Autologous Cytotoxicity Research Articles

The sensitivity of placental trophoblast cells to intraplacental and allogeneic cytotoxic lymphocytes.

The NK-susceptibility of trophoblast cells to allogeneic and autologous intraplacental natural killer (NK), antibody-dependent (K), and mitogen-induced cell-mediated cytotoxicity was studied, using untreated and neuraminidase-treated trophoblast cells from normal, full-term deliveries. The work was preceded by systematic studies of placental cell separation and labelling techniques, and the effects of these techniques on the NK target, K562. The results indicated that maternal NK cells are present among intraplacental lymphocytes, but that their activity is lower than that of peripheral blood lymphocytes and they are not stimulated by interferon to the same extent as peripheral blood lymphocytes (PBL). Trophoblast cells were rarely susceptible to allogeneic NK cells, with low cytotoxicity at high effector-target cell ratios in only two of five experiments. Interferon (IF)-boosted NK cells mediated some cytolysis of trophoblasts in three of four experiments, but high effector/target cell ratios were also required for the effect to be observed. The trophoblast cells could be lysed, however, by K cells and lectin-induced cytotoxicity. Removal of surface sialic acid by neuraminidase treatment of the trophoblast cells had little effect on the susceptibility of these cells to unstimulated NK cells (one of four experiments), but resulted in susceptibility to IF-boosted NK cells in four of four experiments. Normal trophoblast cells did not compete in IF-NK(K562) assays and neuraminidase-treated cells competed weakly in only one of three such experiments, indicating that the NK "target structure" is only weakly expressed on human trophoblast cells. Intraplacental lymphocytes lysed autologous trophoblast cells to a lower extent than allogeneic PBL.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the growth of Epstein-Barr virus-infected B cells. I. Growth regression by E rosetting cells from VCA-positive donors is a combined effect of autologous mixed leukocyte reaction and activation of T8+ memory cells.

Regression of B cell proliferation in co-cultures of EBV-infected B cells (BEBV) and autologous T cells at 1:4 ratio was studied. 3H-TdR incorporation was used to measure proliferation by the participating lymphocyte populations and a 51Cr release assay was used to document the generation of cells capable of killing autologous EBV-transformed B lymphoblastoid cell lines (LCLEBV). EBV-infected B cells cultured alone transformed to blasts by culture day 10, and continued to proliferate throughout the 22 day observation period. When EBV-infected B cells were co-cultured with E rosetted cells from VCA-positive donors, there was a characteristic proliferative response on day 10 (an augmented autologous mixed lymphocyte reaction; AMLR), followed by the development of T8+ cells capable of killing autologous LCLEBV, as well as over 90% suppression of EBV growth by day 22 as assessed by 3H-TdR incorporation, and confirmed in a visual outgrowth assay. Negative and positive selection techniques were used to define the regulatory components in the T cell population. Depletion of T8+ cells from the blood lymphocytes of VCA-positive donors did not significantly reduce the 10 day proliferative response, but the subsequent development of cytotoxic cells and the regression of BEBV outgrowth was not observed. Thus, the circulating T8+ cells are required for the subsequent appearance of autologous LCLEBV cytotoxicity and BEBV growth regulation. However, when the responder population consisted only of T8+ cells, the augmented AMLR response was absent, cytotoxic cell development was weak or absent, and there was no regression of EBV outgrowth. Therefore, the cells participating in the AMLR, as well as T8+ memory cells from VCA-positive donors, are necessary for the control of the in vitro EBV infection. Growth regression is dependent on the proliferation of the regulatory T cells. Mitomycin C treatment of fresh E rosetting cells or those exposed to BEBV for up to 10 days in culture abrogates growth regression and the subsequent appearance of LCLEBV killer cells. However, E rosetting cells exposed to BEBV for 14 days or more already have developed the ability to kill LCLEBV and no longer need to proliferate to induce growth regression when cultured with newly infected BEBV. These results lend additional support to the view that the control of EBV-induced B cell expansion requires a AMLR-dependent clonal amplification of EBV-specific, T8+ cytotoxic cells.

