Suppressed in vitro blastogenic responsiveness of rat spleen cells after continuous infusion of endotoxin by an implanted osmotic pump

Continuous infusion of a gram-negative bacterial endotoxin in relatively small doses into rats by means of an implanted osmotic pump was studied. The model system was designed to examine the effects of endotoxin on the blastogenic response of spleen cells to the endotoxin itself and to a nonspecific T-cell mitogen, concanavalin A (Con A). Rats were implanted with an osmotic pump which delivered saline for the first 42 hr to provide postsurgical recovery before the onset of endotoxin infusion. Previous studies had shown that during the first 1-4 days after administration of endotoxin marked alterations of metabolism and some changes in physiologic parameters such as blood pressure and in vitro myocardial performance occurred. In the present study the blastogenic responsiveness of spleen cells to endotoxin itself as well as to the nonspecific T-cell mitogen Con A was markedly decreased after several days of continuous administration of endotoxin. Control animals receiving only saline for the same period of time showed a similar depression of blastogenic responsiveness to the lipopolysaccharide (LPS), as well as to Con A, however, with a delay of 2-4 days before comparable levels of suppression became evident. These results indicate that marked alterations of immune competence as measured by blastogenesis of spleen cells to Escherichia coli LPS and to a mitogen such as Con A may occur after implantation of an osmotic pump, with or without continuous infusion of endotoxin. Further studies seem warranted to determine the role of the foreign body reaction to the osmotic pump as well as to the endotoxin administered by the pump.     

Splenocyte blastogenesis suppressed in rats implanted with an osmotic pump.

Rats implanted subcutaneously with an empty osmotic pump connected by a polyethylene catheter to a jugular vein for 5 to 10 days evinced a decreased splenocyte responsiveness to blastogenic stimulation in vitro to bacterial lipopolysaccharide, a known B cell stimulator, as well as to the plant mitogens pokeweed mitogen (PWM), a known stimulator of T and B cells, and Concanavilan A, a known T cell stimulator. The surface of the implanted pumps became infiltrated with lymphoid cells, especially macrophages. Suppression of blastogenic responsiveness after implantation for 10 days with an empty pump or even a pump dispensing pyrogen free saline only in a continuous manner was nearly as marked as that which occurred at 2-5 days after continuous infusion with endotoxin. These depressed blastogenic responses, although less, were also evident when rats were implanted with a catheter into a jugular vein connected by means of a swivel to either an empty pump or one dispensing pyrogen free saline. Suppression of blastogenic responsiveness was not related to alteration in serum complement or corticosteroid levels. Since administration of immunomodulatory substances systemically to individuals often involves implantation of an osmotic pump, investigation into the mechanisms of lymphoid cell suppression associated with an implanted pump itself has potential significance.     

Rapid acquisition of an enhanced capacity to produce tumor necrosis factor, alpha/beta interferon, and interleukin 6 after implantation of tumor cells.

This study shows that the ability of mice to produce tumor necrosis factor (TNF), alpha/beta interferon (IFN-alpha/beta), and interleukin 6 (IL-6), but not interleukin 1 (IL-1), in response to endotoxin was dramatically augmented within 24 h of intradermal implantation of 10(6) tumor cells. Tumor cell implantation also caused endotoxin-independent appearance of IFN-alpha/beta and IL-6 in serum within 24 h. Priming for endotoxin-induced TNF production was not evident during the first 12 h of tumor cell implantation and it had decreased by 72 h. However, this decrease was followed by a second peak of priming on day 6 of tumor growth. Priming for endotoxin-induced TNF production was not induced by injection of dead tumor cells, the products of live tumor cells, or syngeneic or allogeneic splenocytes. Priming for TNF production was associated with an increased susceptibility of mice to endotoxin toxicity. These data suggest the existence of a cytokine-dependent host defense mechanism that is rapidly elicited in response to tumor cell implantation.     

