Interleukin-2 receptor monoclonal antibodies have no effect on anti-CD3 monoclonal antibody-mediated cytotoxicity in CD3+ leukemic large granular lymphocytes

We studied the role of Fc receptors and interleukin-2 (IL-2) receptor subunits in anti-CD3 monoclonal antibody (MAb)-mediated cytotoxicity of CD3+ leukemic large granular lymphocytes (LGL). Peripheral blood mononuclear cells from four patients with CD3+ LGL leukemia were cultured with 1 microgram/ml of anti-CD3 MAb. Anti-CD3 MAb-mediated cytotoxicity was not inhibited when K562 target cells were preincubated with heat-aggregated human IgG, suggesting that binding of the effector cell-bound anti-CD3 MAb to Fc receptors of target was not involved in cytotoxicity. Induction of cytotoxicity was not blocked by the addition of either anti-p55 or anti-p75 IL-2 receptor MAbs. These results show that the induction of cytotoxicity by anti-CD3 MAb is not mediated through IL-2 receptor subunits in CD3+ leukemic LGL.     

BCG—PPD的制备及应用

用三氯醋酸(TCA)和硫酸铵(AS)综合法,由卡介菌苗(BCG)培养滤液中提纯制得卡介菌素纯蛋白衍生物(BCG—PPD)。BCG—PPD的纯度和结核菌素纯蛋白衍生物国际标准(PPD-S)及中国标准(PPD—C)相近,高于加拿大标准(PPD—CT68)和丹麦标准(PPD—RT23)。在BCG免疫豚鼠中,BCG—PPD的皮肤迟发型变态反应(DTH)大于结核菌素纯蛋白衍生物(PPD)的DTH反应。在结核菌感染豚鼠组中,BCG—PPD的DTH反应小于PPD的DTH反应.在检查333名新生儿接种BCG12周后的免疫     

Subsets of human large granular lymphocytes (LGL) exhibit accessory cell functions

The present study shows that human large granular lymphocytes (LGL) depleted of OKT3 (T lymphocytes) and Leu-M1-positive (monocytes) cells exhibit accessory cell function for the T lymphoproliferative responses to the soluble stimulants Staphylococcus protein A (SpA) or Streptolysin O (SLO), as well as to surface antigens in the autologous and allogeneic mixed leukocyte reaction (MLR). Fractionation of LGL into subsets according to their reactivity with alpha OKT11, alpha DR, and alpha OKM1 MoAb led to the identification of the subset(s) of LGL with OKT11+, DR+, OKM1+ phenotype as the antigen-presenting cell (APC), whereas the DR-, OKM1- subset(s) of LGL was completely ineffective. Furthermore, virtually all the natural killer (NK) activity of LGL was associated with OKT11+ and OKM1+, DR+ LGL that exerted the observed APC function, suggesting that NK-active cells may also act as effective APC for T lymphocyte activation. These results indicate that human LGL with NK activity may exert other noncytotoxic functions and may play a major role in immunoregulation.     

The effect of IL-2 and IFN-beta on autologous tumor cell kill by Percoll separated LGL fractions

Low density fractions of Percoll density gradient centrifugation of peripheral mononuclear cells contained the majority of large granular lymphocytes (LGL). LGL were used for 5-hr 51Cr release cytotoxic assay against autologous tumor cells in 20 patients with hematological malignancies (9AML, 4ALL and 7NHL). Mean % cytotoxicity (% CTX) was 6.0%, and the addition of IFN-beta and IL-2 in the medium induced the significant increase of % CTX to 15.0% and 26.1%, respectively. When LGL cultured in medium containing IFN-beta and IL-2 were assessed for cytotoxicity daily for 8 days, the enhancement of % CTX by IFN-beta was declined in a few days, while the enhancement by IL-2 was sustained for more than 8 days. The pretreatment of LGL with anti Leu-11 (CD16) plus complement abrogated the enhancing effect by IFN-beta or IL-2, but not with anti Leu-1 (CD5) plus complement. When this treatment was done on day 8 of IL-2 cocultivation, anti Leu-11 plus complement suppressed cytotoxicity significantly, and anti Leu-1 plus complement also induced mild suppression. The phenotypic characteristics of cells revealed the significant increase of anti Leu-19+ cells in IL-2 stimulated day 8 cells. High density fractions of Percoll gradient contained mostly T lymphocytes and showed no cytotoxicity against autologous tumor cells. However, cocultivation with IL-2 for 8 days induced the cytotoxicity, associated with increased number of anti Leu-19+ cells. These results suggested that IL-2 induced cytotoxic activity against autologous tumor cells might be related to the increase of anti Leu-19+ cells.     

