Hyperthermia improves cellular immune response to human hepatocellular carcinoma subsequent to co-culture with tumor lysate pulsed dendritic cells

Dendritic cells play a major role in cellular immunity. The crucial steps of antigen presentation and processing by DCs may be limiting factors for adoptive cellular immunotherapy. Here, we investigated whether hyperthermia of human hepatocellular carcinoma (HCC) cells induces enhanced cytotoxic cellular immune response. Peripheral blood mononuclear cell (PBMC)-derived DCs were pulsed with tumor cell lysate of the human HCC cell line HepG2, which had been heat shocked prior to incubation for 5 h. Subsequent to TNFalpha-induced maturation DCs were co-cultured with autologous CD4+ and/or CD8+ cells, and T cell mediated cytolysis of HepG2 cells was assessed. We observed enhanced CD4+/8+ cellular cytotoxicity against HepG2 cells subsequent to co-culture with the heat shocked tumor lysate pulsed DCs as compared to pulsing DCs with lysate of non-heat shocked tumor cells. The improved cellular immune response can be related to enhanced expression of HSP 70 and 90 in HepG2 cells upon hyperthermia.     

Intracellular distribution of 73,000 and 72,000 dalton heat shock proteins in HeLa cells

Intracellular localization of 73,000 and 72,000 dalton heat shock proteins (HSP73/72) in HeLa cells that were heat shocked or treated with chemical stressors was investigated using indirect immunofluorescent staining. The antiserum used specifically recognized the HSP73/72 in HeLa cells, and HSPs were increased by heating cells at 42 degrees C for 2 or 4 h and by prior treatment with chemical stressors (sodium arsenite, cadmium chloride, 8-hydroxyquinoline and ethanol). There was diffuse cytoplasmic staining at 37 degrees C, whereas nucleoli were stained brightly when cells were heated at 42 degrees C for 2 h. This rapid accumulation of HSP73/72 in the nucleoli was not inhibited by cycloheximide (50 micrograms/ml). Translocation of HSPs to the nucleoli was specific for heat because no translocation was induced by treatment with chemical stressors. When the cells were returned to 37 degrees C after heating, the HSPs in their nucleoli disappeared rapidly and diffuse cytoplasmic staining was present after 6-9 h. Our results suggest that the transient accumulation of HSP73/72 in HeLa cell nucleoli that is induced by heat shock is not correlated with the development of thermotolerance obtained in other cell systems.     

Thermotolerance and heat shock protein induction by slow rates of heating

The magnitude of thermotolerance and the level of heat shock protein (HSP) expression have been measured in Chinese hamster ovary cells after gradual temperature transients from 37 degrees or 39 degrees to 42 degrees or 43 degrees C. When the level of thermotolerance was measured by clonogenic survival after challenging temperatures between 42 degrees and 43 degrees, substantial thermotolerance was observed. However, when the challenging temperature was raised to 45 degrees C, proportionally less thermotolerance was apparent. Heat shock proteins were quantitated by scanning densitometry of radiographs and, in the case of HSP 70, by immunoassay. Scanning densitometry revealed that low levels of heat shock proteins were synthesized during the heating gradients, but less than after a heat shock at 45 degrees C that delivered an equivalent heat dose. The immunoassay of HSP 70 levels measures both pre-existing and newly synthesized protein, and showed that there was net increase in HSP 70 during two of the heating gradients tested, despite the increase in synthesis noted on the gels. Higher turnover of HSP 70 at the elevated temperatures possibly accounted for the failure to detect a net gain in total protein. In contrast, the total amount of HSP 70 doubled during the 6 hr following a heat shock of 45 degrees for 10 min, an equivalent heat dose to one of the gradients where no net increase in HSP 70 was measured by immunoassay. It appears, then, that tolerance to hyperthemia at 43 degrees C or below may occur under some conditions in the absence of elevated levels of HSP 70, but tolerance to higher temperatures is more closely correlated with increased levels of heat shock proteins. However, even at higher temperatures, our data show disparities between the levels of HSP measured and the thermotolerance expressed.     

Effects on the expression of heat shock proteins by step-down heating and hypothermia in rat hepatoma cells with a different degree of heat sensitivity.

