Cytotoxicity in patients with different clinical forms of Chagas' disease

There are few studies on cell-mediated cytotoxicity in human Chagas' disease. In the present study, we evaluated peripheral blood mononuclear cell (PBMC) cytotoxicity activity from chagasic patients with different clinical forms of disease. To verify the cytotoxic response, we performed cell lysis assays using ⁵¹Cr-labelled K562 cells as targets. Results are reported as lytic units (LU=number of cells required for 30% lysis) per million PBMC. Exposure of patients’ PBMC to Trypanosoma cruzi antigen led to an increase in cytotoxic activity compared with unstimulated patient cells against K562. Asymptomatic cardiomyopathy patients had higher responses (37.8±5.0 LU/10⁶ PBMC; mean±s.d.) than indeterminate (11.5±3.6 LU/10⁶) and symptomatic cardiomyopathy (7.8±2.5 LU/10⁶). Normal control PBMC stimulated with T. cruzi antigen had 4.36±1.31 LU/10⁶ PBMC against K562. Addition of recombinant interferon-gamma (IFN-γ) did not lead to significant increase in cytotoxicity in any group of patients. On the other hand, recombinant human IL-12 significantly increased cytotoxic responses from symptomatic cardiomyopathy patients and normal controls who presented low levels of cytotoxicity induced by T. cruzi antigen. The combined use of IL-12 and a neutralizing anti-IFN-γ antibody did not change IL-12-induced cytotoxic responses, showing the direct role of this cytokine on natural killer (NK) cells. NK cells were the main cells responsible for the lysis of K562 target cells as evidenced by testing cell subsets following magnetic cell sorting. These data demonstrate that chagasic patients with different clinical forms of disease have PBMC which respond to T. cruzi antigen with a cytotoxic response, and this response is up-regulated by IL-12.     

Cytotoxic activity of V beta 8+ T cells in Crohn's disease: the role of bacterial superantigens.

In Crohn's disease, disease-related stimuli could alter the T cell receptor (TCR) repertoire. To examine the possibility that changes in function may occur in T cell subsets without obvious changes in expression of TCR, we analysed the TCR repertoire of cytotoxic T lymphocytes in Crohn's disease peripheral blood. Furthermore, we examined the effect of bacterial superantigens, staphylococcal enterotoxin B (SEB) and E (SEE) on the cytotoxic function of T cell subsets bearing different TCR V genes using MoAbs specific for CD3 and TCR V gene products in a redirected cytotoxicity assay. There was no difference between patients and controls in the cytotoxicity measured in concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) with anti-CD3 or with six of seven anti-TCR V gene MoAbs. However, the cytotoxicity of V beta 8 T cells was decreased in Crohn's disease patients. This was not due to a decrease in total or CD8+ T cells expressing V beta 8. Furthermore, in normal subjects, PBMC stimulation with SEE and SEB selectively expanded and increased the cytotoxicity of V beta 8 and V beta 12 T cells, respectively. In Crohn's disease, although SEB stimulation increased the number and cytolytic function of the V beta 12 subset, SEE stimulation failed to increase cytolytic activity of V beta 8+ T cells in spite of the expansion of V beta 8+ T cells. These results suggest that the changes in cytotoxic function observed in V beta 8 T cells in Crohn's patients may reflect previous exposure to a V beta 8-selective superantigen.     

Human recombinant IL-4 suppresses the induction of human IL-2 induced lymphokine activated killer (LAK) activity.

The effect of recombinant human interleukin 4 (rhIL-4) on the induction in vitro of human lymphokine activated killer cell (LAK) activity was investigated. Peripheral blood mononuclear cells (PBMC) from normal healthy donors were incubated for 4 days with or without recombinant human interleukin-2 (rhIL-2) in the presence or absence of rhIL-4. LAK activity was measured against the NK-resistant colon adenocarcinoma cell line SW742, and NK mediated cytotoxicity was determined using NK sensitive K562 cells. Unlike previous reports using mouse effector cells, rhIL-4 neither induced LAK activity nor augmented the cytotoxic response induced by rhIL-2. In four out of six experiments there was a significant reduction of rhIL-2 induced LAK in the presence of rhIL-4, accompanied by a reduction of Tac antigen expression by rhIL-2 activated cells. Recombinant hIL-4 failed to influence the effector phase of the activated PBMC against SW742 or K562 targets.     

