Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

Autologous tumor cell killing activity of tumor-associated lymphocytes in patients with head and neck carcinomas

In order to select the most cytotoxic effector cells for adoptive immunotherapy, lymphokine activated killer (LAK) cells, tumor infiltrating lymphocytes (TILs) and autologous mixed lymphocyte tumor cell culture (MLTC) cells derived from peripheral blood mononuclear cells (PBMC) in the same subject with head and neck carcinomas were prepared. The autologous tumor cell killing activity and cell surface phenotypes of each of the three effector cells were studied. MLTC cells cultured with interleukin-2 (IL-2) showed the strongest cytotoxic activity among these three different effector cells. Although TILs had suppressed killing activity immediately after isolation, after successive cultivations with IL-2, a cytotoxic activity against autologous tumor cells stronger than that of LAK cells appeared. Both IL-2 stimulated MLTC cells and TILs showed an enrichment of CD8 positive and CDU negative cells in a CD3 positive subpopulation.Abbreviations CD cluster differentiation - IL-2 interleukin-2 - LA lymphokine activated - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - NK natural killer - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Biological significance of autologous tumor-killing activity and its induction therapy

Conclusion The overall results presented in this review demonstrate that positive ATK activity at the time of surgery predicts a favorable clinical course in patients who have primary localized solid tumor and receive curative operation. The strong correlation of ATK activity with disease-free interval and total survival (a) indicates that ATK activity is a meaningful prognostic indicator and (b) provides evidence for immunological control of tumor growth and metastasis. According to these data, it is unlikely that cancer patients who remain tumor-free after 5 years of follow-up will develop recurrence or die from the disease. Although there is no direct evidence that ATK effector cells play a critical role in regression of tumor and prevention of tumor regrowth, the lack of ATK activity in patients who relapsed and died after surgery may not result from factors related to their poor performance status since no differences have been observed in background factors between ATK-positive and-negative groups. The prognostic value of ATK activity in patients with documented metastatic tumors has not been established yet. In this respect, however, the induction of ATK activity by BRM has positively correlated with prolonged survival time, while such a correlation is not observed with other parameters such as NK cells or LAK cell activity.Based on the possible biological significance of ATK activity, clinical trials have been conducted to determine whether the induction of ATK activity before surgery by administration of BRM could improve the clinical outcome in patients who naturally have no such potential. The preliminary data indicate that the presence of both natural and induced ATK activity is strongly associated with longterm survival. Thus, considerable emphasis should be placed on a strategy that induces ATK activity in vivo. Such an approach may provide a new focus for cancer immunotherapy.     

Clinical therapeutic effect of adoptive immunotherapy using IL-2-cultured autologous lymphocytes

Our method of adoptive immunotherapy (AIT) using autologous IL-2-cultured lymphocytes differs from so-called LAK therapy in several points. We (1) obtain cultured lymphocytes from effusion lymphocytes (EL) or regional lymph-node lymphocytes (RLNL), when possible, rather than peripheral blood lymphocytes (PBL), (2) use crude IL-2 to induce T cell proliferation and to maintain killer activity, (3) use sonicated autologous tumor extract as antigen (Ag) to stimulate proliferation of cytotoxic T cells, and (4) pretreat the patients with local administration of OK-432 before AIT to induce effector cells that act synergistically with transferred killer cells. Surface marker analysis showed that OKT3, IL-2 receptor, Leu 2+15- cells were elevated while Leu 11a and Leu 3+8+ cells were decreased. Culture of RLNL augmented the expression of Leu 3+8- marker. Both of PBL and RLNL responded to Ag, and their auto-tumor killing activities were augmented in about half of the patients while rarely decrease by the addition of Ag. Response rates of patients with pleural effusion due to breast cancer and those with liver metastasis of breast cancer were 94% and 60%, respectively. Moreover, the survival was prolonged in the treated patients with pleural effusion or gastric cancer patients with peritoneal dissemination.     