Altered interleukin production during Friend leukemia virus infection

Spleen cells from BALB/c mice, infected 14 to 28 days earlier with Friend leukemia virus (FLV), were shown to be inhibited in their ability to produce interleukin 2 (IL-2) when stimulated with mitogen. Likewise, these spleen cell populations failed to respond following mitogenic stimulation or exogenous addition of recombinant IL-2. By contrast, the FLV-infected spleen cell populations produced normal levels of interleukin 1 (IL-1) and thymocytes from FLV-infected mice responded normally to addition of exogenous IL-1. This suggests that FLV infection selectively affects the ability of spleen cells to produce cytokines. Spleen cell populations enriched for T lymphocytes and depleted of tumor cells by density gradient centrifugation in Ficoll were unable to produce IL-2. This indicates that the failure to detect IL-2 in cells from FLV-infected mice was not due to a dilution of T lymphocytes by tumor cells but was a functional inability to produce IL-2. Furthermore, enriched T lymphocytes from FLV-infected mice failed to respond blastogenically to exogenous IL-2. Additional studies indicate that tumor cells, but not macrophages or T lymphocytes from FLV-infected spleens, suppressed the blastogenic response to mitogens and IL-2 production by normal splenic T lymphocytes.     

Alcoholic liver disease in rats fed ethanol as part of oral or intragastric low-carbohydrate liquid diets

The intragastric administration of ethanol as part of a low-carbohydrate diet results in alcohol hepatotoxicity. We aimed to investigate whether comparable liver injury can be achieved by oral diet intake. Male Sprague-Dawley rats were fed ethanol as part of low-carbohydrate diets for 36-42 days either intragastrically or orally. Liver pathology, blood ethanol concentration, serum alanine amino transferase (ALT), endotoxin level, hepatic CYP2E1 induction, and cytokine profiles were assessed. Both oral and intragastric low-carbohydrate ethanol diets resulted in marked steatosis with additional inflammation and necrosis accompanied by significantly increased serum ALT, high levels of CYP2E1 expression, and production of auto-antibodies against malondialdehyde and hydroxyethyl free radical protein adducts. However, cytokine profiles differed substantially between the groups, with significantly lower mRNA expression of the anti-inflammatory cytokine interleukin 4 observed in rats fed low-carbohydrate diets orally. Inflammation and necrosis were significantly greater in rats receiving low-carbohydrate alcohol diets intragastrically than orally. This was associated with a significant increase in liver tumor necrosis factor alpha and interleukin 1beta gene expression in the intragastric model. Thus, oral low-carbohydrate diets produce more ethanol-induced liver pathology than oral high-carbohydrate diets, but hepatotoxicity is more severe when a low-carbohydrate diet plus ethanol is infused intragastrically and is accompanied by significant increases in levels of proinflammatory cytokines.     

Immunological activity of lipopolysaccharide of Helicobacter pylori on human peripheral mononuclear blood cells in comparison to lipopolysaccharides of other intestinal bacteria

Abstract Lipopolysaccharide of Helicobacter pylori was tested for its mitogenicity and for its ability to stimulate cytokine release in human peripheral blood mononuclear cells (PBMC) of healthy and H. pylori -infected blood donors. Mitogenicity in PBMC induced by H. pylori LPS was similar to that induced by Campylobacter jejuni lipopolysaccharide, but lower than that induced by Escherichia coli lipopolysaccharide in the H. pylori negative blood donor group. Furthermore, H. pylori LPS was able to induce tumour necrosis factor (TNF) interleukin 1 (IL-1) and interleukin 6 (IL-6) secretion of PBMC. Compared with the ability of C. jejuni and E. coli lipopolysaccharides to stimulate cytokine release, H. pylori lipopolysaccharide induced a significantly lower TNF and IL-1 secretion of PBMC than the other tested bacterial lipopolysaccharides. Similar amounts of IL-6 release were obtained by stimulation of PBMC with H. pylori and C. jejuni lipopolysaccharides, whereas a higher IL-6 release was measured by stimulation with E. coli lipopolysaccharide. The results of this study suggest that H. pylori lipopolysaccharide has a lower immunological activity than lipopolysaccharides of other intestinal bacteria. This is probably due to its unusual acylation and phosphorylation pattern of lipid A.     