Endothelial cell lysis induced by lymphokine-activated human peripheral blood mononuclear cells

In vitro exposure of human peripheral blood mononuclear cells (PBMC) to interleukin 2 results in the generation of lymphokine-activated killer (LAK) cells. Such LAK cells exhibit cytotoxicity against a spectrum of tumor target cell lines whereas they apparently do not affect normal tissues. In this report we show that PBMC that have been activated with T cell growth factor lyse trypsinized human umbilical cord venous endothelial cells as well as endothelial cell monolayers in a dose-dependent manner. Microscopic analysis showed that during the 4-h incubation period cell clumps containing detached endothelial cells and LAK cells were formed. When these clumps were evaluated with trypan blue the endothelial cells stained positive whereas LAK cells excluded the dye. No lysis occurred when fresh PBMC were added to target endothelial cells. The endothelial cell kill could not be blocked with an anti-LFA-1 antibody nor with intact OKT3 or F(ab')2 fragments of WT32. We conclude that lymphokine-activated PBMC exhibit cell-mediated endothelial cell detachment and lysis.     

Reduced LAK cytotoxicity of peripheral blood mononuclear cells in patients with bladder cancer: Decreased LAK cytotoxicity caused by a low incidence of CD56+ and CD57+ mononuclear blood cells

The cytotoxicity of unstimulated peripheral blood mononuclear cells (US-PBMC), phytohemagglutinin (PHA)-stimulated PBMC (PS-PBMC) and interleukin-2 (IL-2)-activated PBMC (LAK cells) was assessed in patients with noninvasive and invasive transitional-cell bladder cancer and compared with those determined in healthy controls. The differences in the cytotoxicities were correlated with specific changes in the subsets of peripheral blood mononuclear cells (PBMC). PBMC from 37 patients and 13 healthy controls were tested against the bladder cancer cell line T24 in⁵¹Cr-release assays. The PBMC subsets were analyzed using monoclonal antibodies against T cells, natural killer (NK) -cells, monocytes, and activation markers. The cytotoxicities of US-PBMC, PS-PBMC, and LAK cells were all significantly lower in the cancer patients than in the controls (P<0.05). The percentages of PBMC positive for the NK-cell markers CD56 and CD57 were lowest in the patients and were correlated to the decrease in cytotoxicity. Depletion of CD56⁺ or CD57⁺ cells from PBMC prior to or after 2 days stimulation with IL-2 demonstrated that these cells are the major source of LAK-cell cytotoxicity and showed that the reduced ability of bladder cancer patient PBMC to develop LAK-cell cytotoxicity is a result of a low incidence of CD56⁺ and CD57⁺ cells in the blood. These findings indicate that IL-2 therapy alone might not be a sufficient therapy of bladder cancer patients.     

Peripheral blood leukocytes of patients with Argentine hemorrhagic fever as effectors of antibody-dependent cell-cytotoxicity.

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMNL) of patients with Argentine hemorrhagic fever (AHF) were tested as effectors (E) of antibody-dependent cell cytotoxicity (ADCC). 51Cr labeled chicken red blood cells (CRBC) coated with anti-CRBC or normal rabbit serum were used as targets (T). Three ADCC assays were performed with both effectors from patients: on admission (I), 4 days after the transfusion of immune plasma (II), and 30 days after the clinical onset (III). The ADCC values obtained displayed high variation between individuals. From the linear portions in the curves representing specific 51Cr release vs. E:T ratio plots, extrapolations were made to determine lytic units (LU), defined here as effector concentrations required to lyse 50% of the targets. The results were expressed as LU in 10(6) effector cells. The killing activity ranges of patients' PMNL (I = 1.04 +/- 0.34; II = 2.22 +/- 0.66; and III = 2.08 +/- 1.18) were not significantly different from that of 21 normal controls (1.19 +/- 0.36), except for range II (P less than 0.01). ADCC activity ranges of patients' PBMC (I = 3.40 +/- 1.06; II = 3.16 +/- 1.60; and III = 1.93 +/- 0.42) were not significantly different from that determined in 12 healthy subjects (1.86 +/- 0.40). These results demonstrate that patients' PBMC and PMNL can perform ADCC with efficiency comparable to normal effector cells, during the acute period of AHF, and in early convalescence. Consequently, ADCC can be a relevant mechanism in the clearance of Junin virus-infected cells.     