Interleukin-2-induced production of interferon-gamma by resting human T cells and large granular lymphocytes: requirement for accessory cell factors, including interleukin-1

Between 5 and 20% of normal human lymphocytes were found to synthesize interferon-gamma (IFN-gamma) in primary cultures with recombinant interleukin-2 (rIL-2). After 22 hr, IFN-gamma-producing cells included CD5+ T lymphocytes, CD16+ large granular lymphocytes (LGL), and a population of CD5-, CD16- blast cells. Only a small proportion (0-7%) of IFN-gamma-synthesizing cells expressed HLA-DR. The production of IFN-gamma by all rIL-2-responding lymphocyte subsets was shown to require the presence of DR+ accessory cells, probably including nonadherent, esterase-negative monocytes and/or dendritic cells. Accessory cell function in lymphocyte preparations depleted of DR+ cells, or in purified (greater than or equal to 95%) suspensions of LGL, was fully replaced either by addition of 2% autologous, adherent monocytes or by monocyte culture supernatant. The activity of monocyte supernatant was greatly reduced by treatment with antiserum specific for human interleukin-1 beta (IL-1 beta), although a combination of rIL-1 beta and rIL-2 failed to stimulate IFN-gamma production in DR- lymphocytes. These results indicate that rIL-2-induced IFN-gamma synthesis in both T cells and LGL requires the synergistic activity of IL-1, and possibly of one or more other monokines, as yet unidentified.     

Use of estimated breeding values in a selection index to breed Yorkshire pigs for high and low immune and innate resistance factors

Abstract

A random bred population of Yorkshire pigs (G_o) was characterized using fourteen various indicators of immune and innate resistance. Based on initial heritability estimates and correlations between these traits, two measures of antibody (serum IgG, and antibody response to HEWL), and cellular activity (blastogenic response to Con A and cutaneous DTH to BCG/PPD), and one indicator of innate monocyte function (uptake and killing of S. typhimurium) were chosen as breeding criteria to be used in a composite selection index. Based on these five traits a combined estimated breeding value (EBV) was calculated for each animal and pigs were assigned to High, Low or Control breeding groups. Approximately 120 first generation piglets (G₁) were then similarly evaluated. Based on G_o plus G₁ heritability estimates were 0.25, 0.23, 0.08, 0.08 and zero for secondary antibody response to HEWL, blastogenic response to Con A, cutaneous DTH to BCG/PPD, serum IgG, and monocyte function, respectively. Least squares means reflected these estimates in that there were significant (p ≤ 0.05) differences between High and Low line G₁ pigs in antibody, blastogenic, and DTH responses. However, there were no significant line differences in serum IgG or uptake and killing of S. typhimurium. Response to selection was determined both by differences in least squares means and differences in average EBV between the High and Low lines. After one generation of selection, lines were separated by 1.508 (least squares) and 1.205 (EBV) index points, or slightly more than half a standard (phenotypic) deviation.     

Terbium as a fluorescent probe for DNA and chromatin.

Terbium reacted with DNA and chromatin to form a complex in which terbium acted as a sensitive fluorescent probe. By measuring the narrow-line emission of Tb-3+ when DNA is selectively excited, the relative amount of Tb-3+ bound to the DNA can be calculated. Terbium was bound to DNA until one Tb-3+ was present for each phosphate group. After this point no more terbium was bound. TbCl3 was bound to chromatin in a linear manner until approximately 0.48 TbCl3 was added for each phosphate group in the chromatin-DNA solution. From these data it appears that 52% of the phosphate groups in chromatin were unavailable for binding. The binding of Tb-3+ to DNA can be reversed by prolonged dialysis against 0.5 M NaCl and chelating agents. The terbium ion is ideal in that it binds DNA tight enough so that completion of the reaction can be assumed but loose enough so that it can be removed by gentle means. Low concentrations of salt (up to 2 mM NaCl) enhance the quantum efficiency. Below pH 3 and above pH 7 the DNA-terbium complex will not form. Between pH 3 and pH 7 the quantum efficiency of the DNA terbium complex increases from either pH to a maximum at pH 5.5 to 5.6. Several biochemical uses for Tb-3+ ion are suggested.     