Host immune responses to tumor cells augmented by bleomycin and their therapeutic effects on rat fibrosarcoma

The administration--timing-dependent therapeutic effects of bleomycin (BLM) were observed on a fibrosarcoma implanted SC in WKA rats. Five consecutive IP administrations of BLM (5 mg/kg/d) were found to be more effective when BLM was given from Day 8 than when it was given from Day 1 for tumors implanted on Day 0. The therapeutic effects correlated well with antitumor immune responses, which were examined on Day 13 when the tumor had not yet regressed even in surviving rats. The tumor-neutralizing activity of spleen cells was augmented in rats treated with BLM from Day 8 to Day 12, and the suppressor cell activity detected in the spleen cells of tumor-bearing rats was eliminated by the BLM treatment. The tumoricidal activity of peritoneal exudate cells (PEC) was detected in rats treated from Day 8 but not in rats untreated or treated from Day 1. The in vitro treatment of KMT-17 cells with BLM (20 micrograms/ml) for two hours enhanced the sensitivity of the tumor cells to the activity of tumoricidal PEC. This suggests that the direct action of BLM on tumor cells also plays an immunologic role in BLM treatment. The findings reveal that the therapeutic effect of BLM is elicited by its ability to augment the host immune responses to tumor cells.     

The effects of in vivo administration of endotoxin on the functions and interaction of hepatocytes and Kupffer cells

It was the purpose of this study to determine the effects of the in vivo administration of endotoxin on certain in vitro hepatocyte and Kupffer cell functions. An Alzet osmotic pump that contained endotoxin (LPS, 2.5 mg/100g) was implanted into the peritoneal cavity of 300g guinea pigs and delivered the endotoxin over a period of four days. In vivo administration of LPS did not cause a change in the in vitro release of albumin by isolated hepatocytes. However, when hepatocytes were co-cultured with Kupffer cells there was a significant decrease in albumin release for both control and LPS-treated animals. There was no difference between control and LPS-treated animals in the release of C3 by hepatocytes. However, there was a significant increase over the control group in C3 release by Kupffer cells from LPS-treated animals. When hepatocytes and Kupffer cells were cultured together, their release of C3 was not additive. Kupffer cells from LPS-treated animals released significantly greater amounts of PGE2 than control animals when stimulated in vitro with LPS. Thus, these Kupffer cells appeared to be primed to respond to an in vitro challenge of LPS. Kupffer cells from LPS-treated animals had significantly depressed antibody dependent cellular cytotoxicity (ADCC). This endotoxin model is useful for determining the in vivo effects of endotoxin on cellular function and gives some indirect evidence for the detrimental effects of LPS on the immune system and host defense.     

Immune (y) interferon production by murine T cell lymphomas

Various cloned murine T cell hybridomas and T cell lymphomas were evaluated for their ability to produce interferon (IFN). Two T cell tumor clones, L12-R1 and L12-R4, derived from the spontaneously in vitro transformed cell lines L12 originally established from fetal calf serum-primed C57BL/6 spleen cells were found to produce high IFN amounts upon mitogen stimulation. Phorbol myristate acetate led to maximal IFN production (2187 IU) by L12-R4 cells at concentrations of 2 x 10(-7) M, whereas concanavalin A and phytohemagglutinin induced lower levels of IFN synthesis (160 to 243 IU). None of the cell lines tested produced IFN constitutively or upon lipopolysaccharides stimulation. The IFN was characterized as immune (y) by being labile at pH 2 and neutralized by two rabbit anti-murine IFN-y antisera but not by antiserum to murine leukocyte (alpha) and fibroblast (beta) IFN. Phenotypic characterization of IFN-y-producing cells showed the L12 clones to be Thy-1.2+, Lyt-1+, 23-, and Ig-. The L12-R4 tumor cell therefore provide a unique source of IFN-y for purification, and may represent a useful model for studying the molecular mechanisms involved in T cell differentiation leading to IFN-y production.     