Thermosensitization induced by pretreatment at supra- and subnormal temperatures, rate of protein synthesis and expression of the major heat shock proteins under such conditions was investigated in relation to intrinsic heat sensitivity of rat hepatoma cells, i.e. Reuber H35 and HTC. The high degree of heat susceptibility of H35 cells was reflected by a high degree of thermosensitization after pretreatment by heat (step-down heating) at temperatures of 42-44 degrees C for 30 min or cold for 16 h at temperatures ranging from 0 to 25 degrees C. Sensitization under step-down heating conditions was found to be paralleled by a delayed recovery of protein synthesis. Despite an increased relative rate, enhancement of the absolute rate of synthesis of the major heat shock proteins, HSP28, HSP60, HSP68, HSP70, HSP84 and HSP100, was less pronounced during step-down exposure. Comparable results were obtained during recovery of sensitized H35 cells at 37 degrees C after exposure to heat following pretreatment at 0 degrees C. Furthermore, clear differences in the regulation of the specific HSP synthesis, depending on the particular treatment protocol, were observed.     

Sensitization of human Ewing's tumor cells to chemotherapy and heat treatment by the bioflavonoid quercetin

BACKGROUND: The bioflavonoid quercetin, a polyphenolic compound widely distributed in the plant kingdom, has been demonstrated to exert cytostatic activity against a variety of tumor cells in vitro and in vivo. It may be useful in cancer therapy as a thermosensitizer by increasing the cell killing effect of hyperthermia and chemotherapy because of its ability to suppress heat-shock protein expression. MATERIALS AND METHODS: We investigated the effect of quercetin combined with two cytotoxic agents, cDDP (cis-diamminedichloroplatinum II) and VP-16 (etoposide), under various heat-shock conditions in two Ewing's tumor cell lines SK-ES-1 and RD-ES, using XTT-assay and Western blot analysis. RESULTS: Induction of thermotolerance by a sublethal heat-shock (42 degrees C, 1 hour) led to a transient resistance against subsequent heat treatment alone or combined thermochemotherapy with the crosslinking agent cDDP or the topoisomerase II inhibitor VP-16. Quercetin (> or = 50 microM) applied for 24 hours inhibited cell proliferation, increased the cytotoxic activity of cDDP or VP-16 alone or combined with simultaneous hyperthermia and suppressed the development of thermotolerance. Hyperthermia (43 degrees C, 45 degrees C for 1 hour) induced high expression of the inducible form of HSP70, whereas HSP27, which is constitutively expressed at normothermic conditions, is only slightly induced by 43 degrees C and nearly completely suppressed at 45 degrees C. Induction of thermotolerance is accompanied by an elevated expression of both HSP70 and HSP27. Quercetin (> or = 50 microM), alone as well as in combination with thermochemotherapy, inhibited the expression of both HSP70 and HSP27. CONCLUSION: These data suggest that the bioflavonoid quercetin potentially may be useful in clinical trials for optimizing the efficacy of hyperthermia in combination with chemotherapy.     

热休克诱导人肝癌HepG2细胞HSP70和P-gp的表达及槲皮素对二者的抑制作用

背景与目的:研究提示热休克蛋白70(heatshockprotein70,HSP70)可抑制细胞凋亡,降低肿瘤热疗、化疗的疗效,但其与细胞化疗耐受性的内在关系尚不甚明确;P-糖蛋白(P-glycoprotein,P-gp)是一种耐药蛋白,在肿瘤耐药中起重要作用。本研究拟通过热休克诱导人肝癌HepG2细胞表达HSP70和P-gp,并观察槲皮素(quercetin,Qu)对二者表达的抑制效应。方法:恒温水浴法,42℃热休克HepG2细胞90min,分别采用免疫组化、流式细胞术检测热休克前及热休克后2h、4h、8h、12h、24h时HSP70和P-gp的表达情况;并于热休克前1h应用不同浓度槲皮素作用于HepG2细胞,观察其热休克后4h二者的表达。结果:(1)热休克诱导后,免疫组化显示细胞内HSP70表达增多,“核内迁移”;流式细胞术示HSP70阳性细胞数升高,4h达到最高(11.47%),较对照组(6.64%)升高近一倍(P<0.01),24h后回落到低值;P-gp阳性细胞数随HSP70升高后升高,12h达到最高值(96.31%),较对照组(33.95%)升高近两倍(P<0.01),24h后回落到低值。(2)热休克前应用槲皮素能有效抑制热休克诱导HSP70和P-gp的过量表达,以100～200μmol/L槲皮素作用明显(P<0.01),呈浓度依赖性。结论:热休克可诱导HepG2细胞HSP70和P-gp的过量表达,P-gp的表达可能与HSP70的表达有关;槲皮素可阻断热休克诱导HepG2细胞HSP7     

