The immune defense system in gastric cancer tissue, regional lymph node and spleen--aspects from subsets and functions of lymphocytes

Tumor infiltrating lymphocytes (TIL) and lymph node lymphocytes (LNL) were thought to play an important role in local immunity against cancer. But the natural cytotoxicity (NC) of TIL and LNL was very weak, and was not augmented by beta-IFN. This low cytotoxicity was augmented by IL-2, and especially LNL were activated to have higher lymphokine activated killer (LAK) activity than that of PBL. So it was proven that TIL and LNL had an potential of immunological defence system. In order to bring out these potential, immunomodulators (OK-432, PSK) were injected intralesionally under endoscopy one week prior to surgery. The cytotoxicity of TIL and LNL was augmented by the intralesional injection of OK-432 or PSK. There was no significant difference in LAK activity of LNL among the OK-432-, PSK-injected group, and the control. The 2-year survival rate of the OK-432-injected group was much longer than that of the control. From above results, intralesional injection of immunomodulators was thought to be a promising candidate for the immunotherapy of gastric cancer.     

Anti-tumor effect of intravesical instillation of OK432 against rat bladder tumor induced by N-butyl-N-(4-hydroxybutyl) nitrosamine

Summary The anti-tumor effect of OK432 instilled into the bladder was evaluated in rat bladder tumors induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). In experiment I, the rate of the natural killer (NK) activity was determined with cells from spleen and mesenteric lymph nodes. Intravesical OK432 instillation enhanced NK activity; however, this activity was not dose-dependent and was not augmented by OK432 inoculation into the foot pad. In experiment 2, the therapeutic effect of intravesical OK432 instillation was examined in rat bladder tumors induced by BBN. OK432 was instilled weekly for six weeks. Rats given BBN for 10 weeks were divided into six groups: 1) control; 2) saline; 3) OK432 0.05 KE/ml; 4) OK432 0.05 KE/ml bladder instillation with 0.01 KE/ml foot pad inoculation; 5) OK432 0.05 KE/ml, every other week; and 6) OK432 0.5 KE/ml. Weekly OK432 instillation significantly reduced tumor weight and the incidence of tumor development; however, this inhibition was not dose-dependent and was not enhanced by OK432 inoculation into the foot pad. In rats given OK432 weekly, the augmentation of NK activity and increase in tissue infiltrating lymphocytes were significant. These results suggest that intravesical OK432 instillation is effective in the management of superficial bladder tumors. The study further emphasizes that the dose and method of administration are critical variables in determining the efficacy of immunotherapy.     

Augmentation of cytotoxicity of tumor infiltrating lymphocytes by interleukin-2 in gastric cancer patients

Lymphocyte infiltration into tumor has been regarded as an expression of host reaction against tumor, but the natural cytotoxicity of tumor infiltrating lymphocytes (TLL) is often very low. In order to augment this low cytotoxicity, TIL of gastric cancer patients were cultured with interleukin-2 (IL-2) in vitro. On the other hand, immunopotentiators (OK432, PSK) were injected into gastric cancer intralesionally under endoscopy. By the in-vitro culture with IL-2, the cytotoxicity of TIL was augmented against both targets of K562 and MNN28 (gastric carcinoma cell line). In particular, the augmentation of cytotoxicity against MKN28 was more obvious in TLL than PBL (peripheral blood lymphocytes). In the ascitic lymphocytes, the in-vitro culture with IL-2 induced autologous tumor cell killing. Intralesional injection of OK432 or PSK augmented the natural cytotoxicity of TIL, and the ratio of OKT8 and Leu7 cells increased in the TIL of OK432-injected group.     

Effects of the oral or intratumoral administration of OK432 on the immuno-reactivities of regional lymph nodes in gastric cancer patients.

