Different pattern of T cell receptor δ gene rearrangement in tumour-infiltrating lymphocytes and peripheral blood in patients with solid tumours

Tumor-infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL) from four patients with renal-cell carcinoma (three paired with blood), two colon carcinomas (both paired with blood) and two melanomas (blood was not available) were analysed for the T cell receptor (TCR) gene repertoire. Polymerase chain reaction analysis, employing a panel of specific primers for TCR gene segments, showed different gene rearrangement patterns in TIL and PBL in all patients. Simultaneous analysis of TIL and PBL revealed the presence of lymphoid cells in the tumour tissue that were not present in the periphery. These results demonstrate that, although tumor-infiltrating lymphocytes contain / T cells within the range observed in peripheral blood, these cells differ from those in peripheral blood in their gene repertoire and this may account for selective accumulation or/and in situ amplification of / lymphocytes at the tumour site, indicating a unique type of host reaction against tumors.     

CD4+ Th0 cell clones,isolated from a metastatic lymph node of a melanoma patient,possess cytolytic function

In the present study T lymphocytes isolated from a metastatic lymph node (T-LNL) of a melanoma patient have been cloned. In the attempt to verify whether T-LNL may acquire in vitro functional activities in the absence of tumour-associated antigens, they were cloned utilizing allogeneic lymphocytes as feeder cells. Nineteen clones generated from T-LNL proved to be CD4+ and, among these, five were able to kill autologous and allogeneic human melanoma cells in HLA-class-II-restricted way. On the basis of their cytokine production, these CD4+ cytolytic T-LNL clones were shown to belong to the Th0 subset and three of them expersseed the V17 chain of the T cell receptor. These results suggest the presence of melanomaspecific but functionally inactive lymphocytes with T cell receptor oligoclonality in the lymph node environment. These specific T cells may acquire in vitro the capacity to kill autologous and allogeneic tumours without any induction by autologous melanoma cells.     

Impairment in proliferation,lymphokine production and frequency distribution of mitogen-responsive and interleukin-2-producing cells in Hodgkin's disease

Summary In this paper, we have correlated the ability of peripheral blood lymphocytes (PBL) from Hodgkin's Disease patients to proliferate in response to a mitogen, phytohaemagglutinin (PHA), with production of lymphokines interleukin-2 (IL-2) and interferon (IFN), accumulating in the activated lymphocyte culture supernatants. We have also studied the frequency distribution of PHA-responsive and IL-2-producing T cells from PBL using limiting-dilution analysis. We observed that the levels of IL-2 and IFN in the supernatants of activated lymphocytes from patients with Hodgkin's disease were significantly reduced compared to those of healthy donors. Substage-B patients showed marked reduction in the ability to produce IFN. Levels of IL-2 and IFN in the culture supernatants of PBL from Hodgkin's disease patients correlated positively with proliferative responses, when analysed by linear regressison (r = 0.79 andr = 0.60 respectively). However, production of the two lymphokines by activated lymphocytes from the same patients did not correlate (r = +0.04). Further, the frequencies of PHA-responsive cells and IL-2-producing cells in the PBL of patients with Hodgkin's disease (ranges 1/111–1/554 and 1/3009–1/6709 respectively) were also less than those of the healthy donors (ranges 1/80–1/181 and 1/761–1/1828 respectively). Proliferation, IL-2 production in bulk cultures and frequencies of PHA-responsive and IL-2-producing cells correlated well in individual healthy donors. Whereas, one patient (BC 11 214) with a frequency of PHA-responsive cells within normal limits had a very low frequency of IL-2-producing cells. Taken together, the results indicate abnormalities in cytokine production and frequency distribution of cells required for amplification of immune response in patients with Hodgkin's disease.     