HeLa细胞HSP-70多肽体外诱导免疫活性细胞及其抗瘤机理

[目的]探讨用HeLa细胞HSP 70多肽诱导免疫活性细胞 (immunecompetentcells ,ICC)的特异性抗肿瘤机制。[方法]用热休克处理的HeLa细胞提取的HSP 70多肽剌激正常人外周血单个核细胞 ,在剌激扩增前后测定T淋巴细胞表型 ,并测定扩增的免疫活性细胞对HeLa细胞的杀瘤活性。[结果]在扩增的细胞中CD3+、CD8+淋巴细胞百分率明显增高 (P<0.01)。该免疫活性细胞对Hela细胞和来源于病人的宫颈癌细胞都具有较高的杀伤活性 ,而对经HSP 70单抗封闭后的HeLa细胞的杀伤率则明显下降。[结论]HeLa细胞HSP 70多肽能有效刺激PBMCs诱导活化免疫活性细胞 ,该免疫活性细胞具有特异性抗肿瘤作用     

热休克蛋白70的纯化及其抗小鼠肝癌作用的研究

目的　建立热休克蛋白 70 (hsp70 )的分离方法 ,观察其在小鼠体内的特异抗瘤效应。方法　用经 43℃热处理 12h的H2 2细胞 ,制备裂解液 ,用DEAE5 2、ConA和ADP柱进行序贯分离 ,所得蛋白经电泳及Westernblot进行蛋白分子量及性质鉴定。用纯化的hsp70蛋白免疫A组C3H及C组C5 7BL/ 6小鼠 ,RPMI 16 40液免疫B组C3H及D组C5 7BL/ 6小鼠。第 15天A、B组小鼠用H2 2攻击 ,C、D组小鼠用EL 4攻击 ,观察各组小鼠肿瘤发生率及肿瘤大小。结果　纯化蛋白经鉴定确系相对分子质量约为 70 0 0 0的hsp70蛋白。A组小鼠肿瘤发生率为 0 / 6 ,而B组小鼠肿瘤发生率为 6 / 6。C组和D组小鼠的肿瘤发生率均为 6 / 6 ,两组小鼠第 35天的肿瘤大小差异无显著性。结论　用上述蛋白层析法可分离获得纯度高、并保留肿瘤抗原信息的hsp70蛋白。该蛋白免疫后的小鼠能抵抗同种肿瘤细胞的攻击 ,而对异种肿瘤细胞的抵抗能力有限。提示纯化蛋白中的抗原信息可能不是hsp70蛋白本身 ,而是其所携带的小分子多肽。     

The effect of calmodulin antagonists on hyperthermic cell killing and the development of thermotolerance

The role of calmodulin (CaM) in hyperthermic cell killing, and the development of thermotolerance in rat 13762NF mammary adenocarcinoma cells, was investigated by using the CaM antagonists W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and W-13 [N-(4-aminobutyl)-2-chloro-naphthalenesulphonamide] and their less active analogues W-5 [N-(6-aminohexyl)-1-naphthalenesulphonamide] and W-12 [N-(4-aminobutyl)-2-naphthalenesulphonamide]. The CaM antagonists W-7 and W-13 potentiated 43 degrees C cell killing (and the less active analogues did not) at a concentration compatible with CaM inhibition, thus hyperthermic perturbation of CaM-regulated processes may contribute to cellular lethality. The potentiation of hyperthermic killing by antagonists appeared to be temperature-dependent, sensitizing much more effectively to 43 degrees C than to 42 degrees C killing. The effect may be related to differing primary mechanisms of hyperthermic killing activated at the two temperatures, or simply to differences in incorporation or localization of the antagonists. The presence of the CaM antagonists throughout fractionated 42 degrees C or 43 degrees C heating, or during continuous 42 degrees C heating, did not significantly inhibit or potentiate the triggering and development of thermotolerance or alter the rates of heat stress protein (HSP) synthesis. Studies using CaM-agarose isolation of CaM-binding proteins indicated that binding of some HSP to CaM-agarose occurred and was Ca2+-dependent. The specificity and physiological relevance of these HSP binding to CaM was not clear, since their affinity was not high in these cells. Presumably W-7 would perturb any physiologically relevant CaM-protein interactions in cells but W-7 concentrations that reduced HSP and other protein binding to CaM-agarose columns by 50 per cent or more, had no effect on thermotolerance development in cells. These observations, combined with the studies that showed little effect of CaM antagonists on HSP synthesis at concentrations which potentiated cell killing, suggested that events leading to triggering or developing thermotolerance were not strongly dependent on any putative HSP binding to CaM. These studies also suggest some targets of hyperthermic cell killing at 43 degrees C are different from those that lead to the triggering and development of thermotolerance.     