The effects of OK432, a streptococcal preparation, administered either orally (PO-OK432) or intratumorally (IT-OK432) on the immuno-reactivities of regional lymph nodes were investigated in gastric cancer patients. Although native lymph node lymphocytes (LNL) from untreated patients did not show any cytotoxicities against K562 and Raji cells, enhanced activities were found in LNL from patients administered OK432. Augmenting effects on the cytotoxicities of LNL by in vitro additional OK432, interleukin 2 or gamma-interferon were remarkable in the patients given IT-OK432. Moreover, the cytotoxicities of peripheral blood lymphocytes were augmented in vitro more strongly in patients given IT-OK432 than in those given PO-OK432. Flow cytometric analysis of LNL revealed a decrease in CD4+ cells by PO-OK432 and an increase in CD8+ cells by IT-OK432. An increase in CD4+2H4+ cells and a decrease in CD4+2H4- cells were observed in the patients given OK432, though CD8+CD11+ cells decreased by PO-OK432 while CD8+CD11+ cells increased by IT-OK432. Thus, it is suggested that LNL reactive to OK432 immunotherapy may differ between PO- and IT-OK432, and that the immunoreactivities of local lymph nodes and systemical immuno-reactivities may be highly potentiated by IT-OK432 rather than PO-OK432.     

Anti-tumor effect of LAK (lymphokine activated killer) cells against human bladder tumors transplanted to nude mouse

The effect of lymphokine-activated killer (LAK) cells on bladder tumor was examined in vivo and in vitro. In the in vitro experiment, 51Cr-cytotoxic assay was performed for which PBL were used as effector cells. A LAK activity of 26.6% was observed in PBL cultured with IL2 for 4 days, whereas OK-432-induced LAK activity was 22%. Furthermore, in the in vivo experiment, the anti-tumor effect of LAK cells was evaluated in human bladder tumor transplanted into nude mouse. IL2, OK432-induced LAK cells were injected intratumorally. In the LAK-treated group, inhibition of tumor growth was seen. Histologically, it was demonstrated that infiltrating lymphocytes were scattered around tumor cells. The augmentation of NK activity in spleen cells was observed in the LAK-treated group. Although further studies are required to establish its full significance, these findings suggest that immunotherapy against bladder tumors is hopeful.     

Natural killer activity of lymphocytes infiltrating human lung cancers following preoperative systemic recombinant interleukin 2

Tumor-infiltrating lymphocytes (TILs) show depressed natural killer (NK) activity compared with peripheral blood lymphocytes (PBLs). To determine if TIL NK function can be reactivated in vivo, 11 patients with tumors metastatic to the lung were treated with systemic recombinant interleukin 2 (rIL-2). Spontaneous TIL NK activity and NK activity after three days' incubation with 100 U/mL of rIL-2 were increased in patients receiving 15,000 U/kg of rIL-2 preoperatively compared with those receiving between 1000 and 10,000 U/kg. Histologically, higher doses of rIL-2 increased the number of intratumoral lymphocytes, the level of peritumoral lymphocytic transferrin, and the expression of HLA-DR. Spontaneous PBL NK activity in patients receiving between 10,000 and 15,000 U/kg of rIL-2 was also increased and was further increased by in vitro culture with rIL-2. Thus, PBL NK activity and TIL NK function in vivo can be augmented with 15,000 U/kg of systemic rIL-2. Both TIL- and PBL-inducible cytotoxicities were further enhanced by in vitro culture with rIL-2.     

Immunology of tumor infiltrating lymphocytes.

Frequently peripheral blood lymphocytes (PBL) do not reflect the tumor host relationship and cell mediated immunity in the PBL does not often correlate with prognosis. The tumor infiltrating lymphocytes (TIL) interact most closely with the tumor cells and are likely to more accurately reflect tumor host interactions. These studies indicate that TIL from pulmonary tumors are similar to PBL so far as their cell surface markers are concerned. The percentage of T-cells, B-cells, helper cells, suppressor cells, and NK cells are similar in the two compartments. However, the TIL are markedly suppressed in their functional capacity as measured by their proliferative and cytotoxic activity. In addition, natural killer (NK) cell activity is markedly diminished in TIL as opposed to the PBL. In addition, the direct injection of BCG into these tumors reverses this phenomenon by significantly increasing T-cell and NK cell functional activity. Thus, the microenvironment of the tumor profoundly affects the immunologic relationship between the tumor and the host.     