Characterization of the antitumor activities of human tumor necrosis factor-alpha and the comparison with other cytokines: induction of tumor-specific immunity

We have investigated the in vitro and in vivo antitumor activities of recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) against Meth A sarcoma. Meth A sarcoma cells were found to a) be relatively insensitive in vitro to rHuTNF-alpha, and b) express low numbers of TNF-alpha receptors. Intraperitoneally implanted Meth A sarcoma was insensitive to the antitumor effects of rHuTNF-alpha. In contrast, rHuTNF-alpha was highly efficacious against subcutaneously implanted Meth A sarcoma. Biodistribution studies with 125I- or 3H-labeled rHuTNF-alpha demonstrated that, after intravenous administration, the majority of the labeled rHuTNF-alpha localized in the kidney, lungs, and liver. Only low levels of radiolabel were found in subcutaneous Meth A implants. These results support the in vitro data on the low number of TNF-alpha receptors on Meth A sarcoma cells. The ability of rHuTNF-alpha to induce regression of established (7 days) subcutaneous Meth A implants, positively correlated with the degree of both macroscopic and microscopic tumor necrosis. In addition, recombinant human tumor necrosis factor-beta (lymphotoxin) and recombinant murine tumor necrosis factor-alpha induced similar levels of necrosis. Other lymphokines with known antitumor activities, recombinant human interferon-gamma, murine interferon-gamma, and human interleukin 1 alpha, failed to induce detectable necrosis of Meth A sarcoma. Mice which had rejected subcutaneous Meth A sarcoma implants after rHuTNF-alpha treatment and which were later challenged subcutaneously with Meth A sarcoma or other noncross-reacting chemically induced sarcomas were found to be specifically immune to Meth A sarcoma. In addition, low levels of cytotoxic antibodies reactive to Meth A sarcoma were detected in the sera of 21 of 30 Meth A immune mice. Histological evaluation of the hemorrhagic tumor necrosis induced by rHuTNF-alpha suggests that the primary lesion is vascular, possibly directly on the endothelial cells. The mechanisms involved in the generation of specific cell-mediated antitumor immunity in this model are at present unknown.     

Dietary folate improves age-related decreases in lymphocyte function

Although low folate status is thought to be fairly common in the older population, its implication on immunity has not been adequately investigated. Using 11-month-old and 23-month-old male rats (Fisher 344), the present study was undertaken to examine the modifying effects of feeding a control diet (NIH-07) supplemented with folate (35.7 mg/kg) for 3 weeks on the immune cells of spleen and mesenteric lymph node (MLN) origin. The serum concentrations of folate along with vitamin B(12) were elevated in response to the folate supplementation (P<.05). These results were accompanied by an improved proliferative response (stimulation index) to mitogens in both the spleen and MLNs (P<.05). The proportion of T cells in the MLNs, but not in the spleen, was significantly increased in rats fed a diet supplemented with folate. In the spleen, the folate-supplemented diet prevented the age-associated decrease (P<.05) in the production of interferon (IFN)alpha by unstimulated cells and the decrease in T-helper (Th)1/Th2-type response after stimulation with phorbol myristate acetate and ionomycin. In the MLNs, on the other hand, the folate-supplemented diet failed to influence any age-related increase in interleukin (IL)-2, tumor necrosis factor alpha and IFNgamma following stimulation but did result in a significantly increased production of IL-4 (P<.05). Overall, this study provides data suggesting that aging is associated with changes in the proportion of T cells, the ability of immune cells to proliferate and the production of cytokines after stimulation. Supplementing a folate-sufficient diet with additional folate improves proliferative response to mitogens, the distribution of T cells in the MLNs and the age-related changes in cytokine production in the spleen. These results suggest that the dietary folate requirement may be higher in the older population than in the younger population to support immune functions.     