Restoration of Cisplatin sensitivity by mild hyperthermia in radiation-induced Cisplatin-resistant mouse fibrosarcoma cells

Moderate cisplatin resistance has been induced in murine fibrosarcoma cells SSK-R3 by low-dose irradiation without associated changes in radiosensitivity. Resistance can be reverted selectively by stimulation of the cGMP-dependent transduction pathway with sodiumnitroprussid (SNP, 1). In the present study combined thermo-chemotherapy is demonstrated to overcome cisplatin resistance at mild hyperthermic temperature. Between 37 degrees C and 43 degrees C, heat alone has almost the same cytotoxic effect on SSK-R3 cells and the parental SSK cells. If cisplatin exposure is carried out at 40 degrees C for 1 hour, there is an increase in drug sensitization for both cell lines, but the thermal enhancement ratio (TER) is higher in the resistant cells. At 42 degrees C, the survival curves of the resistant SSK-R3 cells and the parental SSK cells almost coincide, resulting in thermal enhancement factors of 5.4 and 3.2, respectively, and restoration of the original cisplatin sensitivity in the SSK-R3 cells. Upon further rise of the exposure temperature to 43 degrees C, the cytotoxic effect of heat alone dominates in both cell lines. The radiosensitivity can be increased to the same extent in both cell lines after one hour exposure to 42 degrees C. SNP, which selectively reverses cisplatin resistance at 37 degrees C, does not exhibit additional differential cisplatin sensitization on SSK-R3 cells compared to the SSK cells at 42 degrees C. These results demonstrate a dominant role of mild, clinically relevant hyperthermic temperature to enhance cisplatin sensitivity and selectively revert cisplatin resistance in SSK-R3 cells. Possible mechanisms underlying this radiation-induced cisplatin resistance are discussed.     

Enhancement of cytotoxicity by hyperthermia after a long-term culture with 5-fluorouracil in transformed cells.

The cytotoxic effect of 5-FU (5-fluorouracil) was demonstrated to be enhanced by hyperthermia after treatment with 5-FU at even a comparatively low dose over fairly long periods. The cytotoxic effect of the combined treatment with 42 degrees C-hyperthermia and 1 microgram/ml 5-FU for 48 hrs, the cytotoxic effect of 42 degrees C-hyperthermia and 5 micrograms/ml 5-FU for 24 hrs, and the cytotoxic effect of 43 degrees C-hyperthermia and 1 and 5 micrograms/ml 5-FU for 8 hrs were studied. The maximally enhanced rate of 42 degrees C-hyperthermia after 5-FU treatment for 96 hrs was 48% after a 1 microgram/ml 5-FU treatment and 150% after 45 hrs with the 5 micrograms/ml 5-FU treatment. The maximally enhanced rate of 43 degrees C-hyperthermia after 5-FU treatment was 170% after 45 hrs with 1 microgram/ml 5-FU treatment and 180% after 24 hrs with 5 mg/ml 5-FU treatment. When V-79 cells were treated at the same temperature, the maximally enhanced rate with 1 microgram/ml 5-FU was almost equal to that of 5 micrograms/ml. Moreover, when each maximally enhanced rate was equalized, each concentration of FU (RNA)/RNA practically became equal, i.e., when the maximally enhanced rates were approximately 150 and 170-180%, FU (RNA)/RNA concentrations were about 40 and 15 ng/mg RNA, respectively. We thus concluded that FU (RNA)/RNA concentration might play an important role as an indicator of the effect of the combined treatment of 5-FU and hyperthermia.     