Intratumoral administration of OK432 in rats with liver tumor

The approach to the treatment of unresectable liver tumor involves immunotherapy. Systemic administration of OK432 has been widely used in the treatment of malignant neoplasms. However, the most potent antitumor activity of the drug may be expected when it is administered intratumorally. The author evaluated the effect of intratumoral injection of OK-432 on the survival time, the immunological parameters (such as the NK activities of spleen cells and peritoneal exudate cells and the interferon production by spleen cells) and the tumor infiltrating lymphocytes (TIL) in the rats with liver tumor induced by feeding the hepatocarcinogen, 3'-methyl-4-di-methyl-aminoazobenzene. The mean survival time was significantly longer in the rats injected with OK432 intratumorally (I.T. group) than in the rats injected with OK432 intraperitoneally only (I.P. group) and in the rats injected with normal saline intratumorally (Control group). The immunological parameters significantly improved in the rats of I.T. group than in the controls. Intratumoral injection of OK432 increased the number of TIL, especially NK cells and suppressor/cytotoxic T cells. These beneficial effects could be responsible for the better survival time in the rats of I.T. group. The author concluded that the intratumoral OK432 administration therapy is effective for the treatment of the patients with unresectable liver tumor.     

Effects of the oral or intratumoral administration of OK432 on the immuno-reactivities of regional lymph nodes in gastric cancer patients

The effects of OK432, a streptococcal preparation, administered either orally (PO-OK432) or intratumorally (IT-OK432) on the immuno-reactivities of regional lymph nodes were investigated in gastric cancer patients. Although native lymph node lymphocytes (LNL) from untreated patients did not show any cytotoxicities against K562 and Raji cells, enhanced activities were found in LNL from patients administered OK432. Augmenting effects on the cytotoxicities of LNL byin vitro additional OK432, interleukin 2 or γ-interferon were remarkable in the patients given IT-OK432. Moreover, the cytotoxicities of peripheral blood lymphocytes were augmentedin vitro more strongly in patients given IT-OK432 than in those given PO-OK432. Flow cytometric analysis of LNL revealed a decrease in CD4⁺ cells by PO-OK432 and an increase in CD8⁺ cells by IT-OK432. An increase in CD4⁺2H4⁺ cells and a decrease in CD4⁺2H4⁻ cells were observed in the patients given OK432, though CD8⁺CD11⁺ cells decreased by PO-OK432 while CD8⁺CD11⁺ cells increased by IT-OK432. Thus, it is suggested that LNL reactive to OK432 immunotherapy may differ between PO- and IT-OK432, and that the immunoreactivities of local lymph nodes and systemical immuno-reactivities may be highly potentiated by IT-OK432 rather than PO-OK432.     

肿瘤浸润性淋巴细胞在白细胞介素-2、-4协同下局部过继免疫治疗膀胱癌的实验研究

目的　观察膀胱癌肿瘤浸润性淋巴细胞 (TIL)联合不同细胞因子瘤灶内过继免疫抗癌的效应及对机体全身抗肿瘤免疫机制的影响。方法　建立BTT73 9动物模型 ,分离、培养TIL。采用正交设计实验方法 ,将TIL、白细胞介素 (IL) 2、 4及三因素交互组合悬液分别直接注射至瘤体内 ,定期测量肿瘤体积 ,免疫治疗 2周后检测NK细胞活性、T淋巴细胞转刺激指数 ,观察组织学及超微结构变化。结果　比较对照组 ,治疗 2周时各TIL相关组均不同程度抑制了膀胱肿瘤体积的增长 ,且NK细胞活性及T淋巴细胞转化增殖能力得以提高 (P <0 .0 5 )。TIL/IL 2疗法明显抑制了瘤体的增长 ,免疫治疗 1周后即表现出协同增强效应 (P <0 .0 5 ) ,而NK细胞活性及T淋巴细胞转刺激指数也显著提高 (P <0 .0 5 )。TIL/IL 2 /IL 4组获得了较强的抗癌功效 ,但与TIL/IL 2组差异无显著性 (P >0 .0 5 )。超微结构变化显示出TIL强烈的溶癌现象。结论　TIL在细胞因子特别是IL 2协同下瘤灶内注射的局部免疫疗法 ,具有较强的抗膀胱癌效应 ,并显著提高了机体全身抗瘤免疫功能。     