In vivo elimination by specific effector cells of an established syngeneic rat moloney virus-induced sarcoma.

BN rats immunized subcutaneously with a viral induced tumor (MST) or with a chemical-induced fibrosarcoma (BC5) were donors of immune spleen cells. Samples of immune spleen cells were tested in vitro against MST and BC5 in a 51Cr release assay before culturing and after 7 days of culture with mitomycin C-treated MST and/or BC5 tumor cells (MSTMit, BC5Mit). These spleen cells were infused in vivo i.v. into x-rayed (400 R) and nonirradiated BN recipients that bore a vascularized and progressive (1 to 1.5 cm in diameter) subcutaneous MST or BC5. Spleen cells from untreated BN donor rats were also tested in vitro and in vivo as controls. Established MST were specifically eliminated by spleen cells immune to MST after culture with MSTMit, but not by spleen cells immune to MST without further culture nor by cultured or uncultured BC5 immune spleen cells and control spleen cells. Also, the growth of BC5 was not affected by MST immune spleen cells cultured for 7 days with MST and/or BC5. Elimination of Moloney sarcoma (MST) in vivo occurred in less than 35 days and was correlated with the generation of cytotoxicity in vitro since only MST immune spleen cells cultured with MSTMit were able to augment significantly their cytotoxic capability in vitro.     

Macrophage activation in vivo and in vitro

Morphology, lysosomal enzyme activity and phagocytic ability were tested in peritoneal macrophage cultures after stimulation in vivo or in vitro with endotoxin, mineral oil or latex particles, and compared to the same parameters in normal peritoneal macrophages. Treatment with latex did not give changes in the parameters tested after in vivo or in vitro stimulation. In all other types of stimulation the cells displayed varying degrees of spreading and changes in granule content. Extensive ruffling of cell membrane was obvious in endotoxin-stimulated cells. The pattern of lysosomal enzyme activity was complex and depended on the means of stimulation. Acid phosphatase showed the greatest increase after both in vivo and in vitro stimulation, N-acetyl-glucosaminidase could not be increased in vitro. Internalization of opsonized red cells mediated by the Fc receptor increased after in vivo stimulation. No such change was observed after in vitro stimulation. Normal peritoneal macrophages do not internalize significantly via the C₃ receptor. In vivo stimulation triggered the capacity to internalize up to 45% of the attached red cells. A similar reaction was obtained in vitro when both endotoxin and FCS were added to the culture medium, but not when endotoxin or FCS were used alone. We conclude that the use of the term activation of macrophages should always be based on quantitative changes in well defined parameters. Changes in one parameter will not necessarily be accompanied by the whole range of biochemical and morphological perturbations. The capacity to ingest via the C₃ receptor may be the most useful parameter.     

Metabolic effects of chronic obestatin infusion in rats

Obestatin is purported to be a peptide hormone encoded in preproghrelin. We studied the metabolic effects of continuous infusion of obestatin via subcutaneously implanted osmotic mini-pumps. Administration of up to 500nmol/kg body weight/day obestatin did not change 24h cumulative food intake or body weight in rats. Similarly, no effects were observed when obestatin was infused at 1000nmol/kg body weight/day for seven days. This dose of obestatin infused during a 24h fast did not alter weight loss, suggesting that obestatin has no effect on energy expenditure, and this dose did not alter glucose or insulin responses during an IPGTT. Obestatin was originally proposed to interact with GPR39 and subsequently the receptor for GLP-1. While both receptors are expressed in pancreatic islets, incubation with obestatin did not alter insulin release from islets in vitro. Moreover, obestatin did not bind to INS-1 beta-cells or HEK cells overexpressing GLP-1 receptors or displace GLP-1 binding to these cells. Our findings do not support the concept that obestatin is a hormone with metabolic actions.     