Auto-tumor recognition following in vitro induction of MHC antigen expression on solid human tumors: stimulation of lymphocytes and generation of cytotoxicity against the original MHC-antigen-negative tumor cells

Summary Expression of major histocompatibility complex (MHC) class I antigens was induced in eight out of nine freshly prepared tumor cell suspensions by exposure to interferon (IFN) and tumor necrosis factor (TNF) in vitro. The untreated, class-I-antigen-negative, and the treated, antigen-positive, cells of three tumors (one breast carcinoma, one plasmocytoma and one ovarian carcinoma) were compared for the capacity to stimulate autologous and allogeneic blood lymphocytes, to generate auto-tumor cytotoxicity and for sensitivity to the lytic effect induced in autologous mixed lymphocyte tumor cell culture (MLTC). The MHC class I-negative cells did not stimulate, while the cells induced for expression of antigens did. On the other hand, when the autologous cytotoxic cells were generated in the MLTC by the class I antigen-positive tumor cells the class I-negative tumor cells were also damaged. Lysis of the class-I-positive tumor cells was abrogated by the W6/32 monoclonal antibody directed against the monomorphic part of the class I molecules.     

Chemical induction of interleukin-8, a proinflammatory chemokine,in human epidermal keratinocyte cultures and its relation to cytogenetic toxicity

Tumor promoters, proinflammatory cytokines, endotoxins, and protein synthesis inhibitors can modulate cell cycle kinetics of various cell types, stimulate production of reactive oxygen species, and induce keratinocytes to produce interleukin-8 (IL-8), a potent chemotactant for polymorphonuclear neutrophils and T lymphocytes. The aim of this study was to determine whether perturbations of cytogenetic responses correlated with the induction of IL-8 expression. Cultures of primary human keratinocytes were grown in serum-free medium with 5 mol/L bromodeoxyuridine to label DNA and exposed either to phorbol-13-myristate-12-acetate (PMA) (0.0001–100 ng/ml), cycloheximice (CHX) (0.01–50 g), lipopolysaccharide (0.1–100 g/ml), tumor necrosis factor- (TNF) (3.13–50 ng/ml), or interleukin-1 (IL-1) (1–182 pg/ml). Metaphase chromosome preparations were stained by a fluorescence-plus-Giemsa technique to differentiate sister chromatids. For IL-8 production, keratinocytes were grown to 70% confluency and then exposed to chemicals for 24 h. Immunoreactive IL-8 was quantitated from the supernatants by ELISA. With the exception of benzo(a)pyrene used as a positive control, none of the agents induced sister chromatid exchanges. However, PMA and TNF induced IL-8 production that coincided with significant cell cycle inhibition. IL-1 had no effect on cytogenetic endpoints, yet stimulated a 6.3-fold increase in IL-8. CHX inhibited cell cycle progression and mitotic activity at concentrations that were 200 times lower than required for IL-8 induction; however, puromycin (0.31–10 g/ml), another protein synthesis inhibitor, did not induce IL-8. At all concentrations tested, TNF reduced the mitotic index by 45%, slowed cell cycle progression by 3.5 h, and induced a flat, albeit large, IL-8 response at concentrations 12.5 ng/ml. These agent-specific response patterns suggest that induction of IL-8 production is not always the inevitable result of cell cycle perturbations or genetic damage.Abbreviations B(a)P benzo(a)pyrene - BrdU 5-bromo-2-deoxyuridine - CHX cycloheximide - ICAM intercellular adhesion molecules - IL-1 interleukin-1 - IL-8 interleukin-8 - KGM keratinocyte growth medium - LPS lipopolysaccharide - PKC protein kinase C - PMA phorbol-13-myristate-12-acetate - PMN polymorphonuclear neutrophil - ROS reactive oxygen species - SCE sister chromatid exchange - TNF tumor necrosis factor      

In vitro expansion and analysis of T lymphocyte microcultures obtained from the vaccination sites of cancer patients undergoing active specific immunization with autologous Newcastle-disease-virus-modified tumour cells