Immunoresponse of tissue infiltrating lymphocytes in bladder tumors

Local immunocompetence was evaluated immunohistochemically in patients with bladder tumors before and after local injections of an immunomodulator. The subpopulations of tissue infiltrating lymphocytes (TIL) were examined by staining six serial sections with Leu4, Leu7, Leu10, LeuM3, OKT4, and OKT8 antibodies. T cells predominated over B cells in 19 of 25 bladder tumors. T cell infiltration was prominent around tumor cells, and it was marked in non-invasive tumors. B cells were rare in the stroma. In patients with low-stage tumors, OKT8 cells were more prominent than OKT4 cells. NK cells accumulated within cancer nests but their infiltration was scanty in invasive bladder tumors. Before surgery, immunomodulators (OK-432, IL-2) were injected intratumorally. Their administration resulted in marked increase of T and NK cells, irrespective of the stage of disease; there was a slight increase in B cells. These findings suggest that local immunosurveillance plays a role against bladder tumors. Further studies are required to elucidate host immune responses in the microenvironment of the cancer site, as well as the systemic immune reaction.     

Augmentation of cytotoxicity of regional lymph node lymphocytes of gastric cancer after intratumoral injection of OK-432

Although regional lymph node lymphocytes (LNL) are thought to be a barrier of the immunological surveillance, their natural cytotoxicity is strongly suppressed. In this study, an immunopotentiator OK-432 (10K.E.) was injected into gastric cancer lesions under endoscopy 1 week before operation, and the effects on the cytotoxicity of LNL were examined by single cell assay. This assay is characterized by assessment of killer cell frequency in preventing the killer cells from recycling by their fixation in agarose, and by direct microscopic observation of both binding and killing phases. NK sensitive cell line K562 was used as target cells. By intratumoral injection of OK-432, the killer cell frequency of LNL in 8 out of 20 cases was elevated to almost equal level as that of peripheral blood lymphocytes. In clinical stages these 8 cases belonged to early stages (stage I 7 cases, stage II 1 case). In LNL subsets of OK-432 injected cases, the percentage of OKT8 positive cells was decreased and the ratio of OKT4 and OKT8 was significantly increased compared with non-injected group.     

OK432 inhibits experimental hepatic metastasis of colon adenocarcinoma ACL-15 in F344 rats

The effect of OK432 on hepatic metastasis, induced by inoculating 1 X 10⁶ ACL-15 cells from a rat colon adenocarcinoma cell line into the ileocolic vein of male F344 rats, was investigated in this study. Metastases were detected 14 days after inoculation in the control rats, however, pretreatment 3 days prior to the tumor cell inoculation with an anti-asialoGM1 antibody, which eliminates natural killer (NK) cell activity in vitro, increased the number of hepatic metastases, shortened the survival time, and decreased the NK activity of the nonparenchymal liver cells (NPC). In contrast, pretreatment with OK432 2 days prior to tumor inoculation significantly decreased the number of hepatic metastases, prolonged the survival time, and augmented the NK activity of the NPC, although treatment with OK432 3 or 7 days after inoculation did not decrease the number of hepatic metastases. Moreover, NPC from the OK432-pretreated rats had a marked antitumor effect against ACL-15 cells in the Winn's neutralization test. The results of this study indicate that pretreatment with OK432 before tumor cell inoculation inhibits hepatic metastasis in this experimental model, possibly by augmentating liver-associated NK activity.     