Effects of weak laser radiation (632.8 nm) on isolated mouse immune cells

The dose dependence of in vitro effects of low-intensity radiation of a He-Ne laser (632.8 nm, 0.2 mW/cm²) on the functional activity of peritoneal macrophages and lymphocytes of mouse spleen was studied. The exposure of isolated cells was varied from 5 to 180 s. If the exposure did not exceed 60 s, stimulation of secretory activity was observed: increased production of interleukin 2, interferon γ, and interleukin 6 in lymphocytes; increased production of tumor necrosis factor α, nitric oxide, and interleukin 6 in macrophages; and enhanced activity of natural killer cells. A longer exposure (up to 180 s) either had no effect on the synthesis of certain cytokines (interleukin 2 in lymphocytes and interleukin 6 in macrophages) or inhibited it, which was expressed in decreased production of interleukin 6 and interferon γ in lymphocytes and nitric oxide in macrophages, as well as in suppression of the activity of natural killer cells. Conversely, the production of interleukin 3 decreased after a short-term exposure but increased after 180-s irradiation. The high sensitivity of cells to extremely weak laser light also manifested itself as a considerable increase in expression of the inducible heat shock protein 70; this effect was observed at all doses studied, including the 5-s exposure. In contrast, expression of the heat shock protein 90 slightly decreased after irradiation of cells with laser light.     

Gonadotropin-releasing hormone alters the T helper cytokine balance in the pregnant rat

The interactions between immune-endocrine and reproductive systems are heightened during pregnancy as an adaptive mechanism, and are regulated by a complex array of hormones and cytokines that control the survival of a semiallogeneic conceptus. GnRH can exert direct effects on the immune system via its receptor (GnRH-R) on lymphoid cells. In the present study, we employed in vitro, ex vivo, and in vivo approaches to investigate the role of GnRH in the modulation of T helper cytokines in pregnant rats undergoing termination of pregnancy. Day 8 pregnant rats were infused with a GnRH agonist (GnRH-Ag) for 24 h using an osmotic minipump. Sham control rats were infused with the vehicle, saline. Lymphocytes were isolated from sham and treated rats and polyclonally stimulated with immobilized anti-CD3 antibody. The levels of the signature T helper 1 (Th-1) cytokines (interferon-gamma [IFN-gamma] and interleukin-2 [IL-2]) and Th-2 cytokines (IL-4 and IL-10) were measured in culture supernatants. Using immunoflourescence confocal microscopy, we demonstrated for the first time the spatial localization of GnRH-R protein on the surface of lymphocytes. We observed a marked increase in IFN-gamma and inhibition of IL-4 production from lymphocytes of pregnant rats treated in vitro with different doses of GnRH-Ag. Further, the responsiveness of lymphocytes to produce IFN-gamma was markedly increased in cells cultured ex vivo from GnRH-Ag infused rats, whereas the capacity of lymphocytes to produce IL-4 was significantly inhibited. In addition, GnRH-Ag infusion in pregnant rats induced a shift toward Th-1 cytokines in the serum. We did not observe any significant difference in IL-2 and IL-10 production in response to GnRH-Ag. Our results suggest an additional function for GnRH as a Th-1 inducer and Th-2 inhibitor. GnRH can thus skew the cytokine balance to predominantly Th-1 type in pregnancy, leading to the termination of pregnancy in rats.     