LAK-cell-mediated cytotoxicity against tumor cell targets used to monitor the stimulatory effect of interleukin-2: cytotoxicity, target recognition and phenotype of effector cells lysing the Daudi, T24 and K562 tumor cell lines.

The T24 transitional bladder carcinoma cell line, the Daudi Burkitt lymphoma cell line and the K562 erythroleukemia cell line have all been used as target cells in 51Cr release assays to measure the in vivo induced lymphokine-activated killer (LAK) cell cytotoxicity during interleukin-2 (IL-2) therapy of cancer patients. However, different relationships between the clinical response to IL-2 treatment and the LAK cytotoxicity have been reported using these three different target cells. The purpose of the present study was to evaluate whether the LAK cytotoxicities measured against these target cells represent similar effector-to-target-cell interactions, so similar conclusions may be drawn of 51Cr release assay results in which the cell lines are used as target cells. The cytotoxicity of peripheral blood mononuclear cells (PBMC) and PBMC depleted of different natural killer and T cell subsets was measured against the three targets. LAK cell recognition of targets was evaluated by cold target inhibition experiments, and the development of LAK-cell-mediated lysis with time was evaluated in 51Cr release assays of varying duration. This study shows that LAK-mediated lysis of T24 and Daudi cells was closely related and LAK cytotoxicity measured in 51Cr release assays against these two target cells may be measurement of similar effector-to-target cell interactions.     

Interferon-alpha (IFN-alpha) enhances cytotoxicity in healthy volunteers and chronic hepatitis C infection mainly by the perforin pathway.

Cell-mediated cytotoxicity is exerted via perforin and Fas ligand (FasL). We have recently shown that IFN-alpha up-regulates FasL expression in T cells isolated from healthy volunteers and augments activation-induced T cell death. Since the Fas/FasL system is implicated in the pathogenesis of hepatic failure and both molecules have been shown to be up-regulated in hepatitis C virus (HCV) infection, we studied whether cytotoxicity via the FasL system is enhanced by IFN-alpha and therefore could contribute to hepatic injury. We investigated FasL and perforin expression in peripheral blood mononuclear cells (PBMC) derived from HCV+ donors by Northern analysis and soluble FasL synthesis by ELISA. Natural killer (NK) cell and cytotoxic T lymphocyte (CTL) cytotoxicity was studied by 51Cr-release assays. IFN-alpha up-regulates FasL mRNA and protein synthesis in mitogen-activated PBMC of HCV+ individuals, as previously found in healthy subjects. Stimulation with IFN-alpha increases perforin mRNA levels in PBMC. In NK cytotoxicity assays, the enhancement of cytotoxicity by IFN-alpha is mainly due to the perforin pathway, while the FasL pathway plays only a minor role. In CTL cytotoxicity experiments neither the FasL nor the perforin pathway is further enhanced by IFN-alpha. Our data suggest that up-regulation of perforin by IFN-alpha results in elevated cytotoxicity, suggesting that IFN-alpha might support elimination of virally infected cells via this pathway. In contrast, the major effect of IFN-alpha on the Fas/FasL system might be the enhanced elimination of activated T cells as a means of finally limiting a T cell response.     

Relation between the increase of circulating CD3+ CD57+ lymphocytes and T cell dysfunction in recipients of bone marrow transplantation.

Some patients undergoing bone marrow transplantation (BMT) persistently present increased proportions of circulating CD57+ T cells. We analysed the cell surface phenotype in peripheral blood mononuclear cells (PBMC) from 69 allogeneic and 11 autologous BMT recipients. In parallel samples from 49 patients, the proliferative response to T cell mitogens was assessed, either in the presence or absence of exogenous interleukin-2 (IL-2). PBMC samples from long-term allogeneic BMT patients with increased proportions of CD57+ cells displayed significantly (P less than 0.001) lower proliferative responses, compared with samples from patients with normal proportions of CD57+ cells and from healthy subjects. Elimination of the CD57+ population by C'-dependent lysis did not normalize the proliferative response. After positive selection by cell sorting, CD57+ cells responded poorly, but in the presence of IL-2 the proliferation appeared to be similar to that displayed by the CD57- subset and still suboptimal compared with normal controls. These data suggest that the hyporesponsiveness to mitogenic stimuli in the presence of exogenous IL-2 of PBMC from allogeneic BMT recipients cannot be simply attributed either to the putative suppressor activity of CD57+ cells, or to a poor proliferative capacity of this subset. Supporting this notion we report that PBMC from long-term autologous BMT recipients containing high proportions of CD57+ T cells respond normally to T cell mitogens.     