Adriamycin-mediated potentiation of cytotoxicity against freshly isolated bladder cancer cells by autologous non-activated peripheral blood lymphocytes and tumor infiltrating lymphocytes

PURPOSE: Previous studies indicated that cancer patients lack functional anti-tumor cytotoxic lymphocytes. However, anti-tumor cytotoxic lymphocytes may coexist with immunoresistant tumor cells. We reasoned that anti-tumor cytotoxic activity of lymphocytes may be revealed if the tumor cells are sensitized to killing. It has been reported that adriamycin (ADR) exhibits various immunomodulating activities. In the present study, we investigate the effect of ADR on the susceptibility of freshly isolated bladder cancer cells to lysis by autologous non-activated peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL). MATERIALS AND METHODS: Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. RESULTS: Treatment of ADR-resistant fresh bladder cancer cells with ADR at 0.1 microg./ml. or more for 3 hours or more enhanced their susceptibility to lysis by autologous PBL. This ADR-induced enhancement of susceptibility of fresh bladder cancer cells to lysis by PBL was also observed when lymphokine activated killer cells, purified natural killer cells and T lymphocytes were used as effector cells. Furthermore, while cytotoxicity of freshly derived TIL against autologous bladder cancer cells was minimal, significant cytotoxicity was observed with ADR-treated bladder cancer cells. The ADR analogs, epirubicin and pirarubicin, also enhanced the susceptibility of bladder cancer cells to lysis by autologous PBL. Treatment of bladder cancer cells with ADR had no effect on the expression of MHC class I on the cancer cells or the frequency of bladder cancer target cell conjugates to autologous PBL. Treatment of bladder cancer cells with ADR augmented their sensitivity to anti-Fas monoclonal antibody and tumor necrosis factor-a. Pretreatment of effector cells with ADR had no effect on their cytotoxic function. CONCLUSIONS: These findings demonstrate that PBL and TIL in patients with bladder cancer exhibit anti-tumor cytotoxic function, but their function is not manifested due to development or acquisition of tumor cell resistance to killing. However, the resistance of bladder cancer cells to killing by cytotoxic lymphocytes is overcome if cancer cells are sensitized by subtoxic concentrations of ADR. These findings suggest that treatment of bladder cancer patients with low doses of ADR may sensitize the cancer cells to killing by autologous circulating and tumor infiltrating lymphocytes and may be a novel immunotherapeutic modality for the treatment of drug-resistant and/or immune-resistant bladder cancer.     

The regulation of natural killer cell activity by splenic nonspecific suppressor cells and its modification in cancer patients

Spleen cells (SC), splenic venous blood lymphocytes (SVL) and peripheral blood lymphocytes (PBL) from gastric and esophageal cancer patients were simultaneously tested for natural killer (NK) and nonspecific suppressor (Ts) cell activities. Furthermore, the influence of Ts activity on the augmentation of NK activity by a biological response modifier (BRM) was also investigated. Positive Ts activities were frequently detected in the SC, SVL and PBL of advanced cancer patients. The NK activities of SC and SVL were maintained even in advanced cancer patients, though significantly depressed NK activities were observed in the PBL of advanced cases. Cancer patient SC, SVL and PBL with positive Ts activity showed low NK activities. Moreover, the NK activities of SVL and PBL were low in the patients with positive Ts activity in SC. The NK activity of normal control PBL was strongly augmented by interleukin 2, interferon and OK-432. These BRMs exhibited comparable capacities to augment the NK activities of SC, SVL and PBL with negative Ts activity in cancer patients, however, the effects of these agents seemed to be low in cells with a positive Ts activity. These results suggested that NK activity might be regulated by nonspecific suppressor cells and the presence of suppressor cells might affect the augmentation of NK activity through BRM in circulating blood lymphocytes and also in spleen cells.     