负载腮腺腺样囊性癌抗原的树突状细胞诱导的抗肿瘤效应

目的：探讨腺样囊性癌肿瘤抗原负载的树突状细胞通过淋巴细胞介导的免疫反应,体外杀伤腺样囊性癌细胞的细胞毒性效应。方法：外周血单核细胞在GM—CSF＋IL-4的诱导下体外培养,用肿瘤细胞抗原冲击后,流式细胞仪检测树突状细胞抗原负载前后CD1a、CD83表达量的变化。MTT比色法检测同种异体的混合淋巴细胞反应和诱导细胞毒淋巴细胞CTL杀伤肿瘤细胞。结果：凋亡肿瘤抗原刺激后,CD83表达增加（P〈0．01）,而CD1a表达量下调（P〈0．05）。负荷肿瘤抗原树突状细胞体外诱导出明显的细胞不良反应,并刺激同种T淋巴细胞增殖。结论：GM—CSF＋IL-4诱导的单核细胞来源的树突状细胞,能在体外摄取肿瘤抗原而进一步成熟,通过激活淋巴细胞杀伤癌细胞。     

Effects of PKC inhibitors on suppression of thermotolerance development in tsAF8 cells.

Effects of protein kinase C (PKC) inhibitors (H7, staurosporine, calphostin C) on thermotolerance development were investigated in temperature sensitive tsAF8 cells derived from Syrian hamster BHK21 cells. Cells were pre-heated at 45 degrees C for 20 min, incubated at 34 degrees C with PKC inhibitors for varying lengths of time, i.e. 1.25-10.0 h, and then heated at 45 degrees C for 30 min. Increasing survival fractions after the second heat treatment was inhibited by the treatment with H7 (40-160 microM), with staurosporine (0.05-1.0 microM), and with calphostin C (0.8, 1.2 microM) in a concentration dependent manner. When the concentrations of these PKC inhibitors were low, the restraint of increasing survival fractions was temporary, since survival fractions increased 3-7.5 h after pre-heating. However, the survival fractions were almost constant by the treatment with 160 microM H7 and 1.0 microM staurosporine. Induction of HSP72 after heat stress was investigated in tsAF8 and BHK21 cells. Cells were heated at 45 degrees C for 20 min and incubated at 34 or 39.7 degrees C (tsAF8), at 37 degrees C (BHK21). Intensity of intracellular fluorescence from HSP72 was measured by flow cytometry. HSP72 was induced in BHK21 cells, but there was no definite induction of HSP72 in tsAF8 cells at either 39.7 or 34 degrees C. These results suggest that PKC is related with the thermotolerance development in tsAF8 cells; however, HSP72 is not involved in the thermotolerance development in tsAF8 cells.     

β-榄香烯诱导的抗肿瘤免疫保护作用机理初探

目的 探索中药莪术有效成分β－榄香烯的抗肿瘤作用及其分子机理。方法 用免疫荧光法和流式细胞仪检测不同因素处理的小鼠肝癌Ｈ２２细胞膜ＨＳＰ７０蛋白表达的变化,并用处理后的Ｈ２２细胞作为抗原进行体仙抗肿瘤免疫保护实验。结果 各实验组Ｈ２２细胞胞膜ＨＳＰ７０蛋白的表达率及表达强度均较对照组细胞有显著增加。     

Induction of leukemia-specific antibodies by immunotherapy with leukemia-cell-derived heat shock protein 70

Cancer immunotherapy using heat shock protein (HSP) derived from autologous tumor requires cluster of differentiation (CD)4⁺ as well as CD8⁺ T-cells for the prolongation of patient survival, suggesting that a humoral immune response through CD4⁺ T-cells is important in addition to cellular immunity. However, the role of humoral responses in HSP-based autologous tumor immunotherapy remains unclear. In the present study, we investigated whether leukemia-specific antibodies and antibody-mediated cytotoxicity against autologous leukemia cells have a crucial role in a mouse A20 leukemia model by immunizing A20-derived HSP70. Immunization with A20-derived HSP70 induced the production of anti-A20-antibodies and the antibodies recognized HSP70-binding peptides derived from A20. One of those was a major histocompatibility complex (MHC) class-I binding peptide, which has been clarified as the target peptide of CD8+ cytotoxic T-cells (CTL) against A20. The anti-A20-antibodies produced by immunization with A20-derived HSP70 induced complement-dependent cytotoxicity (CDC) against A20 in vitro . In addition, immunization with A20-derived HSP70 increased intracellular interleukin-4 (IL4)-production of CD4⁺ T-cells, confirming the activation of type-2 helper T-cells. Taken together, immunization with leukemia-cell-derived HSP70 induces antibodies against leukemia-cell-specific peptides and might play a crucial role in the eradication of leukemia cells by CDC in mice. These findings will enable future establishment of a novel therapeutic strategy using antileukemia antibodies in HSP-based autologous tumor immunotherapy. ( Cancer Sci 2008; 99: 1427–1434)     