In order to understand further the effects of Newcastle-disease-virus(NDV)-modified tumour vaccines we investigated the feasibility of isolating lymphocytes from the site of injection of patients undergoing postoperative active specific immunization (ASI) with autologous NDV-modified tumour cells. Delayed-type-hypersensitivity(DTH)-like reactions from five cancer patients were surgically removed, minced and the tissue particles were digested with collagenase and DNase. Lymphoid cells recovered were expanded in a highly efficient limiting-dilution analysis system optimized for T cell growth [Moretta et al. (1983) J Exp Med 157: 743] and lymphocyte microcultures (clonal probability >0.8) could be grown for up to 1 year. Analysis of the microcultures for phenotype and function showed that the majority were positive for CD4 (92%) and TCR (96%). Concanavalin-A-induced production of interleukin-2 (IL-2), IL-6, interferon and tumour necrosis factor was detected in more than 70% of the microcultures. Lectin-dependent cytotoxicity was only very rarely observed. The general characteristics of the microcultures obtained support the notion of a DTH-like reaction taking place at the site of tumour cell challenge. The possibility of in vitro expansion and cultivation of T lymphocytes from ASI vaccination sites should help to elucidate further the role of these cells in active specific immunization against autologous tumour cells.This study was supported by Dr.-Mildred-Scheel-Stiftung and the Tumorzentrum Heidelberg     

T lymphocytes can mediate lysis of autologous melanoma cells by multiple mechanisms: Evidence with a single T cell clone

Summary The specificity analysis of a CD3⁺, WT31⁺, CD8⁺ cytotoxic T lymphocyte (CTL) clone (CTL 49), isolated from peripheral blood lymphocytes of a melanoma patient (no. 665) after mixed lymphocyte culture with an HLA-A2⁺ allogeneic lymphoblastoid cell line (VSKB-LCL), revealed that CTL 49 could lyse, in addition to HLA-A2⁺ lines, autologous HLA-A2^– melanoma (Me665/2) and K562 targets. Killing of VSKB-LCL, but not of Me665/2, could be inhibited by anti-CD3 and by anti-HLA-A2 antibodies or by modulation of the CD3 complex. Cold-target competition studies showed that K562, but not VSKB-LCL, could compete with Me665/2 for lysis by CTL 49. However, unlike K562, Me665/2 could be lysed by CTL 49 in a Ca²⁺-independent fashion in 4 h and 18 h assays. CTL 49 expressed mRNA specific for tumor necrosis factor (TNF) and, to a lesser extent, for lymphotoxin (TNF). Exposure of the clone to anti-CD3 antibodies induced the expression of interferon(IFN)--specific and the up-regulation of TNF- and TNF-specific mRNA. Antibodies to TNF, TNF and IFN reduced the lysis of Me665/2, but not of K562, by CTL 49 in 18-h cytotoxic assays. Antibodies to TNF and to IFN almost completely inhibited the lysis seen on Me665/2 (but not on K562), in 96-h assays, by supernatants isolated from VSKB-LCL- or anti-CD3-stimulated CTL 49 cells. Taken together, these data indicate that major-histocompatibility-complex-independent lysis of autologous tumor cells and of natural killer reference targets by the same alloreactive T cell clone are activities related at the level of target recognition but distinct at the level of the lytic hit. Thus, efficient lysis of autologous tumor cells results from a complex mechanism based upon direct effector-target interaction as well as on cytokine-mediated cytolytic effects.     

Growth effects of nagilactone E on cultured cells of lettuce

Nagilactone E, a norditerpene dilactone isolated from a gymnosperm, Podocarpus nagi (Podocarpaceae), was able to stimulate the growth of cultured cells of Lactuca sativa cv. Grand Rapids at 0.1 g ml^-1 but not at 0.01 or 1 g ml^-1. Cell wet weight/unit time and cell number/unit wet weight were increased when they were recorded at the end of the 14-day incubation period. Also, the population of cells treated with that concentration of nagilactone E had a higher percentage of cells with shorter cell length compared to the control (untreated).     

Induction of glycanolytic activities by Streptomyces sp. QM-B814 growing on different glucopolymers

The actinomycete Streptomyces sp. QM-B814 is able to use polymeric glucans as sole carbon source by the induction of specific extracellular enzymatic systems. Carboxymethylcellulose and barley -glucan were the most effective inducers of endoglucanase activities towards -(1,4) and mixed -(1,3)-(1,4) linkages. Avicelase activity was induced by Avicel and carboxymethylcellulose. The higher activity towards filter paper (cellulase) was measured in cultures growing in Avicel, carboxymethylcellulose, and barley -glucan. Laminarinase activity was induced only by laminarin and lichenan failed to induce significant activity levels against any of the substrates tested.     