The regulation of natural killer cell activity by splenic nonspecific suppressor cells and its modification in cancer patients

Spleen cells (SC), splenic venous blood lymphocytes (SVL) and peripheral blood lymphocytes (PBL) from gastric and esophageal cancer patients were simultaneously tested for natural killer (NK) and nonspecific suppressor (Ts) cell activities. Furthermore, the influence of Ts activity on the augmentation of NK activity by a biological response modifier (BRM) was also investigated. Positive Ts activities were frequently detected in the SC, SVL and PBL of advanced cancer patients. The NK activities of SC and SVL were maintained even in advanced cancer patients, though significantly depressed NK activities were observed in the PBL of advanced cases. Cancer patient SC, SVL and PBL with positive Ts activity showed low NK activities. Moreover, the NK activities of SVL and PBL were low in the patients with positive Ts activity in SC. The NK activity of normal control PBL was strongly augmented by interleukin 2, interferon and OK-432. These BRMs exhibited comparable capacities to augment the NK activities of SC, SVL and PBL with negative Ts activity in cancer patients, however, the effects of these agents seemed to be low in cells with a positive Ts activity. These results suggested that NK activity might be regulated by nonspecific suppressor cells and the presence of suppressor cells might affect the augmentation of NK activity through BRM in circulating blood lymphocytes and also in spleen cells.     

Activation by the protein-bound polysaccharide PSK (krestin) of cytotoxic lymphocytes that act on fresh autologous tumor cells and T24 human urinary bladder transitional carcinoma cell line in patients with urinary bladder cancer

PSK, a protein-bound polysaccharide Kureha, was tested for its ability to modulate the cytotoxicity of lymphocytes that act on autologous tumor cells and T24 human urinary bladder tumor cells in urinary bladder cancer patients in a 6-h 51Cr release assay. In vitro treatment of peripheral blood lymphocytes (PBL) with PSK for 18 hours resulted in an augmentation or induction of cytotoxicity against relatively resistant T24 cells in previously reactive and nonreactive cases, respectively. The PSK-treated PBL were able to kill more effectively tumor cells that were freshly isolated from the same cancer patients than non-treated PBL. The effects of PSK were noted with PBL as well as tumor infiltrating lymphocytes (TIL) and with PSK at concentrations of 10 to 100 micrograms./ml., while PSK at higher doses reduced their lytic activities. The addition of PSK to the assay at the same concentrations also enhanced the cytotoxicities. Autologous tumor killing (ATK) activities of both large granular lymphocytes (LGL) and T lymphocytes were enhanced by PSK. Treatment of PBL with PSK did not effect on the proportion of PBL binding to the tumor cells, while it augmented the cytotoxic activity. Cell-free supernatant of PSK-stimulated lymphocyte culture did not contain any detectable amounts of interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). In addition, anti-IFN-alpha monoclonal antibody (MAb), anti-IFN-gamma MAb and anti-IL-2 MAb did not inhibit PSK-induced augmentation of cytotoxicity against T24. Oral administration of PSK (three gm./day) to patients with urinary bladder cancer daily for seven days before operation resulted in an augmentation of the cytotoxicity against T24 cells in five out of 10 patients and no change of the cytotoxicity in the other five patients. ATK activity was also enhanced by oral administration of PSK in three out of five patients. These results indicate that the antitumor activity of PSK may be in part mediated through activation of tumor killing system independent of IFN-alpha, IFN-gamma and IL-2.     