Hyperthermia induces differentiation without apoptosis in permissive temperatures in human erythroleukaemia cells.

PURPOSE: The aim of the present study was to investigate whether induction of differentiation by hyperthermia is accompanied by apoptosis and necrosis to further evaluate the benefits of using hyperthermia as a differentiation inducing physical modality. MATERIALS AND METHOD: Differentiation was evaluated in K562 erythroleukaemia cells by measuring haemoglobin synthesis and flow cytometric measurement of glycophorin A expression. Apoptosis was measured by Annexin-V-FITC and Propidium Iodide (PI) double staining assay. Apoptosis and necrosis was also evaluated morphologically using staining with acridine orange/ethidium bromide (AO/EtBr) by fluorescence microscopy. Heat shock protein 70 (HSP70) level was measured by ELISA kit. RESULTS: Hyperthermia (43 degrees C) induced differentiation as judged by increased haemoglobin synthesis and glycophorin A expression. No sign of apoptosis or necrosis could be detected at this temperature. Cell viability did not change due to heat treatment, and cellular proliferation was reduced in a dose (heating time) dependent manner. At 45 degrees C, hyperthermia induced apoptosis and necrosis with minimal or no sign of differentiation. HSP70 level was significantly increased at 43 degrees C along with differentiation of leukaemic cells, while at 45 degrees C no significant effect on HSP70 production could be observed. CONCLUSIONS: The encouraging results obtained here indicate that by heat treatment at 43 degrees C, hyperthermia can be used alone or in combination with other modalities as a differentiation inducing agent without any detectable apoptotic activity. Positive correlation between HSP70 production and induction of differentiation and lack of apoptosis by hyperthermia confirm the possible role of HSP70 in the heat-induced differentiation and apoptosis in leukaemic cells.     

Triple combination of retinoic acid + low concentration of cytosine arabinoside + hexamethylene bisacetamide induces differentiation of human AML blasts in primary culture

Differentiation induction therapy provides an alternative therapeutic approach for patients with acute myeloid leukemia (AML) who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid (RA) + low concentration of cytosine arabinoside (Ara-C) + hexamethylene bisacetamide (HMBA) on differentiation of blasts from 24 AML patients was studied. Non-adherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells per ml in 24-well tissue culture plates containing RPMI 1640 culture medium with 20 per cent fetal calf serum and 10 per cent 5637-conditioned medium and incubated with 10(-6) M retinoic acid, 10(-6) M cytosine arabinoside and/or 2 mM hexamethylene bisacetamide for six days at 37 degrees C in a humidified incubator under 5 per cent CO2. Morphological, cytochemical and functional differentiation into mature cells were induced in blasts from 22 out of the 24 AML patients following exposure to the triple combination of 10(-6) M RA + 10(-6)M Ara-C + 2 mM HMBA in primary culture. These effective results justify a clinical trial of such triple combination for AML patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy.     

Functional analysis of clonally expanded CD8, TCR gamma delta T cells in a patient with chronic T-gamma lymphoproliferative disease

Leukemic cells from a patient with T-gamma lymphocytosis were found to have the surface phenotype, CD3+, CD4-, CD8+, Leu19+, TCR delta 1+, WT31-. The clonal nature of the TCR gamma delta T cell proliferation was documented by flow cytometry and Southern blot analysis. Morphologically, they were large to medium-sized mature lymphocytes with cytoplasmic granules. Functionally, the cells revealed strong cytotoxic activities against NK-sensitive target cells, but had neither killer T cell activity nor suppressive activity on PWM-driven immunoglobulin synthesis by B cells. Interestingly, both suppressive and cytotoxic T cell activities were recovered with the depletion of CD8+ T cells. These studies may suggest some functions of the CD8+ population of human TCR gamma delta T cells in a normal immune system.