Intrapleural administration of OK432 in cancer patients: Augmentation of autologous tumor killing activity of tumor-associated large granular lymphocytes

Summary Ten patients with carcinomatous pleural effusions were treated with single intrapleural (i.pl.) injections of the streptococcal preparation OK432 on day 0 and the effects of i.pl. OK432 on the lysis of fresh or cryopreserved autologous tumor cells isolated from the pleural effusions were observed on day 7. In eight patients tumor cells in the effusions had decreased or disappeared by day 7. The other two patients, however, had no clinical evidence of therapeutic benefit from i.pl. OK432. Effusion tumor cells were relatively resistant to lysis by autologous lymphocytes when tested in a 4-h ⁵¹Cr-release assay. Positive reactions were recorded for blood and effusion lymphocytes in two of ten untreated patients. Injection of OK432 i.pl. resulted in an induction or augmentation of cytotoxicity against autologous tumor cells and K562 in the effusions of seven of ten subjects by day 7. In contrast, autologous tumor killing activity of blood lymphocytes was not always modified by i.pl. OK432. Purification of large granular lymphocytes (LGL) by discontinuous Percoll gradient centrifugation enriched autologous tumor killing activity, with no reactivity in LGL-depleted, small T lymphocytes. Significant lysis of autologous tumor cells was observed with effusion LGL from seven of ten untreated patients. Seven days after i.pl. OK432 injection, effusion LGL expressed enhanced cytotoxicity against autologous effusion tumor cells, whereas T cells were still not cytotoxic to autologous tumor cells on day 7. The frequency of LGL among effusion lymphocytes was not altered by i. pl. OK432. Adherent effusion cells were not involved in lysis of autologous effusion tumor cells in either untreated or OK432-treated patients. In vitro treatment of blood and effusion lymphocytes with OK432 induced an enhancement of autologous tumor-killing activity in patients who subsequently responded to i.pl. OK432 treatment. OK432 augmented in vitro autologous tumor killing activity of LGL, whereas T cells failed to lyse autologous tumor cells even after in vitro activation with OK432. These results indicate that i.pl. administration of OK432 to cancer patients will result in an augmentation of autologous tumor killing activity of LGL in the pleural effusions, and that this could be responsible for the antitumor activity of i.pl. OK432 therapy.     

Autologous tumor killing and natural cytotoxic activity of tumor-associated macrophages in cancer patients

Summary Tumor-associated macrophages (TAM) isolated from pleural effusions and ascites fluids of cancer patients were tested for cytotoxicity against freshly isolated autologous tumor cells and K562 in a 4-h⁵¹ Cr-release assay, and in vitro effects of OK432 (a streptococcal preparation) and partially purified human leukocyte interferon (IFN) on their cytotoxicities were examined. Positive cytotoxicities against K562 were recorded for TAM samples from 2 of 23 pleural effusions and 3 of 10 ascites specimens. Tumor-associated macrophages were not cytotoxic to autologous tumor cells, while low but significant lysis was observed with tumor-associated lymphocytes (TAL) samples from 2 of 13 pleural effusions and 1 of 6 ascites specimens. In vitro treatment with OK432 resulted in an enhancement of natural cytotoxicity in 4 of 13 TAM and 10 of 15 TAL samples. An induction or augmentation of autologous tumor killing activity by OK432 was observed in 2 of 10 TAM and 8 of 11 TAL samples. In contrast, IFN failed to induce autologous tumor killing activity, although IFN-enhanced lysis of K562 was detected in 1 of 7 TAM and 2 of 9 TAL samples. These results indicated that autologous tumor killing and natural cytotoxic activities were defective in macrophages and lymphocytes at the site of the tumor growth, and both activities were strongly enhanced by OK432 rather than IFN.     