Immune reactivity in cattle with ocular squamous cell carcinoma after intralesional BCG immunotherapy

Summary Lymphocyte stimulation with Con A and specific immune reactivity to BCG (antibody formation to BCG and DTH reaction to PPD) were determined in BCG-treated, surgically treated and untreated cows with ocular squamous cell carcinoma. In tumor-bearing cows the Con A-induced proliferation of lymphocytes was reduced when compared to healthy controls. This suppression consisted of a reduced blastogenic response to Con A of lymphocytes from tumor-bearing cows, and the presence of a factor in the sera of these animals, as these sera suppressed the blastogenic response of lymphocytes from healthy cows. BCG had only a minor influence on the suppressive activity. Antibodies to BCG were demonstrated in 50% of the BCG-treated animals. The formation of antibodies was not influenced by intradermal injection of PPD of Mycobacterium bovis. Absorption of a BCG antibody containing serum with BOSCC tumor extracts did not reveal the existence of cross reacting antigens between BCG and BOSCC. Pretherapeutic and posttherapeutic Con A reactivity could not be correlated with clinical response. Of the 30 BCG treated cows 29 developed a positive DTH reaction to PPD. Correlation between clinical response and immune reactivity was seen only with regard to the DTH reaction to PPD: this reaction remained positive for a longer period after treatment in animals with a favorable clinical outcome than in nonresponding animals.Animals were maintained under the guidelines laid down by the Faculty of Veterinary Medicine, State University, Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation) Abbreviations used: BCG, Bacillus Calmette-Guerin; BOSCC, bovine ocular squamous cell carcinoma PBL peripheral blood leukocytes; PPD, purified protein derivative of Mycobacteria; DTH, delayed type hypersensitivity Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen     

IL-7 regulation of cytotoxic lymphocytes: pore-forming protein gene expression, interferon-gamma production, and cytotoxicity of human peripheral blood lymphocytes subsets.

The effects of IL-7 on the generation of cytolytic human peripheral blood lymphocytes (PBL) were investigated. Induction of T-cell pore-forming protein (PFP) mRNA and cytotoxic potential by IL-7 was both slow and minor compared with that observed in IL-2-cultured T cells. IL-7 and suboptimal doses of IL-2 (10 U/ml) were found to costimulate PFP mRNA expression and cytotoxic potential in T cells. Clearly, however, both IL-7 and IL-2/IL-7 induced the PFP gene expression and cytotoxic potential of CD8+ T cells and not CD4+ T cells. In addition, neither monoclonal antibodies (mAb) to the p55 or p75 IL-2-receptor subunits had any effect upon IL-7 induction of CD8+ T-cell cytotoxicity, indicating that IL-7 induction of cytotoxic CD8+ T cells was IL-2 independent. IL-7 induction of CD3- large granular lymphocyte (LGL) and PB gamma delta T-cell cytotoxicity was also delayed and reduced compared with that effected by IL-2. IL-7 (10 or 1000 U/ml, 72 hr) enhanced the NK and LAK cytotoxic of LGL and PB gamma delta T cells. By contrast IL-7 or IL-2 augmented the redirected cytotoxic potential of PB gamma delta T cells, but not that of LGL, and neither lymphokine had any effect on constitutive PFP mRNA expression in either lymphocyte subset. In addition, IL-7 induction of LGL IFN-gamma production was weak and delayed compared with that effected by IL-2 and neither IL-2 nor IL-7 stimulated IFN-gamma production in PB gamma delta T cells. Therefore, overall the effects of IL-2 and IL-7 on various cytotoxic human PBL were qualitatively similar, but quantitatively and kinetically different.     

Fractionation, morphological and functional characterization of effector cells responsible for human natural killer activity against cell-line targets