HLA-DR-antigen-associated restriction of EBV-specific cytotoxic T-cell colonies

EBV-specific cytotoxic T cells can be generated in vitro in a secondary response. Several previous studies with bulk cultures provided evidence that cytotoxicity was restricted by HLA-A,B-related antigens. In the present family study, the EBV-specific cytotoxic T-cell response of a normal EBV-seropositive donor was analysed in detail by T-cell colony formation. Peripheral blood mononuclear cells were stimulated by the gamma-irradiated autologous lymphoblastoid cell line (LCL) and 3 days later seeded into agarose. Colonies were harvested, amplified by addition of interleukin-2 (IL-2), and analysed for T-cell markers and specificity in 51Cr-release assays. Twenty-two colonies were studied: all colonies were OKT3+, five were predominantly OKT4+, 9 were OKT8+ and 8 were mixtures. As expected from previous work, the OKT8+ colonies were cytotoxic for the autologous LCL target and cytotoxicity was blocked by monoclonal antibody (W6/32) to the nonpolymorphic determinants of HLA-A,B,C antigens. Significantly, the OKT4+ colonies tested also showed specific cytotoxicity, but lysis of the autologous LCL was blocked by the monoclonal antibody (OKlal) to the non-polymorphic determinants of HLA-DR antigens. Two interesting patterns of specificity were seen in cytotoxicity tests on sibling LCL targets. In one pattern, targets bearing the A11, B5, DR7 haplotype were lysed, while those bearing the A1, B8, DR3 were not, indicating haplotype preference. In the other pattern, there was lysis of the autologous cell line but not of the sibling targets. These results including HLA-DR-associated restriction, haplotype preference and strict self-preference, further illustrate the complexity of the EBV-cytotoxic T-cell response.

Relationship between expression of hepatitis B virus antigens in isolated hepatocytes and autologous lymphocyte cytotoxicity in patients with chronic hepatitis B virus infection.

Previous studies demonstrated that peripheral blood lymphocytes are cytotoxic to autologous hepatocytes in patients with chronic hepatitis B virus infection. We examined whether cytotoxicity is specifically directed against hepatocytes expressing HBsAg or HBcAg. Viral antigens were detected by immunofluorescence in isolated hepatocytes before and after exposure to T and non-T lymphocytes from 28 patients with chronic HBV infection in an autologous cytotoxicity assay. There was significant reduction in the percentage of HBcAg-positive hepatocytes after exposure to T lymphocytes and, to a lesser extent, after exposure to non-T cells; hepatocytes expressing HBsAg were not affected. Other studies showed that many hepatocytes containing HBcAg have IgG of anti-HBc specificity bound to their nuclei. In the present study, there was significant association between the presence of HBcAg and nuclear IgG in isolated hepatocytes. The ratio between the percentages of HBcAg-positive and IgG-positive hepatocytes (reflecting the proportion of hepatocytes containing free-core antigen) correlated significantly with reduction in HBcAg-positive liver cells after exposure to T cells, but not to non-T cells. These results suggest that hepatocytes in which viral replication is occurring and which express core determinants are susceptible to cell-mediated cytotoxicity and that T-cell cytolysis may be modulated by anti-HBc which is bound to hepatocytes.

Interferon-induced autologous reactivity of human natural killer cells.

The reactivity of human natural killer (NK) cells against normal autologous target cells was studied. The following major observations were made: (i) non-stimulated NK cells did not lyse autologous target cells, although allogeneic cells were killed to some extent. However, treatment of effector cells with interferon (IFN) resulted in induction of autologous cytotoxicity, and both autologous and allogeneic fibroblast targets were efficiently killed without preference. This was demonstrated both with unfractionated lymphocytes and with effector cells highly enriched in large granular lymphocytes (LGL). (ii) NK cells bound to autologous cells as efficiently as to allogeneic cells. The cells binding to autologous cells were able to lyse K562 cells, demonstrating their NK nature. After stimulation with IFN autologous cells were also killed. (iii) The exemption from autologous cytotoxicity by the non-stimulated NK cells seemed to result from a block at a post-binding stage, and there did not appear to be any need for accessory cells in the induction of autologous reactivity by IFN treatment of the LGLs. The results thus point to important post-binding-stage regulatory mechanisms saving normal cells from the cytotoxic action of circulating NK cells and may help to understand the suggested role of NK cells in certain autoimmune diseases.

Role of autologous lymphocyte cytotoxicity in colonic neoplasia.