Th-1 lymphocytes induce dendritic cell tumor killing activity by an IFN-γ-dependent mechanism

Dendritic cells (DCs) encompass a heterogeneous population of cells capable of orchestrating innate and adaptive immune responses. The ability of DCs to act as professional APCs has been the foundation for the development and use of these cells as vaccines in cancer immunotherapy. DCs are also endowed with the nonconventional property of directly killing tumor cells. The current study investigates the regulation of murine DC cytotoxic function by T lymphocytes. We provide evidence that CD4(+) Th-1, but not Th-2, Th-17 cells, or regulatory T cells, are capable of inducing DC cytotoxic function. IFN-γ was identified as the major factor responsible for Th-1-induced DC tumoricidal activity. Tumor cell killing mediated by Th-1-activated killer DCs was dependent on inducible NO synthase expression and NO production. Importantly, Th-1-activated killer DCs were capable of presenting the acquired Ags from the killed tumor cells to T lymphocytes in vitro or in vivo. These observations offer new possibilities for the application of killer DCs in cancer immunotherapy.     

Role of human circulating and tumor-infiltrating lymphocytes in cancer defense and treatment

We have analyzed the anticancer efficacy of various subsets of human circulating and tumor-infiltrating lymphocytes (TIL). These studies showed that circulating natural killer (NK) cells mediate the most potent oncolytic activity against a variety of tumor targets, after enrichment or stimulation with interleukin-2 (IL-2). Interestingly, NK cell oncolytic activity was directed also against tumor targets frequently designated as 'NK-resistant'. This indicates that NK cells display a broader spectrum of killing than is commonly recognized. TIL did not display any tumoricidal activity when unstimulated, but acquired cytotoxic potential after activation with IL-2. Comparative studies of TIL and circulating lymphocytes from patients with ovarian cancer demonstrated that these two groups of lymphocytes manifested similar levels of cytotoxicity and the same spectrum of target cell killing. No specificity in autologous tumor cell killing was displayed by TIL; instead, TIL were effective against autologous as well as allogeneic tumor targets. The lack of TIL tumor specificity was not detected only in ovarian tumors, but was manifested also in renal- and squamous-cell cancers. Characterization studies demonstrated that the primary oncolytic cells in the periphery and among TIL are NK cells. T lymphocytes displayed some, but rather negligible cytotoxic activity. In contrast, when IL-2-activated NK and T cells were analyzed for lytic activity against normal hematopoietic cells, T cells displayed high levels of bone marrow killing. The anti-bone marrow lytic activity of IL-2-activated T lymphocytes may be harmful after therapy with conventionally prepared lymphokine-activated killers. In light of these observations, new directions to adoptive immunotherapy are discussed.     

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

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

EGFR-Targeted Granzyme B Expressed in NK Cells Enhances Natural Cytotoxicity and Mediates Specific Killing of Tumor Cells

Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells'' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.     

Natural and activated cytotoxic lymphocytes which act on autologous and allogeneic tumor cells

Summary A considerable proportion of human blood lymphocytes that express natural cytotoxic potential belong to the T subset. They are heterogenous with regard to their surface receptor characteristics. Results obtained with the exceptionally sensitive cell lines (such as K562, Molt-4) and freshly harvested cells as targets reveal different types of cytotoxicity. While certain cell lines may be killed without the involvement of antigen recognition, the freshly harvested cells are recognized and killed by lymphocytes that carry receptors for surface antigens on these targets. In accordance, such tests can reveal the cytotoxicity of T cells that carry receptors for histocompatibility antigens. In view of the high frequency of these cells in the lymphocyte population, enlargement of the clone is not always necessary and activation suffices.Cytotoxicity against autologous tumor cells derived from surgical specimens of sarcomas and carcinomas was demonstrated only in a few patients when the blood lymphocytes were used directly after collection. Cytotoxic cells could be generated in a proportion of cases when a stimulus was given, e.g., co-cultivation with allogeneic lymphocytes in the conventional MLC test. Co-cultivation of autologous lymphocytes and tumor biopsy cells was the most efficient measure for the generation of auto-tumor cytotoxicity. This condition allows the enlargement of the reactive clone. When this was inhibited, e.g., by the presence of interferon in the mixed culture no auto-tumor killing was generated.We would like to emphasize that tumor cells seem not to share the membrane properties of the in vitro model lines, which exhibit high sensitivity to the natural or activated killer cells without the involvement of antigen recognition.     