Human natural killer cells cytotoxic against cell-line target cells (NK-CLT) were isolated and characterized by utilizing adsorption-elution of the effector cells from the K-562 target cells. The cell associated with the cytotoxicity was a large lymphocyte with pale and characteristically granular cytoplasm. Thus, its morphology was identical with that of the large granular lymphocyte (LGL) previously shown to be the principal cytotoxic NK cell against fetal fibroblasts (NK-FF). The association of LGL with natural killer activity was verified with contact analysis from mixtures of unfractionated effector cells and target cells, which revealed that the number of contact of LGL with K-562 was correlated to the level of the individually expressed intensity of natural cytotoxicity. The ANAE-staining distribution of LGL was intensively positive with granular or diffuse staining pattern. In direct surface marker analysis LGL were E-rosette forming but, in contrast to NK-FF, heterogenous in regard to the Fc receptors. During in vitro incubation after elution from the target cells, the cytotoxic activity of LGL increased several fold. Also, the presence of K-562 among unfractionated effector cells caused an augmentation of cytotoxicity. This phenomenon was not observed as a result of effector cell-fetal fibroblast coculturing. Evidence from fetal fibroblast adsorption-elution and aggregated IgG blocking experiments suggested that the LGL with strong expression of Fc receptors were initially cytotoxic “mature” NK-cells, whereas the LGL with a weak expression of Fc receptors were initially noncytotoxic, but contact with K-562 “augmented” or “recruited” them to nonselective cytotoxicity.     

Human large granular lymphocytes express high affinity receptors for murine monoclonal antibodies of the IgG3 subclass

We have evaluated the ability of murine monoclonal antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) by human lymphoid cells. Purified large granular lymphocytes (LGL) and interleukin 2-dependent cloned LGL lines having a CD2+/CD16+/CD3- phenotype were tested as effector cells in an ADCC assay by using a family of IgG isotype switch variant anti-Thy-1.1 antibodies against 51Cr-labeled Thy-1.1+ murine SL2 thymoma target cells, a system in which human cells have no spontaneous cytotoxicity. Cytotoxicity was greatest when using the IgG3, followed in rank order by the IgG2a and IgG2b. No cytotoxicity was observed with the IgG1 antibody. Because the antigen-binding regions of the antibodies are identical, the differences in cytotoxicity directly reflect the relative affinity of LGL Fc receptors for each antibody isotype.     

Characterization of human large granular lymphocyte subpopulations: comparison of the phenotype of NK cells and of interleukin 2-dependent progenitors of cytolytic effector cells

Antigenically different subpopulations of human large granular lymphocytes (LGL) were identified according to their reactivity with monoclonal antibodies (MoAb). Antigen-positive and -negative subsets were isolated by immunoaffinity columns using a Sepharose 4B gel coupled with F(a')2 goat anti-mouse IgG or by flow cytometry cell sorting. The distinct LGL subsets were tested for natural killer (NK) activity against a panel of tumor targets: K562, Daudi, Alab; and for antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated RL male 1 cells. LGL positively selected for any of the following phenotypic markers: B73.1+, OKM1+, OKT11+, and OKT10+ were highly cytotoxic, while B73.1- and OKM1- cells were completely devoid of NK activity. The OKT10- and OKT11- LGL subsets were occasionally cytotoxic, with low levels of reactivity. LGL subpopulations were also tested in a limiting dilution assay (LDA) for their capacity to proliferate in medium supplemented with interleukin 2 (IL-2) and to develop NK-like cytotoxic activity. The majority of proliferative progenitors have the following phenotype: OKT11+, OKM1-, B73.1-, and OKT10-, while the majority of progenitors for cytotoxic cells were OKT11+, OKM1+/-, OKT10+, and B73.1-. Results indicate that although B73.1+ cells can grow, the mature B73.1+ NK cells seem to be primarily derived in vitro from a small subset of less differentiated B73.1 pre-NK progenitors in the peripheral blood lymphocytes.     

CD3 negative "small agranular lymphocytes" are natural killer cells

We describe here that CD3-, CD16+ and/or CD56+ small lymphocytes, in a highly reproducible fashion, mediate a significant level of K562 killing that is, on a "per cell" basis, comparable to the cytolytic activity of CD3- LGL. The CD3- small lymphocytes appeared to have no granules based on light and electron microscopy and lack of right-angle scatter on the FACS; we thus refer to them as small "agranular" lymphocytes (SAL). The lytic activity against K562 is inhibited by treatment with either L-leucine methyl ester or EGTA, which are reported to effect granule-dependent killing. We suggest that the SAL have lytic molecules in their cytoplasm (which are sensitive to these treatments) but that these molecules are not organized into discrete granules as found in LGL. The CD3- SAL are phenotypically very similar to LGL and both SAL and LGL mediated equal and reproducible antibody-dependent cell-mediated cytotoxicity. These observations force redefinition of the concept of NK cells to include both CD3- LGL and CD3- SAL.     