Cytotoxic T lymphocyte clones from peripheral blood and from tumor site detect intratumor heterogeneity of melanoma cells. Analysis of specificity and mechanisms of interaction

CTL clones isolated from PBL or from tumor-infiltrating lymphocytes (TIL) of a melanoma patient (pt665) were screened for specificity on a panel including autologous tumor cells from two distinct metastases (Me665/1, Me665/2), autologous EBV-transformed B cells and 15 allogeneic cell lines of different histology. Each clone displayed a peculiar cytolytic activity ranging from lysis of most targets (PBL clone 4C4) to preferential reactivity on the two autologous metastases (TIL clone 8B3). Blocking and modulation experiments, revealed that the lysis of autologous-Tu cells by TIL clone 8B3, but not by PBL clone 4C4, could be inhibited by mAb to HLA-class I and to CD3 Ag or by CD3 complex modulation. Clone 8B3 was tested also on a panel of 25 tumor clones from Me665/2, revealing that only 4 neoplastic clones were lysed (2/4, 2/14, 2/17, and 2/51). Cold target competition experiments indicated that the uncloned autologous melanomas and one tumor clone (2/17), but no two other tumor clones (2/10, 2/15), could compete with one another for lysis by 8B3. Determination of melanin content of tumor clones from Me665/2 revealed that the four neoplastic clones recognized by 8B3 possessed much lower melanin levels than all the other 20 clones not lysed by this effector.     

Lymphocytes infiltrating colorectal cancer have low proliferative capacity but can secrete normal levels of interferon γ

Significant numbers of infiltrating mononuclear cells are commonly observed in solid tumours, although their role in restricting tumour growth is not clear. Tumour-infiltrating lymphocytes (TIL) from 38 patients with colorectal cancer, in parallel with peripheral blood lymphocytes (PBL), were assayed to determine their ability to proliferate in response to concanavalin A (ConA), interleukin-2 (IL-2), ConA+IL-2, phorbol 12-myristate 13-acetate (PMA)+ionomycin (IOM), and staphylococcal enterotoxin B (SEB). These reagents were selected to give a range of weak to strong proliferative responses either via or independent of the T cell receptor. Proliferation of TIL was significantly lower than that of PBL in all cultures: ConA (P<0.001), IL-2 (P=0.002), ConA+IL-2 (P<0.001), PMA+IOM (P<0.001), SEB (P=0.002). In addition to the low proliferative capacity of TIL, production of cytokines by TIL may also play a role in control of tumour growth. We have assayed IFN production in the supernatants from 16 paired TIL and PBL cultures, and tumour necrosis factor (TNF) in 6 paired cultures. TNF concentrations were significantly lower in TIL cultures than in PBL cultures stimulated with ConA (P<0.05), but no different in control or IL-2 stimulated cultures. IFN levels did not significantly differ between PBL and TIL cultures, indicating that despite the restricted proliferative capacity of TIL, these cells remain capable of secreting significant amounts of IFN.     

Specific immune recognition of autologous tumor by lymphocytes infiltrating colon carcinomas: Analysis by cytokine secretion

Summary Tumor-infiltrating lymphocytes (TIL) were grown in the presence of interleukin-2 from 19 colon carcinoma specimens, including 1 primary lesion and 18 metastatic lesions. These cultures showed a median proliferation of 606-fold (range 13-fold to 28 000-fold) over 49 culture days (range 26–76 days). By phenotype, mature cultures were 69%–99% CD3⁺ (mean 93%) and contained mixed populations of CD4⁺ and CD8⁺ cells (CD4>CD8 in 10 of 19 cultures). Fresh cryopreserved colon tumors were not lysed by autologous TIL in short-term⁵¹Cr-release assays, and were poorly lysed by lymphokine-activated killer cells. Ten TIL cultures were assayed for cytokine secretion in response to autologous and allogeneic tumors during a 6- to 24-h coincubation. Culture supernatants were tested by ELISA for the presence of granulocyte/macrophage-colony-stimulating factor, interferon , and tumor necrosis factor . Of 10 TIL, 4 secreted at least two of these cytokines specifically in response to autologous and/or HLA-matched fresh allogeneic colon carcinomas, but not to melanomas or HLA-unmatched colon carcinomas. Cytokine secretion was mediated by both CD4⁺ and CD8⁺ TIL, and could be inhibited by mAb directed against the appropriate class of MHC antigen. These data provide evidence for specific, MHC-restricted immune recognition of human colon carcinomas by T lymphocytes.     