Characterization of recirculating lymphocytes in rheumatoid arthritis patients: selective deficiency of natural killer cells in thoracic duct lymph

Five patients with rheumatoid arthritis (RA), who were treated by lymphocyte depletion by using thoracic duct drainage (TDD), provided an opportunity to characterize the phenotype and function of their recirculating lymphocytes. We found that: a) thoracic duct lymphocytes (TDL) were similar in their proportion of T cells (83% +/- 6 OKT3+), OKT4+ subset (65% +/- 8), and OKT8+ subset (22% +/- 6) to peripheral blood lymphocytes (PBL): b) fewer natural killer-like cells were present in TDL (5% +/- 4 Leu-7+; 2% +/- 2 Leu-11+: 8% +/- 2 OKM -1+) than in PBL (20% +/- 10 Leu-7+: 11% +/- 6 Leu-11+; 18% +/- 5 OKM -1) (p less than 0.01); c) TDL differed from synovial fluid lymphocytes ( SFL ) and synovial membrane lymphocytes ( SML ) in that TDL lacked a high percentage of activated lymphocytes (T cells bearing Ia antigen, OKT10 , and transferrin receptor): d) immature T cells (expressing either OKT6 antigen or reactive with peanut agglutinin) were not found in TDL even late in the course of TDD: and e) in vitro functional studies demonstrated that TDL were similar to PBL in their ability to synthesize immunoglobulin after mitogen stimulation and to generate cytotoxic T lymphocytes capable of lysing autologous EBV-transformed B cells. However, natural killer activity, as measured by lysis of K562 cells was significantly lower in TDL than PBL (p less than 0.05). These results demonstrate that natural killer cells defined by phenotype and function are excluded from thoracic duct lymph and thus have a circulation pattern different from most T cells.     

Tumor-specific cytolysis by lymphocytes infiltrating human melanomas

Tumor infiltrating lymphocytes (TIL) were grown in IL-2 from single cell tumor suspensions of 14 human melanomas resected from 12 patients. As a function of time in culture, 4 of 14 TIL cultures eventually expressed highly specific cytolytic activity against fresh autologous melanoma targets in short term chromium release assays, failing to lyse multiple allogeneic tumors or autologous normal cells. These highly specific TIL were identified as CTL by phenotype (CD3+/CD4-/CD8+/Leu7-) and by function (lysis inhibited by antibodies directed against CD3 and MHC class I molecules). Cell separation experiments using immunomagnetic beads identified a highly tumor-specific CTL subpopulation within a nonspecific TIL culture, suggesting that the lytic activity of tumor-specific CTL may be diluted by the nonspecific killer activity present in heterogeneous TIL cultures. These studies provide evidence for specific MHC-restricted human immune responses against autologous tumor in cancer-bearing patients, and may be of importance to ongoing clinical trials using TIL in the immunotherapy of advanced malignancies.     

Induction of cytotoxicity against autologous tumour cells by interleukin-12: evidence for intrinsic anti-tumor immune capacity in curatively resected gastrointestinal tumour patients

