the lungs and the liver. In the lungs clearance of YAC-1 cells began immediately after the cells were arrested. However, the rate of clearance could be almost abrogated by pretreatment of the recipients with anti-asialo GM1 antiserum, which destroys most of the NK cells in vivo and strongly depresses the in vitro NK cell activity. In contrast, YAC-1 cells arrested in the liver were not cleared from this organ during the first 1-2 hours after arrest. After this delay clearance of the cells commenced. Pretreatment of the recipients with anti-asialo GM1 also strongly depressed the clearance of tumor cells from the liver. Although pretreatment with polyinosinic-polycytidylic acid enhanced in vitro NK cell activity, it could augment only slightly the clearance of YAC-1 cells from the lungs and the liver. Thus these results strongly support the hypothesis that the rapid clearance of tumor cells from both the lungs and the liver depends, at least partially, on the NK cell activity within these organs.

Effects of dimethyl sulfoxide treatment on H-2 expression and susceptibility to NK- or cytotoxic T-lymphocyte-mediated lysis of the YAC-1 lymphoma and its beta 2-microglobulin-deficient variant.

The effects of dimethyl sulfoxide (DMSO) on H-2 expression and susceptibility to NK- and cytotoxic T-lymphocyte (CTL)-mediated lysis in the murine T-cell lymphoma YAC-1 and its beta-2-microglobulin (beta 2m)-deficient variant were studied. Fluorescence-activated cell sorter analysis revealed induction of H-2Kk and beta 2m 3 days after culture of YAC-1 with DMSO, whereas optimal H-2Dd induction required more than 1 week. H-2Kk and H-2Dd induction by DMSO was equal to pretreatment of YAC-1 cells with 50-100 and 10-20 U/ml interferon (IFN)-gamma, respectively, but the T-cell differentiation antigens Lyt-1, Lyt-2, Thy-1, and L3T4 remained unaffected. DMSO protected YAC-1 cells from NK lysis as efficiently as 10-20 U IFN/ml, whereas susceptibility to anti-H-2a-, H-2Kk-, and H-2Dd-specific CTLs was augmented as in IFN-treated YAC-1 cells. In contrast, the beta 2m-deficient variant, which remained H-2 negative at the cell surface after DMSO treatment, also remained NK sensitive. Thus DMSO can induce H-2 expression and alter the sensitivity of murine lymphoma cells to different effector cells.

Large granular lymphocytes in the liver.

Considerable numbers of large granular lymphocytes (LGL) were isolated from rat liver by a simple method consisting of sinusoidal lavage at elevated (50 cm water column) perfusion pressure. This method gave a yield comparable with the enzymatic dissociation method commonly used for the isolation of nonparenchymal liver cells, but was shorter in time and had the advantage of avoiding the potentially harmful effects of the dissociating enzymes. The isolated LGL were highly cytotoxic against YAC-1 lymphoma cells and this cytolytic activity was blocked by treatment of the effector cells with an antibody against natural killer cells (anti-asialo GMI). We characterized the hepatic LGL as nonphagocytic, nonadherent, per-oxidase-negative and acid phosphatase-positive cells which could be enriched in the low-density fraction of a Percoll gradient. At the light micro- scopic level, they showed characteristic azuro- philic granules, which corresponded to strongly osmiophilic granules with a specific morphology in electron microscopy. It is concluded that these LGL are identical to the “pit cells” which were formerly described by electron microscopy in situ as normal components of the liver sinusoids and which are easily recognized by their fine structure. It is also proposed that the liver may represent one of the major natural killer organs.

Effect of sex hormones on NK and ADDC activity of mice

The effect of pharmacologic doses of sex hormones on NK and ADCC activity against YAC-1 lymphoma and CRBC target cells was studied. Estradiol (E) and testosterone (T) administration for 2 weeks caused a substantial reduction of splenic NK activity in TA3 mice of either sex. In prolonging the treatment time, the intensity of suppression was gradually increased. Both E and T have apparently no inhibitory effect on ADCC activity of TA3 mice, although the ADCC activity slightly increased in the early stage of T treatment. The ADCC activity of T-treated male mice was slightly higher than that of E-treated males. Passive transfer of the splenic mononuclear cells and serum of treated mice does not affect the NK and ADCC activity of normal recipient mice. Addition of different concentrations of sex hormones into the culture medium or pre-treatment of effector cells for 12h failed to change the NK and ADCC activity of murine splenic cells.