Chemotaxis of large granular lymphocytes

The hypothesis that large granular lymphocytes (LGL) are capable of directed locomotion (chemotaxis) was tested. A population of LGL isolated from discontinuous Percoll gradients migrated along concentration gradients of N-formyl-methionyl-leucyl-phenylalanine (f-MLP), casein, and C5a, well known chemoattractants for polymorphonuclear leukocytes and monocytes, as well as interferon-beta and colony-stimulating factor. Interleukin 2, tuftsin, platelet-derived growth factor, and fibronectin were inactive. Migratory responses were greater in Percoll fractions with the highest lytic activity and HNK-1+ cells. The chemotactic response to f-MLP, casein, and C5a was always greater when the chemoattractant was present in greater concentration in the lower compartment of the Boyden chamber. Optimum chemotaxis was observed after a 1 hr incubation that made use of 12 micron nitrocellulose filters. LGL exhibited a high degree of nondirected locomotion when allowed to migrate for longer periods (greater than 2 hr), and when cultured in vitro for 24 to 72 hr in the presence or absence of IL 2 containing phytohemagluttinin-conditioned medium. The chemotactic LGL was HNK-1+, OKT11+ or HNK-1+, OKT11- on the basis of monoclonal antibody and complement depletion. They did not bear either T cell or monocyte cell surface markers, exhibiting an OKT3-, OKT4-, OKT8-, OKM1-, and MO2- phenotype, and did not form E rosettes at 29 degrees C, which is characteristic of lytic NK cells in contrast to T cells. Furthermore, a rat LGL leukemia (RNK) exhibited a chemotactic response to both f-MLP and casein. LGL chemotaxis to f-MLP could be inhibited in a dose-dependent manner by the inactive structural analog CBZ-phe-met, and the RNK tumor line specifically bound f-ML[3H]P, suggesting that LGL bear receptors for the chemotactic peptide.     

Modulation of CD3- large granular lymphocyte functions by agonist and antagonists of protein kinase C: effects on NK and lymphokine-activated killer activity and production of IFN-gamma

The biochemical mechanisms involved in the activation and killing of tumor targets by large granular lymphocytes (LGL) have not yet been clearly defined. This laboratory has investigated these processes by analyzing the effects of protein kinase C (PKC) inhibitors (1-(5-isoquinolinesulfonyl)2-methyl-piperazine-dihydrochloride and retinol) on LGL cytotoxicity and IFN-gamma production. We now report that PKC inhibitors block the LGL functions of 1) NK activity, 2) IFN-gamma production, and 3) LAK activity induced by IL-2. Complete inhibition of cytotoxic activity occurs rapidly because only 2.5 h treatment of the LGL with the inhibitors was required. However, the inhibition of NK activity by the PKC inhibitors could be reversed by IL-2 or the synthetic diacylglycerol, L-gamma-1-oleyl-2-acetol-sn-3-glycerol (OAG), but not by IFN-alpha. The reversal of inhibition observed with OAG indicates that, in these studies, (1-(5-isoquinolinesulfonyl)2-methyl-piperazine-dihydrochloride is inhibiting PKC activity and not the activity of other cellular kinases. Furthermore, inhibition of LGL functional activity with PGE2 could not be reversed with OAG, supporting the contention that PG inhibition of NK activity is mediated by a pathway that does not directly involve PKC. These results indicate, in addition to IL-2-mediated events, that basal NK activity is under PKC regulatory control.     

Comparison of the effect of IL-2 and IL-6 on the lytic activity of purified human peripheral blood large granular lymphocytes

The effects of IL-6 and IL-2 on highly purified, human peripheral blood large granular lymphocytes (LGL) were investigated and compared. IL-6 enhanced LGL NK activity in a dose-dependent manner against K562, however IL-2 was a more potent stimulus of LGL NK function. Neither IL-2 nor IL-6 increased LGL cytotoxic potential in a parallel estimation of heteroconjugated antibody (anti-CD16 x anti-nitrophenyl mAb)-dependent cytotoxicity against nitrophenyl-modified YAC. Unlike IL-2, IL-6 did not significantly induce LGL lymphokine-activated killer activity, LGL proliferation, or LGL lymphokine production. In particular, IL-6 did not stimulate detectable LGL IL-2 production or IL-2R modulation, and mAb to the p75 IL-2R had no effect on IL-6 induction of LGL NK activity. Therefore, in the absence of T cells, IL-6 provided an IL-2-independent signal to LGL that resulted in augmentation of their NK activity without stimulating their proliferation or other LGL functions.     

Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua

Large granular lymphocytes (LGL) are the most abundant cell type in first trimester human pregnancy decidua. We have shown previously that CD56-positive decidual LGL have cytotoxic activity against the natural killer (NK) target K562, and that this cytotoxicity is augmented by pretreatment with interleukin-2 (IL-2). We now report that flow cytometrically purified populations of CD56-positive decidual LGL have no cytotoxic activity against either the BeWo choriocarcinoma cell line or freshly isolated term trophoblast. Incubation of unfractionated decidual cells with IL-2 induced cytotoxicity against BeWo, but term trophoblast remained resistant to lysis. Both BeWo and trophoblast showed much lower binding frequencies to decidual or peripheral blood cells than K56 targets, and excess trophoblast did not inhibit cytotoxic activity against K562. This suggests that the resistance of trophoblast to lysis by either decidual or peripheral blood LGL is due to the lack of accessible NK target structures on the surface of trophoblast.     

Effects of terbium on the cytotoxicity of cisplatin in FaDu human head and neck squamous cell carcinoma

In this investigation, we report a relationship between the terbium (Tb3+) binding protein and the cytotoxicity of cisplatin in human head and neck cancer cells. In the FaDu cell line, the cytotoxic action of cisplatin was shown to be approximately six times more potent than the cytotoxicity of Tb3+. When cisplatin was combined with 80 microM Tb3+, the IC20 and IC50 values for cisplatin were reduced by 70% and 24%, respectively. The IC80 value, however, was increased by 124%. The results suggest that the cytotoxicity of cisplatin is enhanced by Tb3+ at low cisplatin concentrations. In agreement with previous studies, calcium and cisplatin were found to be mixed-type and noncompetitive inhibitors, respectively, of the Tb3+ -FaDu intensity. These findings imply that the receptor binding of Tb3+ can modulate the cytotoxic activity of cisplatin.     

Lysis of fresh human tumour cells by autologous tumour-associated lymphocytes: Two distinct types of autologous tumour killer cells induced by co-culture with autologous tumour

Summary The specific and natural killer (NK)-restricted nature of auto-tumour cytotoxicity of tumour-associated lymphocytes was studied in cancer patients with malignant pleural effusions. Large granular lymphocytes (LGL) and small T lymphocytes were isolated from carcinomatous pleural effusions by centrifugation on discontinuous Percoll gradients. Tumour cells freshly isolated from pleural effusions were classified according to their susceptibility to lysis by Percoll-purified LGL from the blood of normal donors in a 4-h ⁵¹Cr release assay. Of 12 NK-sensitive tumour samples, 11 were killed by autologous fresh effusion LGL, whereas only 2 were lysed by autologous T cells. Neither LGL nor T cells were cytotoxic to NK-resistant autologous tumour cells. T cells and LGL were each cultured in vitro with autologous tumour cells for 6 days. Effusion LGL maintained their auto-tumour killing activity in 10 of 12 autologous mixed lymphocyte-tumour cultures (MLTC) with NK-sensitive tumour, while LGL lost the activity when cultured alone. Removal of high-affinity sheep erythrocyte-rosetting cells from Percoll-purified LGL enriched effector cells. Autologous MLTC-derived LGL could also kill NK-sensitive allogeneic effusion tumour cells and K562 cells, as did fresh LGL. In autologous MLTC LGL failed to acquire lytic function to NK-resistant autologous tumour cells. In contrast, in vitro activation of effusion T cells with autologous tumour cells induced auto-tumour killer cells in 9 of 12 NK-sensitive tumour samples and 3 of 6 NK-resistant tumour cases. However, cultured T cells were incapable of killing allogeneic tumour cells and K562 cells. In the autologous MLTC effusion T cells proliferated vigorously in response to autologous tumour cells, whereas LGL showed no proliferation. The enrichment of blasts from cultured T cells on discontinuous Percoll gradients resulted in an enhancement of auto-tumour cytotoxicity, with no reactions recorded in blast-depleted, small, resting T cells. These results indicate that two distinct types of auto-tumour-recognising lymphocytes, LGL and T cells, are present in carcinomatous pleural effusions of cancer patients and that each effector type recognises different membrane moieties of autologous effusion tumour cells.