The aim of this study was to investigate the cell-mediated immune response in 14 patients undergoing curative resection for a gastrointestinal tumor by the induction of peripheral blood mononuclear cell (PBMC)-mediated immune activity against autologous tumour cells. PBMC were stimulated by interleukin-12 (IL-12; 100 IU/ml) and IL-2 (1,000 IU/ml) without contact with tumour cells for 36 h. Specific cytotoxic activity against autologous tumour cells (auTu), natural killer (NK)-sensitive cells (K562) and allogeneic tumour cells (RF48/HT29) was determined by fluorescence cytotoxicity assay. Additionally, inhibition experiments using the mononuclear antibodies (mAb) FMC16 and W6/32 against major histocompatibility complex I (MHC I) on autologous tumour cells were performed in order to determine the involvement of specific T lymphocytes. The cytotoxic activity of unstimulated PBMC did not differ between the three target cells. IL-12 caused a 3.2-fold increase in activity against auTu ( P=0.002). In contrast, after stimulation with IL-2, only a slight increase in activity was observed. After IL-12 stimulation, cytotoxic activity against auTu was 2.5- to 2.7-fold higher than the corresponding activity against K562/allogeneic tumour cells ( P=0.002/ P=0.006). After blocking of the MHC I complex on auTu by FMC 16 or W6/32 mAb, a 62.9%/74.4% reduction in the specific cytotoxicity of IL-12-stimulated PBMC was found. In summary, IL-12 induced an effective immune response against auTu, which was partly mediated by specific cytotoxic T lymphocytes (CTL). It was considered that de novo generation of this activity during 36 h incubation without antigen contact was hardly possible, but that the observed induction of effective anti-tumor cytotoxicity was rather based on the re-activation of a pre-existing immune potential from the tumour-host interaction. These findings indicate the existence of an autologous anti-tumor immune response following curative resection in patients undergoing surgery for solid tumours, which might influence the development of tumour recurrence from disseminated tumour cells. Making use of this capacity could constitute an attractive immunotherapeutical approach for curatively operated tumour patients.     

Use of human leukocyte antigen-mismatched allogeneic lymphokine-activated killer cells and interleukin-2 in the adoptive immunotherapy of patients with malignancies

Clinical effects and side effects were studied in the adoptive immunotherapy of patients bearing malignant diseases using human leukocyte antigen (HLA)-mismatched allogeneic lymphokine-activated killer (LAK) cells. Allogeneic LAK cells were induced from peripheral blood lymphocytes (PBL) of normal donors by means of initial stimulation with pokeweed mitogen (PWM). Six of 15 patients applied in the adoptive immunotherapy showed clinical effects such as partial or complete regression of pulmonary metastasis, pleural effusion and primary tumor. All pulmonary metastatic lesions were eliminated in one case by this adoptive immunotherapy combined with chemotherapy. Generally toxic effects were chillness, fever and general fatigue which were reversible, and no allergic side effects occurred even though allogeneic LAK cells were injected frequently except one patient who showed preshock like symptom accompanied with leukocytopenia and continuous hypotension immediately after infusion but was finally rescued. In the patients who received more than 10¹¹ of allogeneic LAK cells, anti-HLA class I antibodies appeared without any evidence of autoantibody. However, immunological side effects were never experienced after injection of allogeneic LAK cells even when the anti-HLA class I antibodies appeared in the patients. Taken together, allogeneic LAK cells could be considered as alternative therapy for patients with malignancies who could not supply sufficient materials of autologous LAK cells.Abbreviations PWM pokeweed mitogen - LAK lymphokine-activated killer - IL-2 interleukin 2 - PEL peripheral blood lymphocytes - TIL tumor-infiltrating lymphocytes - GVHD graft-versus-host disease - HLA human leukocyte antigen     

Immunization of Rhesus monkeys with a SialylTn–mAb17-1A conjugate vaccine co-formulated with QS-21 induces a temporary systemic cytokine release and NK cytotoxicity against tumor cells

Tumor-associated antigens resulting from aberrant glycosylation, such as the SialylTn carbohydrate antigen, are frequently over-expressed on cancer cells and provide potential targets for cancer vaccination. Immunization of Rhesus monkeys with SialylTn coupled to a highly immunogenic carrier molecule and formulated on aluminum hydroxide induced a strong immune response against the carrier protein but only a moderate IgM immune response against the SialylTn carbohydrate antigen. Co-formulation with QS-21 adjuvant dramatically enhanced the anti-SialylTn immune response and resulted in a SialylTn-specific IgG switch. The kinetics of the carbohydrate-specific IgG response correlated with a temporary release of cytokines such as IFNγ, IL-2, IL-1β, TNFα and GM-CSF which was measurable in the immune serum by xMAP Multiplex technology. Furthermore, tumor cell killing by activated natural killer cells was induced. These data demonstrate that immunization with a tumor-associated carbohydrate antigen in a highly immunogenic formulation results in a temporary release of type 1 cytokines which may be required for the induction of a specific IgG immune response against the carbohydrate antigen as well as for activation of effector cells against tumor cells.     