The T-lymphocyte mediated killing of autologous carcinoma colon cells was investigated. There was no change in the incidence of activity with advanced disease, age, or nutritional status of the patient and no difference could be demonstrated in lymphocytes extracted from blood, draining lymph nodes, or the tumour itself. Nevertheless. T-lymphocyte activity did appear to be specific for the patients's own tumour, as it was rarely observed with allogeneic tumours. There was also no correlation with lymphocyte natural killer activity. The in vitro studies demonstrated patient specific T-lymphocyte activity in 23 of 47 patients with carcinoma of the colon, but the results do not correlate with clinical and pathological findings.

Induction of cytotoxic activity in human lymphocytes against autologous and allogeneic melanoma cells in vitro by culture with interleukin 2.

The influence of interleukin 2(IL2) on the cytotoxic activity of lymphocytes from patients with melanoma against autologous and a variety of allogeneic melanoma cells was studied. IL2 was produced from blood lymphocytes cultured for 24 h with phytohaemagglutinin (PHA) and purified by membrane chromatography to exclude PHA. Lymphocytes from 13 patients with melanoma at various clinical stages were cultured fro 6 days with IL2 (2 U/ml) and then tested for cytotoxic activity against autologous melanoma cells, three allogeneic melanoma and three non-melanoma cells. Autologous cytotoxicity was generated by culture with IL2 alone and was not increased by culture with both IL2 and autologous tumour cells. Marked increases in cytotoxic activity were also generated against the allogeneic target cells and were maximal against the NK-insensitive Chang target cells. Similar degrees of cytotoxicity were induced by IL2 stimulation of lymphocytes from melanoma patients, patients with nonmelanoma carcinoma and normal subjects against the allogeneic target cells. Cold target inhibition studies were carried out against IL2 induced autologous cytotoxicity in five patients. In four of five studies the autologous target cells inhibited more than the allogeneic target cells. There was no significant difference between the inhibition produced by allogeneic melanoma cells and that produced by non-melanoma cells. Similarly, in studies against allogeneic target cells, there was no significant difference in the inhibition produced by allogeneic melanoma compared to non-melanoma target cells. This applied irrespective of whether effector cells were from melanoma or non-melanoma subjects. These results suggest that lymphocytes from patients with melanoma are primed against autologous antigens in vivo and that provision of a second signal, IL2, in vitro can induce cytotoxicity against the autologous tumour. The cytotoxicity generated against the allogeneic target cells did not appear to have specificity to melanoma. Several results, such as the pattern of cytotoxicity against the target cells and change in cell surface markers on the lymphocytes during culture, suggested that cytotoxicity was mediated by activated T cells rather than by nature killer cells. These findings appear to have important implications both in the understanding of tumor host relationships and for the use of IL2 in therapy.

In vitro cytotoxicity of lymphocyte subpopulations against autologous human bladder tumor cells.

The in vitro cytotoxicity of unfractionated blood lymphocytes and T and non-T lymphocytes respectively, was tested against autologous tumor cells from 21 patients with grade III-IV urothelial bladder carcinomas. Generally, little or no cytotoxicity was seen. However, some differences in autologous cytotoxicity between T and non-T lymphocytes were detectable. Four of 21 patients had cytotoxic non-T lymphocytes against autologous bladder tumor cells. The results indicate, that the effector cells are primarily non-T lymphocytes, and that cytotoxicity appears at lower levels of T lymphocytes in unfractionated lymphocyte preparations.

Lymphocyte cytotoxicity against autologous bladder tumor cells in humans, investigated by use of a chromium-51 release assay.

A technique for isolation of a single cell suspension of viable tumor cells from bladder tumor biopsies is described. By application of the tumor cells as targets in a 51chromium release assay, the cytotoxicity of autologous lymphocytes was measured in 38 patients with transitional-cell tumors of the urinary bladder. The lymphocyte-medicated cytotoxicity was evaluated in relation to the histological grade and clinical stage of the bladder tumors. No significant correlations were found between the autologous cytotoxicity and the grade or stage of the bladder tumors.

In vitro demonstration of in situ autologous tumour-cell cytotoxicity in MSV-induced tumours in A/SN mice.