Cytotoxic activity and T cell receptor repertoire in tumor-infiltrating lymphocytes of adrenal cell carcinomas

The cytotoxic activity and T cell receptor (TCR) V repertoire in tumor-infiltrating lymphocytes (TIL) of three primary adrenal cell carcinomas were analyzed. Fresh, non-cultured TIL from two of the three tumors showed low but significant lysis of the autologous tumor, and for one of the patients this activity was strongly enhanced upon culture in interleukin-2. An allogeneic adrenal cell carcinoma line and the K562 or Daudi targets included as controls were not killed. Phenotypic analysis of freshly isolated TIL demonstrated that the cells from the two patients that demonstrated cytolytic capacity mainly consisted of CD45RO⁺ T cells. In vitro cultured TIL lines from these patients demonstrated a high percentage of CD8⁺ cells expressing either the V6 gene or the V8 gene product, as measured with a panel of mAb specific for TCR V and V gene products. Analysis of the TCR V gene mRNA expression in freshly isolated non-cultured TIL, using a polymerase-chain-reaction-assisted cDNA-amplification assay, confirmed the strong expression of the genes coding for the TCR V6 or the V8. This assay also demonstrated a more restricted TCR V gene usage in the TIL as compared to peripheral blood lymphocytes from the same patient.This study was supported by the Swedish Cancer Society and by the Cancer Society in Stockholm     

Tissue specificity of chromosomal rearrangements in ataxia-telangiectasia

Summary Cytogenetic studies of lymphocytes and fibroblasts from individuals with ataxia-telangiectasia (AT) demonstrate spontaneous chromosomal breakage. In the AT lymphocytes, this damage results in a high frequency of balanced rearrangements involving chromosome bands 7p14, 7q35, 14q12, and 14q32. The T-cell receptor , , and chain gene complexes and the immunoglobulin heavy chain gene complex, all of which may be functional in lymphocytes, have been localized to these bands. To assess the relationship between genes at these breakpoints and the entirety of the AT phenotype, we undertook a detailed cytogenetic analysis of fibroblasts and lymphocytes from seven AT homozygotes. Our findings indicate that the rearrangements present in the lymphocytes are not commonly observed in the fibroblasts, despite the increased instability of chromosomes from these cells relative to lymphocytes. Furthermore, the changes in the fibroblasts are neither consistent within nor between patients, suggesting that chromosome rearrangement occurs more randomly in this tissue. Therefore, differential site-specific damage in separate tissues may generate the distinct features of the disease in those tissues and may account for the pleiotrophic effects of the AT gene.     

Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute     

Dendritic cells derived from metastatic cancer patients vaccinated with allogeneic dendritic cell–autologous tumor cell hybrids express more CD86 and induce higher levels of interferon-gamma in mixed lymphocyte reactions

Dendritic cells (DCs) are highly effective antigen-presenting cells that, when derived from cancer patients, seem to be functionally deficient. Herein, we show that vaccination with allogeneic DC–autologous tumor cell hybrids affects the phenotype and improves the function of monocyte-derived DCs (Mo-DCs) from cancer patients. Mononuclear cells were isolated from patients peripheral blood by density gradient centrifugation, and adherent cells were cultured in medium containing GM-CSF plus IL-4 and, after 5 days, TNF-. After 2 more days, Mo-DCs were harvested and their CD80, CD86, and CD83 expression was assessed by flow cytometry. They were also used as stimulators in mixed lymphocyte reactions (MLR), where IFN- production was measured by ELISA. Mo-DCs from unvaccinated patients expressed significantly lower levels of CD86, and tended to express lower levels of CD83 than Mo-DCs from healthy donors. However, Mo-DCs generated after hybrid cell vaccination presented increased expression of the same markers and induced significantly higher levels of IFN- in MLR. These results indicate that the use of allogeneic DC–based cancer vaccines induces recovery of DC function in metastatic cancer patients and, therefore, could precede the use of autologous DCs for vaccine preparation. Such an approach could be relevant and should be investigated in clinical trials.     