An active T-cell-independent mechanism enhances MHC class I transcription and expression on a mouse T-cell lymphoma in vivo

The present study focuses on the mechanism underlying changes in H-2 cell surface antigen expression after passage of the in vitro grown YAC-1 lymphoma as an ascites tumor. The increase in cell-surface expression correlated with elevated levels of class I transcripts as revealed by Northern blots. The enhanced H-2 expression was also seen with a cloned YAC-1 line, and not until 2 weeks after in vitro explantation had levels of H-2 decreased to those on the in vitro established YAC-1. Arguing for the necessity of a mature functioning immune system, suckling mice were unable to increase H-2 expression on inoculated lymphoma cells. Also pretreatment with cyclophosphamide or irradiation abolished the capacity of adult mice to increase cell surface H-2 on YAC-1 cells. A functioning T-cell system was not required for H-2 enhancement to occur since athymic nude mice were fully competent. The possible significance of an active T-cell-independent host mechanism which enhances tumor H-2 expression at the transcriptional level is discussed.

In vivo natural antitumor resistance against murine EL-4 lymphoma cells in lethally irradiated syngeneic C57BI/6 mice

Natural resistance has been detected in lethally irradiated C57B1/6 (B6) mice inoculated intravenously with the ascites form of a syngeneic B6 leukemia. EL-4 cells were injected into lethally irradiated (800 R) B6 mice and tumor cell proliferation was evaluated by 125IUdR uptake in different organs 4 days after the challenge. Differential growth of lymphoma cells was observed when young mice were injected as compared with older mice and when mice were treated with agents known to interfere with natural resistance (e.g., poly(I:C), FLV-P, carrageenan, cyclophosphamide, high doses of irradiated cells). Similar results were obtained by measuring rapid clearance of 125IUdR-labeled EL-4 cells from lungs of intact B6 mice. In vivo cold competition studies, employing EL-4 and several other tumor lines of the same or different haplotype, showed that only EL-4 and RBL-5 cells were capable of inhibiting syngeneic resistance against EL-4 tumor. On the contrary, YAC-1 lymphoma cells, the most susceptible target to natural killer-mediated cytotoxicity in vitro, did not compete. These results suggest that EL-4 cells express membrane determinants not detectable on normal H-2 b parental bone marrow cells and are susceptible to natural resistance against hemopoietic tumor cells in lethally irradiated syngeneic B6 mice.

Large granular lymphocytes from SCID horses develop potent cytotoxic activity after treatment with human recombinant interleukin 2.

Peripheral blood mononuclear cells from foals with hereditary severe combined immunodeficiency (SCID) have morphologic characteristics of large granular lymphocytes (LGL). Attempts to demonstrate cytotoxic activity were without success unless the LGL were incubated with 100 U of human recombinant interleukin 2 (rIL 2)/ml for 24 hr. With rIL 2 incubation, low effector to target ratios (10:1) consistently yielded high levels of cytotoxic activity (30 to 50%) in a standard 4-hr 51Cr-release assay using YAC-1 lymphoma or K562 erythroleukemia cell lines as targets. Monoclonal antibody EqT12 reacted with a large percentage of these cells, and the level of cytotoxic activity was directly correlated to the percentage of EqT12+ cells in the preparation. In normal horses, the percentage of circulating EqT12+ cells is low (5%), while at the same time, cytotoxic activity is not usually detectable even with rIL 2 incubation. In contrast, the percentage of EqT12+ cells in blood of SCID horses is high (up to 40%), as is the IL 2-inducible cytotoxic activity. These results indicate that cytotoxic cells with morphologic and functional characteristics of natural killer cells are produced by horses with SCID.