Moloney sarcoma-virus (MSV)-induced tumours in A/Sn mice have been dispersed with collagenase and DNase 8-15 days after virus inoculation, and both "sarcoma" and inflammatory cells separated by sedimentation velocity and adherence techniques. The isolated "sarcoma" cells had the morphological characteristics of atypical cells (i.e. cytoplasmic blebbing, vacuolization and prominent nucleoli) and were easily adapted to in vitro growth. As few as 2 x 10(3) of these cells inoculated i.m. produced new tumours within 8 days of injection in both syngeneic and allogeneic mice. Also, cell-free supernatant from "sarcoma"-cell cultures produced tumours, indicating that the successful transplantation of the "sarcoma" cells was probably due to production of infective virus. Cells cytotoxic in vitro against the "sarcoma" cells were present within both spleen and tumour of the tumour donors, but not in the spleens of normal mice. The cytotoxicity was specific against virus-infected cells, since in a mixture of virus-positive (gp 70) and virus-negative cells, positive cells were removed while negative cells were not affected, as measured by a visual cytotoxicity assay using immunostaining. Although T cells could be isolated from the MSV-induced tumours, these cells did not appear to mediate the cytotoxicity detected against the MSV "sarcoma" cells. These results suggest that early MSV infections might be sensitive to cytotoxic mechanisms distinct from those reported with established MLV- or MSV-induced tumour lines.

頭けい部がんの免疫学的研究

Recent advances in cancer immunology were reviewed in the first half of this paper.Using syngeneic animals, the biological activities of lymphocyte in attacking cancer cells have precisely been analysed. However, several fundamental problems in human cancer immunology still remain obscure. Especially in the case of human solid tumors including head and neck cancer, there has been scarsely clear proof that human autologous lymphocytes respond and specifically attack cancer cells.The last half of this report, explained that human autologous T-lymphocyte cytotoxicity to cultured squamous cell cancer cell had been successfully induced in our laboratory, and the possibility of specific immune therapy of the head and neck cancer was discussed. In addition to those, data found in our laboratory about natural killer (NK) activity and regional lymph node metastasis were briefly demonstrated.

Human tumor-lymphocyte interaction in vitro. VII. Blastogenesis and generation of cytotoxicity against autologous tumor biopsy cells are inhibited by interferon.

In a high proportion of cases blood lymphocytes from cancer patients cultured with autologous tumor biopsy cells undergo blastogenesis. In addition, cytotoxicity against autologous tumor cells is generated. These autoactivated lymphocytes also kill the highly NK-sensitive K-562 and Molt-4 cells. Addition of interferon (IF) at the initiation of the cultures inhibited blastogenesis and the generation of autologous cytotoxicity. On the other hand, lymphocytes already activated for autologous killing in the mixed cultures were not affected by short-term IF treatment prior to the cytotoxic assay. Lymphocytes cultured alone lose their cycotoxicity against K-562. However, when IF was present in these cultures the lymphocytes were cytotoxic for K-562. In addition, lymphocytes cultured for 6 days under conditions which did not result in activation killing of Molt-4 cells could be induced by short-term incubation of the effectors with IF.

Cytotoxicity in vitro of blood lymphocytes from bladder cancer patients and controls to allogeneic or autologous tumor cells derived from established cell lines or short-term cultures.

Blood lymphocytes from small groups of patients with transitional-cell carcinoma of the urinary bladder (TCC), clinical controls (CC) or healthy donors (HD) were tested for cytotoxicity in vitro by a 51Cr-release assay. The target cells were either from TCC or control tissue (long-term cultures) or were from short-term TCC cultures, kept in vitro for 10-20 transfer generations. When tested with allogeneic target cells from long-term cultures, TCC patients' lymphocytes tended to be more cytotoxic to TCC targets than to control targets. For the control lymphocytes this was not seen. A large proportion but not all of the cytotoxicity to these target cells was due to immunoglobulin-dependent cellular reactions, probably mediated by natural and disease-related antibodies of the lymphocyte donors, since it was significantly inhibited by Fab-fragments of rabbit antibodies to human immunoglobulin. Moreover, it was, to a large extent, mediated by lymphocytes with Fc-receptors for IgG. For seven of the TCC target cell cultures (two long-term and five short-term) autologous lymphocytes were also available for testing. While two patients were non-reactive to their own tumor cells, five reacted strongly in the autologous combinations. These autologous reactions were immunoglobulin-independent and were mediated by Fc-receptor-negative effector cells. In some instances, autologous cytotoxicity was accompanied by similar reactions to some of the allogeneic TCC targets but not to the allogeneic non-TCC control targets. On the basis of available information on HLA antigens in this material, the pattern of cross-reactions suggests that the cytotoxicity encountered in the autologous and in some of the allogeneic TCC-combinations may be the expression of antibody-independnet but specific CTL-mediated reactions, regulated by HLA. However, at the present stage of the investigation, other mechanisms must also be considered since the target cells from short-term TCC cultures were sometimes lysed by control lymphocytes in immunoglobulin-independent reactions. Whatever the explanation, the results show that the cytotoxicity observed in the in vitro systems is usually the net result of several different types of reaction. Which effector cell types and which mechanism of recognition will predominate in a given lymphocyte/target cell combination is greatly influenced by the nature and origin of the target cells used.