The therapeutic effect of OK-432-combined adoptive immunotherapy against liver metastases from gastric or colorectal cancers

Twenty-four patients with liver metastases from gastric or colorectal cancer were treated with OK-432-combined adoptive immunotherapy (AIT). Lymphocytes isolated from regional lymph nodes or peripheral blood were cultured with medium containing T cell growth factor and sonicated tumor extract antigen (SE-Ag) for 9–13 days. The cultured lymphocytes were transferred mainly through the hepatic artery after the administration of OK-432, a streptococcal preparation. Sixteen of the 24 patients received a low dose of anti-cancer agents between the OK-432 injection and cell transfer. When cultured without SE-Ag, regional lymph node lymphocytes (RLNL) showed significantly (P<0.05) higher cytotoxic activity against autologous tumor cells and, on the contrary, lower cytotoxic activity against K562 than peripheral blood lymphocytes (PBL). When cultured with SE-Ag, cytotoxicity of RLNL against autologous tumor cells was nearly equivalent to that of PBL. The blastogenesis of fresh PBL to SE-Ag was significantly (P<0.05) augmented after the OK-432-combined AIT. Two patients showed complete response and 4 patients showed partial response among 19 patients who had evaluable lesions. Five patients whose liver metastases were resected were treated with OK-432-combined AIT as an adjuvant therapy. To date they are alive without recurrence in the liver.Abbreviations AIT adoptive immunotherapy - RLNL regional lymph node lymphocytes - SE-Ag sonicated tumor extract antigen     

Neuroimmunomodulation with enkephalins: in vitro enhancement of natural killer cell activity in peripheral blood lymphocytes from cancer patients

To initiate investigations into the effects of enkephalins on immune function in cancer patients, the effect of methionine-enkephalin and leucine-enkephalin on natural killer (NK) cell activity in isolated peripheral blood lymphocytes from cancer patients was investigated. Incubation of lymphocytes with either enkephalin resulted in significant increases in NK cell activity. At effector:target cell ratios of 100:1, 33:1 and 11:1 leucine-enkephalin significantly (p less than 0.05) enhanced NK activity at dilutions of 10(-6), 10(-8), 10(-10), and 10(-14) mg/ml. Similar results were obtained with methionine-enkephalin with the exception that the 10(-6) dilution gave insignificant changes at both the 33:1 and 11:1 cell ratios. The results indicate a difference in dose response to both enkephalins between lymphocytes from cancer patients and normal volunteers.     

Augmentation of antileukemia lytic activity by OKT3 monoclonal antibody: synergism of OKT3 and interleukin-2

We have shown that short-term incubation (45 min) of peripheral blood lymphocytes of normal donors with OKT3 monoclonal antibody (MoAb), directed against T-cell-associated antigen CD3, resulted in an acquisition of lytic activity against fresh leukemic cells. Induction of such antileukemia activity was specific for OKT3, since Leu-1 MoAb (directed against another T cell surface molecule, CD5) did not induce a lytic effect. The OKT3-generated antileukemia effect was displayed against various types of leukemia including chronic myelogenous leukemia and acute myelogenous leukemia of various histological subtypes (M1, M2, M5). We furthermore demonstrated that OKT3 MoAb substantially enhanced leukemia killing by interleukin-2 (IL-2)-activated killer cells obtained from peripheral blood of patients with leukemia. Of most importance was the observation that the combined treatment of effector cells with IL-2 and OKT3 MoAb resulted in the highest levels of lysis of both autologous and allogeneic fresh leukemic cells that have been observed in leukemic patients to date. Of importance was to note that OKT3 treatment was effective in induction of cytotoxic activity also in patients whose effector cells were unresponsive to stimulation with IL-2 alone. All of these observations suggest that IL-2-activated and OKT3-MoAb-treated effector cells may represent the most aggressive population of antileukemia-directed killer cells and may play a significant role in the treatment of human leukemia.