Biotransformation of 5βH-pregnan-3βol-20-one and cardenolides in cell suspension cultures of Nerium oleander L.

Summary In order to demonstrate enzyme activities playing a role in the biosynthesis of cardenolides and 2,6-dideoxysugars, 5H-pregnan-3ol-20-one and cardenolides (digitoxigenin, oleandrigenin/L-oleandrose, oleandrin, neriifolin, digitoxigeninmonodigitoxoside and strospeside) were fed to cell suspension cultures of Nerium oleander L.. It could be shown that cell suspension cultures of Nerium oleander L. are able to oxidize, isomerize and glucosylate 5H-steroidaglycones at C-3. The respective glucosides of the 5H-steroid-aglycones are the main biotransformation products. These cell cultures are an appropriate tool for the production of labelled 5H-steroidglucosides.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - EtOAc ethylacetate - MeOH methanol - MS Murashige & Skoog     

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin -chain was established, completing the analysis of this hemoglobin. Comparisons with other avian -chains show variations smaller than those for the corresponding -chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform -chain, and a total of 35 positions are affected by differences among all avian -chains analyzed (versus 61 for the -chains). At three positions, the Psittacula -chain has residues unique to this species. Three ₁₁ contacts are modified, by substitutions at positions 51, 116, and 125.     

Modulation of natural killer activity by thymosin alpha 1 and interferon

Summary A single injection of -interferon (-IFN) (30 000 units/mouse), a major biological modifier of natural killer (NK) cytolytic activity, strongly stimulated NK activity in normal mice, as expected, while the same treatment did not statistically alter the NK response in cyclophosphamide (CY)-suppressed animals.We investigated the possibility of thymosin ₁ cooperating with -IFN in boosting NK activity in CY-suppressed animals.The results show that treatment with thymosin ₁ (200 g/kg) for 4 days, followed by a single injection of -IFN 24 h before testing, strongly restored NK activity in CY-suppressed mice. Thymosin ₁ was, moreover, able to accelerate the recovery rate of NK activity in bone marrow reconstituted murine chimeras.Taken together the data support the concept that the synergic effect between thymosin ₁ and -IFN could be the result of effects on differentiation of the NK lineage at different levels.     

Lysing of fresh human tumor by a cytotoxic factor derived from autologous large granular lymphocytes independently of other known cytokines

Summary During interaction with autologous tumor cells large granular lymphocytes (LGL) of cancer patients released a soluble cytotoxic factor, termed LGL-derived cytotoxic factor, which mediated lysing of autologous fresh tumor cells. The cytotoxic factor was compared with purified human recombinant cytotoxic cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) , IFN, interleukin-1 (IL-1) and IL-2. The LGL cytotoxic factor exhibited cytotoxicity against autologous and allogeneic fresh human tumor cells in an 18-h⁵¹Cr-release assay, while these target cells were resistant to lysing by any of the recombinant cytokines. Mixtures of recombinant(r) TNF, rLT, rIFN, rIFN, rIL-1 and rIL-2 were still unable to produce cytotoxic effects on fresh human tumor cells. Treatment with monoclonal and polyclonal antibodies directed against rTNF, rLT, rIFN, rIFN, or rIL-1 did not inhibit the cytotoxic activity of LGL-derived cytotoxic factor against fresh human tumor cells. Even a mixture of all the antibodies was incapable of blocking the cytolytic activity of the factor to fresh human tumor cells. Furthermore, intact LGL-mediated lysing of autologous tumor cells was not inhibited by any of the antibodies. These results may indicate that a cytotoxic factor produced by LGL in response to autologous tumor cells mediates lysing of fresh human tumor cells independently of TNF, LT, IFN, IL-1 and IL-2.