Stimulation of human peripheral blood lymphocytes by autologous EBV-infected B cells

EBV carrying lymphoblastoid cell lines (LCL) strongly stimulate DNA synthesis in normal autologous peripheral T lymphocytes in vitro (autologous stimulation—AS), and generate non-specifically cytotoxic T cells. The AS reaction was explored by replacing the stimulating LCLs with recently infected B cells from normal individuals. Autologous B cells infected one day earlier with EBV induced significant DNA stimulation but generated no killer cells. The ability to generate cytotoxic T cells appeared 6 to 8 days after EBV infection. Cytotoxicity was not EBV-specific. The ability to activate the alternate complement pathway appeared at approximately the same time. B-cells of convalescent infectious mononucleosis (IM) patients could generate autologous cytotoxicity already 1 day after EBV-infection. The difference between the AS reactions induced by normal and convalescent IM donors is discussed.

Ultrastructure, karyology and immunology of a cell line originated from a human transitional-cell carcinoma.

A cell line (J82) was derived from a poorly differentiated, invasive, transitional-cell carcinoma, Stage T3. The cells have been propagated in vitro for 5 years and showed 100% aneuploidy and a mixed epithelial-fibroblastic morphology. The majority of cells contained 2Y chromosomes and several distinctive markers. Peripheral-blood lymphocytes from the donor of the J82 cells were tested sequentially for cytotoxicity toward autologous and allogeneic tumour cells. Autologous cytotoxicity was detected against J82 cells in early in vitro passage. Allogeneic lymphocytes from some patients with transitional-cell carcinoma were also cytotoxic to J82 cells in primary culture. However, selective cytotoxicity by lymphoid cells from bladder-carcinoma patients was not detected against J82 cells in long-term tissue culture.

Establishment and characterization of a squamous cell carcinoma of the vulva in tissue culture and immunologic evaluation of the host

A continual epithelial cell culture has been established from a squamous cell carcinoma of the vulva metastatic to the right inguinal lymph node. Initial characterization of the cell line (designated LT2), including morphology, growth properties, karyotyping, and secretion of carcinoembryonic antigen (CEA) and the beta subunit of human chorionic gonadotropin (HCG-β) is reported. The LT2 cell line satisfies the following criteria of malignancy: (1) establishment in culture for more than one and one-half years exceeding more than 70 generations; (2) heteroploid karyotype; (3) abnormal cytology; (4) growth in soft agar suspension; and (5) tumorigenicity in nude mice. This cell line has also been found to be free from contamination by fibroblasts, mycoplasma, bacteria, and fungi. With the LT2 cell line, the specific tumor-host immunorelationship was assayed by autologous (host lymphocyte versus LT2 cells) cytotoxicity and lymphoblast transformation in the presence of tumor extracts or mitomycin C-treated LT2 cells. In vivo patient immunocompetence was evaluated specifically by skin testing with autologous tumor extracts and nonspecifically with bacterial recall skin test antigens and dinitrochlorobenzene. In vitro nonspecific immunocompetence was evaluated by total peripheral white blood cell and lymphocyte counts, T cell, active T cell and B cell determinations, lymphocyte response to the mitogen phytohemagglutinin, and spontaneous lymphocyte-mediated cytotoxicity versus cultured bronchogenic carcinoma cells. Correlation of immunocompetence and the status of the tumor-host relationship with the patient's clinical course, and the potential significance of LT2 CEA and HCG-β production are discussed. The patient's response to bacillus Calmette-Guerin immunotherapy is evaluated.