Endotoxin protection of rats from pulmonary oxygen toxicity: possible cytokine involvement

Treatment with endotoxin protects rats against lung injury during hyperoxia (greater than 98% oxygen at 1 atmosphere absolute for 60 h). This study demonstrates that serum from endotoxin-treated donor rats also protects recipients from oxygen toxicity. Rats treated with serum from saline-treated donors were not protected, and protection was not explained by residual endotoxin in protective sera. Unlike endotoxin-protected rats (where lung antioxidant enzyme activity is elevated after hyperoxia), postexposure superoxide dismutase (SOD) and catalase (CAT) activities in the lungs of serum-protected rats were not affected. Levels of tumor necrosis factor (TNF) and interleukin 1 (IL-1) in protective sera were increased. This study demonstrates that increases in lung SOD and CAT activity are not required for endotoxin protection from hyperoxia and suggests that TNF and IL-1 may participate in the mechanism of endotoxin protection.     

Neutralization of microcystin shock in mice by tumor necrosis factor alpha antiserum

Microcystis aeruginosa is a common cyanobacterium in water blooms that appear widely in nutrient-rich, fresh, and brackish waters, and its toxic blooms cause the death of domestic animals. The administration of a crude toxic cell extract of M. aeruginosa K-139 to mice can produce tumor necrosis factor (TNF) and prompt severe physiological disturbances, especially liver damage, which can lead to death. The in vitro production of TNF-alpha by peritoneal macrophages was observed after stimulation with the cell extract or the purified toxin from K-139 cells. The expression of a TNF-alpha mRNA was also detected in spleen cells and peritoneal macrophages after stimulation with the cell extract. However, a previous injection of rabbit anti-murine TNF-alpha serum could prevent the liver damage to some extent and protect the mice from death. These findings indicate the involvement of TNF in microcystin shock.     

Neutralization of microcystin shock in mice by tumor necrosis factor alpha antiserum.

Microcystis aeruginosa is a common cyanobacterium in water blooms that appear widely in nutrient-rich, fresh, and brackish waters, and its toxic blooms cause the death of domestic animals. The administration of a crude toxic cell extract of M. aeruginosa K-139 to mice can produce tumor necrosis factor (TNF) and prompt severe physiological disturbances, especially liver damage, which can lead to death. The in vitro production of TNF-alpha by peritoneal macrophages was observed after stimulation with the cell extract or the purified toxin from K-139 cells. The expression of a TNF-alpha mRNA was also detected in spleen cells and peritoneal macrophages after stimulation with the cell extract. However, a previous injection of rabbit anti-murine TNF-alpha serum could prevent the liver damage to some extent and protect the mice from death. These findings indicate the involvement of TNF in microcystin shock.     

Functional activity in vivo of effector T cell populations. II. Anti-tumor activity exhibited by syngeneic anti-MoMULV-specific cytolytic T cell clones

The functional activity in vivo of murine tumor-specific cytolytic T lymphocyte populations and clones was studied. Tumor cell destruction induced after the i.v. injection of cytolytic effector cells was quantitated by monitoring the elimination of 131IUdR-labeled tumor cells in the peritoneal cavity by using whole-body counting techniques. Mixed leukocyte-tumor cell cultures were established by using spleen cells from C57BL/6 regressor mice that had rejected an intramuscular tumor induced by the injection of MSV-MoMuLV virus. This effector cell population was observed to eliminate syngeneic MoMuLV-induced tumor cells in a dose-dependent manner. Treatment of the effector cell population with monoclonal anti-Lyt-2 antibodies plus complement totally abrogated their ability to induce tumor cell destruction in the peritoneal cavity. MSV-MoMuLV-specific Lyt-2+ cytolytic T cell clones derived by micro-manipulation of T lymphocyte-tumor cell conjugates were also tested for functional activity in vivo. Several clones induced a rapid, specific elimination of 131I-labeled MBL-2 tumor cells from the peritoneal cavity after i.v. injection, whereas others were inactive. Both active and inactive clones were highly cytolytic and secreted MAF/IFN-gamma lymphokines. In contrast to previous results obtained in a tumor allograft model, the MSV-MoMuLV-specific cytolytic T cell clones that were active in vivo did not proliferate in vitro in response to stimulation with irradiated tumor cells plus filler spleen cells in the absence of an added source of interleukin 2.