Clinical studies of the treatment of bladder tumor and its effects--experience in 86 cases

Prognosis on evaluable 86 patients with primary bladder tumor seen during the 10 years up to 1985 was evaluated in relation to treatment mode and tumor stage. The majority of patients underwent multimodal therapies including surgery, chemotherapy and immunotherapy with picibanil (OK432). Transurethral resection of the tumor was performed as an initial surgical treatment in 49 patients, 9 of whom ultimately underwent total cystectomy. After leaving hospital, these patients were kept on immunotherapy with OK432 and topical chemotherapy with bladder instillation of mitomycin C or adriamycin (ADM) with or without systemic administration of Tegefur as long as possible. The overall actual 5-year survival rate for the patients treated by initial transurethral resection was 80%. Recurrence rate for these 49 patients was 35%. Total cystectomy with urinary diversion was performed in 37 patients who had been placed postoperatively on systemic administration of Tegafur and immunotherapy with OK 432 as long as possible. The overall actual 5-year survival rate for the patients treated with total cystectomy was 54%. The patients with pT2 and lower stage tumor had an actual 5 year survival rate of 72%, while the patients with pT3 and higher stage tumors had a survival of 10%. The high recurrence rate in the patients with superficial tumor and the low actual survival rate of the patients with pT3 and higher stage remain a problem in the treatment of bladder tumor. In recent trials, bacillus Calmette-Gúerin instillation therapy has been initiated to lower the recurrence rate in superficial tumor and we have had a satisfactory 4-year result.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of tumour-infiltrating lymphocytes, regional lymph node lymphocytes and peripheral blood lymphocytes and their reaction to interferon-gamma in patients with renal carcinoma

The immunological distribution of tumour-infiltrating lymphocytes (TIL), regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL) was evaluated by means of immunohistochemical staining using monoclonal antibodies of each subset of lymphocytes (stored in frozen sections) in a series of 22 patients with renal carcinoma. The immunological effect of IFN (interferon)-gamma on these immunocompetent cells was also investigated. The effect of IFN-gamma on TIL was an increase in CD3 (pan-T cells), especially an increase in CD8 (cytotoxic/suppressor-T cells). When examining these cells according to stage and grade, a marked increase in CD3 was found in low stage and low grade patients. With regard to RLNL, there was a tendency towards a decrease in CD3 and an increase in CD20 (B-cells) following the administration of IFN-gamma. No specific effect on stage and grade was observed apart from a reduction in T cell subset ratios in high grade patients. With regard to PBL, no specific trend was noted except for an increase in CD16 (NK cells) when IFN-gamma was administered.     

Antitumor Killer Lymphocytes in the Peripheral Blood of a Patient with Transitional Cell Carcinoma of the Bladder

Background: Peripheral blood lymphocytes (PBL) from patients with bladder cancer also contain cells possessing cytotoxic activity against autologous tumor cells. These cells are phenotypically heterogenous and include natural killer (NK) and cytotoxic T cells. This study investigated the role of cytotoxic lymphocytes directed against autologous bladder cancer cells.
Methods: PBL were obtained at intervals before and after surgery and analyzed for cytotoxic activity against autologous bladder cancer cells in 4-hour⁵¹ Cr release assay. PBL stimulated with autologous tumor cells were also transformed with human T-lymphotropic virus type-1, establishing a cell line (KB31) which was analyzed for phenotype and cytotoxic activity against the autologous tumor cells.
Results: PBL preoperative cytotoxic activity was low, but increased after surgery. Cytotoxic activity was found not only against autologous bladder cancer cells, but also against heterologous bladder cancer (KK-47) and myeloid leukemia (K562) cells, with the highest activity against the heterologous cell lines. The cytotoxic activity of KB31 was 40|X% against autologous tumor cells 6 weeks after initiation of the cell line, but decreased to 5|X% by 6 months. This activity was lower than that against the other cell lines, and was similar to that of PBL in short-term culture. Fluorescence-activated cell sorter (FACS) analysis demonstrated that in KB31 cells at 6 weeks, CD8+ cells were dominant, but CD56+ cells predominated at 6 months.
Conclusion: These results suggest that the presence of cytotoxic activity in the peripheral blood of the patient was due to both cytotoxic T cells and NK cells. The cytotoxic activity was lowest prior to surgery and increased postoperatively.