Cytotoxicity against human peripheral blood mononuclear cells and T cell lines mediated by anti-T cell immunotoxins in the absence of added potentiator.

Several in vitro assays have indicated that anti-T cell immunotoxins (IT), composed of monoclonal antibodies (MoAbs) conjugated to ricin A chain (RTA), are maximally effective against T cells only in the presence of potentiators. It was thought that such IT might not be sufficiently cytotoxic to deplete T cells in vivo upon administration to patients. Therefore, we have re-evaluated the in vitro assays and report herein that even with a short exposure time (2 h), the two anti-T cell IT, H65-RTA (anti-CD5 MoAb coupled to RTA) and 4MRTA (anti-CD7 MoAb coupled to RTA30), were specifically cytotoxic for peripheral blood mononuclear cells (PBMC) in the absence of potentiators. Moreover, as has been reported for IT when tested against T cell lines, prolonging the exposure time of the IT with PBMC from 2 h to as long as 90 h, without added potentiators, enhanced their cytotoxicity from 2- to 40-fold. In contrast, most T cell lines were more sensitive to IT in the presence of potentiator, and IT cytotoxicity was much less enhanced by prolonging the exposure time. Thus, T cell lines may not serve as accurate models to determine the efficacy of IT against PBMC in vitro or in vivo. We conclude that IT-induced cytotoxicity of PBMC can be demonstrated in vitro at pharmacologically achievable concentrations in the absence of added potentiators.     

Hormonal and immunological regulation of 2', 5'-oligoadenylate synthetase activity in human peripheral blood mononuclear cells.

A newly developed method for assaying 2', 5'-oligoadenylate (2, 5A) synthetase activity by polyacrylamide gel electrophoresis was applied to peripheral blood mononuclear cells (PBMC) from normal subjects, HIV-positive subjects, and renal cell carcinoma (RCC) patients. Sex differences were observed in 2, 5A synthetase activity of PBMC from normal young adults, males having eightfold higher activities of this enzyme than females. Moreover, compared to values for postmenopausal (PM) females receiving estrogen replacement, untreated PM females had higher activities. Collectively, these results suggest that estrogen downregulates 2, 5A synthetase activity. Activities of 2, 5A synthetase were investigated in two disease states associated with altered immune function. In one patient with AIDS-related Kaposi's sarcoma, interferon-alpha (IFN-alpha) therapy increased 2, 5A synthetase activity twofold. In addition, combined therapy with interleukin-2 (IL-2) and IFN-alpha increased 2, 5A synthetase activities in eight of nine patients with RCC. Therefore, in patients receiving immunotherapy with IL-2 and IFN-alpha, our new assay could contribute to evaluation of immune stimulation. In general, studies in vitro confirmed these observations; however, exposure of PBMC from RCC patients revealed that in vitro IL-2 failed to induce this enzyme activity as it did in PBMC from normal volunteers.     

Cytotoxic CD4+ T cells specific for Francisella tularensis.

The specific cell-mediated cytotoxicity of tularaemia-immune human T lymphocytes were studied in vitro. Peripheral blood mononuclear cells (PBMC) of six tularaemia-vaccinated healthy subjects were stimulated with F. tularensis LVS whole cell antigen for 6 days and used as effector cells in a conventional 4-h 51Cr release cytotoxicity assay. The target cells were phagocyting autologous monocytes, which were pulsed with F. tularensis or PPD antigen. The specific lysis of the F. tularensis-pulsed cells (42.6% +/- 11.7) was significantly higher (P less than 0.05) than that of the PPD-pulsed ones (22.2% +/- 8.3) or unpulsed control cells (15.9% +/- 5.2). The cytotoxicity was associated with CD4+ F. tularensis-specific T cell clones (TLC), which killed 36.3% +/- 12.3 of the F. tularensis-pulsed targets but only 6.9% +/- 6.5 of the unpulsed control targets. Their lysing was inhibited by monoclonal anti-HLA-DR and anti-HLA-DQ antibodies, but not by CD15 (monocyte/macrophage) antibody. The functional role of CD4+ lymphocytes in tularaemia immunity is discussed.     