Lymphocyte cytotoxicity against autologous tumour biopsy cells in humans.

By the application of separation techniques in a stepwise manner to mechanically prepared cell suspensions from human tumour biopsies it has been possible to isolate tumour cells having high viability and low contamination with host cells. These tumour cells have been used as targets in 51Cr release cytotoxicity assays using autologous lymphocytes from blood or lymph node as effectors. It has been possible to investigate this reactivity in 30 patients with malignant and four patients with benign tumours. Significant autologous cytotoxicity in the blood lymphocytes was detectable in two of 12 lung tumours, nine of 18 other carcinomas and sarcomas but in none of the benign cases. Cytotoxicity was not limited by the presence of obvious residual disease at the time of operation although reactivity was less frequently detectable in patients with secondary involvement. Reactivity in the tumour-draining lymph nodes largely paralleled that in the blood in the limited number of patients examined. The technical problems encountered during this study and the role of natural cytotoxicity in the interpretation of these data are discussed.

Immunotherapy with autologous white cell infusions (“lymphocytes”) in the treatment of recurrent glioblastoma multiforme.A preliminary report

Autologous leukocytes (10(7) to 10(9)), obtained with the Haemonetic's Leukaphoresis apparatus, were inoculated directly into recurrent glioblastoma tumors via indwelling catheters or by direct intratumoral injection through existing craniotomy openings. The rational use for autologous leukocyte (lymphocyte) infusions was based on in vitro autologous lymphocyte cytotoxicity to glioblastoma cells in the absence of serum inhibitory factors. Seven of 17 patients treated had life expectancy under 1 month; all patients had received definitive surgery, and all but two received radiation, nitrosourea chemotherapy and/or dexamethasone, and showed evidence of clinically recurrent disease. Following autologous leukocyte infusion (lymphocyte/granulocyte ratio 1:1), eight patients sustained clinical improvement and were alive up to 17 months later. No neurotoxicity ascribable to the procedure has been observed. One patient, who was comatose at the time of single leukocyte infusion, returned to full activity and lived for 17 months without an increase in tumor mass by brain scan. These results suggest that infusions of autologous leukocytes (lymphocyte-monocytes) directly into glioblastoma may be a viable additional treatment for glioblastoma and certainly warrants further evaluation.

Autologous Human Cellular Cytotoxicity Induced by Mitogenic and Nonmitogenic Lectins

Abstract Autologous systems are of great interest in the study of cellular cytotoxicity. By eliminating considerations of previous allogeneic sensitization, an autologous system allows straight forward study of effector mechanisms. The induction of lymphocytotoxicity by a variety of plant lectins toward xenogeneic, allogeneic, or syngeneic target cells has been described by Holm and Perlmann (1–4) and other investigators (5–7). Although early studies of lectin-induced lymphocytotoxicity demonstrated a close correlation between the degree of mitogenic stimulation of the lymphocytes and subsequent cytotoxicity (2), recent work has suggested that the phenomena of mitogenesis and of cytotoxicity may be independent of one another (5, 7). The ability to fractionate phytohemagglutinin into its major components (8), and the existence of other plant lectins that are nonmitogenic, provide an opportunity to study human autologous cellular cytotoxicity induced by lectins, with particular attention directed to the relationship between cytotoxicity and mitogenicity.

Immunization with irradiated tumour cells and specific lymphocyte cytotoxicity in malignant melanoma.

A microculture technique has been adapted to assay the cytotoxic properties of the lymphocytes from patients with malignant melanoma when cultured with their autologous tumour cells. In a series of patients with established melanoma specific autologous cytotoxicity was extremely uncommon, being detectable in only 3 out of the 22 cases studied. This cytotoxicity did not correlate with clinical staging of the disease but may well have been related to tumour volume. By autoimmunization of patients with an irradiated suspension of their own tumour cells the appearance of cytotoxic lymphocytes could be provoked in 5 out of the 12 patients studied. This cytotoxicity was detectable at the end of the first week after the autograft and disappeared by the third week. Cytotoxic lymphocytes did not correlate in any obvious way with the appearance of specific antitumour antibodies detected by immunofluorescence. So far there has been no evidence of a serum factor capable of blocking the lymphocyte cytotoxicity in these patients.The presence and possible significance of cytotoxic lymphocytes in patients with malignant disease is discussed.