Killing of human leukaemia/lymphoma B cells by activated cytotoxic T lymphocytes in the presence of a bispecific monoclonal antibody (alpha CD3/alpha CD19).

Bispecific antibodies (BsAb) can be used to retarget T cells irrespective of their specificity to certain target cells inducing target cell lysis. We have tested the efficacy of the BsAb SHR-1, directed against the T cell antigen CD3 and the B cell antigen CD19 to induce (malignant) B cell kill by T cells as measured in a 51Cr-release assay. Two cytotoxic T cell clones (CTL), expressing TCR alpha beta or TCR gamma delta, were effective in killing CD19 expressing B cell lines at different stages of differentiation in the presence, but not in the absence, of the BsAb. CD19- target cells were not killed. Fresh CD19+ leukaemia/lymphoma cells were also efficiently killed by SHR-1 preincubated CTL clones. In addition, phytohaemagglutinin (PHA) or CD3-activated IL-2 expanded peripheral blood mononuclear cells (PBMC) of normal donors did so after 2 weeks of stimulation. A concentration of 100 ng/ml of the BsAb was sufficient to obtain optimal lysis of all target cells tested. These results show that fresh human leukaemia/lymphoma cells, freshly derived from active lymphoblastic leukaemia (ALL) as well as non-Hodgkin's lymphoma (NHL) patients, can be effectively killed in the presence of this BsAb by activated T cells.     

Patients with X-linked lymphoproliferative disease have a defect in 2B4 receptor-mediated NK cell cytotoxicity

Patients with the X-linked lymphoproliferative disorder (XLPD) are unable to control Epstein-Barr virus (EBV)-induced infections and lymphoproliferation. This disease is caused by a deficit of SAP, an adapter protein involved in the signal transduction of several cell surface receptors of the CD2 superfamily. One of these receptors, called 2B4, is expressed on NK cells, cytotoxic T cells and myeloid cells and activates NK cell cytotoxicity. Here we show that XLPD patients have a defect of 2B4 receptor-mediated NK cell cytotoxicity. This defect may contribute to the pathogenesis of XLPD by reducing NK cell lysis of EBV-infected B cells.     

Natural cytotoxic cell activity in the snake Psammophis sibilans.

Thymocytes, splenocytes and peripheral blood mononuclear cells (PBMC) of the snake Psammophis sibilans consistently killed the human erythroleukemic cells K562 in a 4 h assay as judged by lactate dehydrogenase enzyme release. PBMC and splenocyte natural cytotoxicity (NC) increased proportionally with increase in the effector/target cell ratio. Spontaneous killer cell activity was consistently 2-3 times higher in peripheral blood (PB) than in spleen. On the other hand, thymocytes displayed low, yet detectable, NC. In an attempt to define the cell subpopulation responsible for natural killer (NK) activity, PBMC were depleted of macrophages or B lymphocytes before use in NK cell assays against K562 cells. Depletion of macrophages did not impair NK activity thus suggesting that macrophages do not mediate spontaneous lysis in the present 4 h assay. Conversely, removal of B lymphocytes by panning onto dishes coated with monoclonal antibody against snake Ig significantly reduced, but did not eliminate, PBMC spontaneous cytotoxicity. These data suggest that T, B and perhaps distinct NK cells participate in spontaneous lysis. This suggestion was confirmed by studies of NC in thymus, spleen and PB the year round. Strong NC was detected during spring and autumn when high numbers of leukocytes including T and B cells can be recovered from spleen and PB. Negligible spontaneous cytotoxicity was observed during early and mid-summer and in winter, periods of the year when snakes are thymus-less and contain few T and B cells in peripheral lymphoid organs. These findings, the first to document natural cytotoxic activity in snakes, were discussed in relation to the issue of NK cell identity in vertebrates.     

Tyrosine phosphorylation in peripheral T cells of kidney transplant recipients: analyses of baseline levels and response to T cell receptor stimulation.

Impaired immunosurveillance in recipients of organ transplants has been attributed to alleviation of T cell functions. We analyzed the phosphorylation of tyrosine residues in T cells of peripheral blood, and after T cell receptor (TCR) stimulation. The TCR was stimulated by OKT3 monoclonal antibody (mAb) in non-separated heparinized blood specimens of patients (n=64) and healthy controls (n=25). After fixation and red cell lysis, lymphocytes were permeabilized by saponin. Subsequently, intracellular phosphotyrosine residues and surface CD3 antigen were stained simultaneously with specific mAbs. We analyzed transplant recipients and healthy donors for baseline levels of total cellular tyrosine phosphorylation and for increase in phosphotyrosine content following stimulation by OKT3. Phosphotyrosine levels were significantly lower in non-stimulated T cells of kidney transplant recipients compared to controls (p=0.004). There was a marked variability in the levels of tyrosine phosphorylation among transplanted patients (p=0.02). T cell receptor stimulation by OKT3 mAb in vitro led to a strong increase of tyrosine phosphorylation in all specimens of patients and healthy controls. In conclusion, we demonstrated decreased phosphotyrosine levels in T cells of kidney transplant recipients compared to healthy donors. However, increase in tyrosine phosphorylation was not impaired in all patients as a result of TCR stimulation.     

Activation of human T cells with NK cell markers by staphylococcal enterotoxin A via IL-12 but not via IL-18

We have reported recently that mouse liver NK cells and NK1 x 1+ T cells were activated by bacterial superantigens via the IL-12 production from Kupffer cells. In the present study, we examined the effect of staphyloccoccal enterotoxin A (SEA) on human T cells with NK cell markers, CD56 or CD57 (NK-type T cells). After stimulating peripheral blood mononuclear cells (PBMC) with SEA, PBMC produced a large amount of IFN- and acquired a potent antitumour cytotoxicity. The in vitro depletion of either CD56+ TCR NK cells, CD56+ T cells or 57+ T cells from PBMC significantly inhibited the IFN- production from PBMC. When purified NK-type T cells, NK cells and regular T cells were cultured with monocytes and SEA they all produced IFN-, while the IFN- amounts produced by both NK-type T cells were greater than those produced by NK cells. NK cells as well as CD56+ T cells showed cytotoxicity against NK-sensitive K562 cells, whereas both NK-type T cells showed a more potent cytotoxicity against NK-resistant Raji cells than did NK cells. The IFN- production from each population as well as from whole PBMC was greatly inhibited by anti-IL-12 antibody but not by anti-IL-18 antibody. The antitumour cytotoxicity of whole PBMC was also significantly inhibited by anti-IL-12 antibody while the SEA-induced proliferation of PBMC was not affected by anti-IL-12 antibody. Furthermore, SEA-activated NK-type T cells as well as NK cells showed cytotoxicities against vascular endothelial cells. Our findings suggest that human NK-type T cells are thus involved in bacterial superantigen-induced immune response.     

Comparison of europium and chromium release assays: cytotoxicity in healthy individuals and patients with cervical carcinoma.

Natural killer (NK) and lymphokine-activated killer (LAK) cell activities were measured in peripheral blood obtained from healthy women to compare a standard 51Cr release assay with a nonradioactive europium (Eu3+) release assay based on time-resolved fluorescence. The two types of cytotoxicity assays were first compared in paired determinations performed on 28 samples of peripheral blood mononuclear cells obtained from healthy women who had normal pap smears or no biopsy evidence of cervical squamous intraepithelial lesions (SIL). Target cells (NK-sensitive K562 and NK-resistant Raji cell lines) were labeled with Eu3+ only, 51Cr only, or both labels and compared in cytotoxicity assays using fresh or interleukin 2 (IL-2)-activated effector cells. Spontaneous release in the Eu3+ release assay was comparable to that observed in the 51Cr release assay, but maximum Eu3+ release always exceeded that of 51Cr. In 4-h assays, specific release of Eu3+ from target cells was more rapid than that of 51Cr, consistently resulting in 30 to 40% higher levels of activity. However, a significant linear correlation (P < 0.001) was observed between cytotoxicity levels based on measurements of Eu3+ and 51Cr release in 4-h assays. The Eu3+ release assay was then used to measure NK and LAK activities in the peripheral blood of women with cervical SIL or cervical squamous cell carcinoma (SCC). Mean NK activity of women with advanced SIL (121 lytic units [LU]) or SCC (93 LU) was found to be similar to that of controls (101 LU) or patients with normal cervical biopsies (90 LU), as was the ability to generate IL-2-stimulated NK activity. However, LAK activity during 18 h of incubation in the presence of IL-2 was reduced in patients with cervical SCC (P < 0.05) compared with that in normal controls. Results of 51Cr assays performed in parallel with patient samples gave comparable results. Advantages of EU3+ release assays for routine evaluation of cytotoxicity are discussed.