Unimpaired ability to generate adherent lymphokine-activated killer (A-LAK) cells in patients with primary or metastatic liver tumors

Summary Adherent lymphokine-activated killer cells (A-LAK cells) obtained from human peripheral blood mononuclear cells represent a population of potent antitumor effectors enriched in interleukin-2(IL-2)-activated natural killer cells. This study shows that A-LAK cells can be successfully generated from the blood of patients with liver cancer not treated with adjuvant chemotherapy or irradiation. Mononuclear cells were isolated from the blood of 33 patients with liver tumors (6 benign, 10 primary malignant, 17 metastatic) at the time of liver resection. A-LAK cells were separated by adherence to plastic following activation of peripheral blood mononuclear cells in 1000 U/ml recombinant IL-2. A-LAK cells (enriched up to 92% in CD3^–CD56⁺ cells) showed better subsequent expansion and two to six times higher antitumor cytotoxicity per cell than unseparated LAK cells cultured under the same conditions. The ability to generate A-LAK cells with superior in vitro cytotoxicity from the blood of most patients with liver cancer indicates that adoptive cellular immunotherapy may be a feasible and new way of treatment for primary and secondary hepatic neoplasms in man.     

In vivo migration and tissue localization of highly purified lymphokine-activated killer cells (A-LAK cells) in tumor-bearing rats

Our laboratory has previously reported that the adoptive transfer of highly purified lymphokine-activated killer cells (adherent-LAK, A-LAK) into Fischer 344 (F344) rats bearing established lung or liver micrometastases effectively reduced the resultant tumor growth more than 90%, leading to significant increases in animal survival (Cancer Res. 49, 1441, 1989). To begin to investigate the mechanism(s) by which A-LAK cells mediate this anti-tumor effect, we studied their migration patterns in F344 rats bearing experimentally induced lung and liver metastases as well as subcutaneous tumors. A-LAK cells which were phenotypically 95 to 100% natural killer cells/large granular lymphocytes were labeled with either 51Chromium or fluorescein diacetate (so as to be visualized microscopically). Intravenous injection of such labeled A-LAK cells did not show significant differences in their tissue distribution patterns in tumor-bearing versus normal rats, even when high levels of exogenous recombinant interleukin-2 (rIL-2) was administered. A-LAK cells first migrated to the lungs and then subsequently migrated to the liver and spleen as early as 2 to 6 hr following iv injection. The kinetics of exit of A-LAK cells from the pulmonary capillary beds was not significantly different in rats bearing 3-day micrometastases or 14-day macrometastases compared to normal rats. Moreover, the presence of metastases in the liver did not alter the extent or kinetics of entry of A-LAK cells into the liver even in the presence of exogenously administered rIL-2. Finally, in rats bearing subcutaneous tumors, no evidence could be obtained that A-LAK cells were selectively localized to the tumor site. Tissue sections of livers from metastases-bearing animals injected with fluorescein diacetate labeled A-LAK cells did not demonstrate significant numbers of A-LAK cells infiltrating tumor nests with or without the administration of exogenous IL-2. These data suggest that A-LAK cells may mediate tumor regression in vivo by direct and indirect mechanisms, possibly through the secretion of cytokines and/or the recruitment of secondary effector cells.     

Generation and characterization of purified adherent lymphokine-activated killer cells in mice

During the incubation of murine spleen, lymph node, or bone marrow cells with IL-2 (1000 U/ml) a small percentage of cells became adherent to the surface of plastic tissue culture flasks. After removal of the non-adherent lymphoid cells, plastic adherent lymphokine-activated killer (LAK) cells could be efficiently expanded in the presence of IL-2. Plastic adherent-derived A-LAK cells were characterized by high rates of proliferation and their cytotoxic activity was more than 10 fold higher than LAK cells generated in the bulk (unfractionated) spleen cell cultures. A-LAK cells could be continuously generated from the non-adherent cell population. Using multiple transfers (every 1 to 2 days) of non-adherent LAK cells into new flasks, new rounds of plastic adherent cells were generated with high expansion capability and high levels of cytotoxic activity. Morphologically, A-LAK cells were large granular lymphocyte and phenotypically expressed markers characteristic of NK cells (asialo GM1+, NK1.1+, Qa5+, Ly-6.2+, Thy-1.2+, but negative for Lyt-2.2 and L3T4). A-LAK cells generated from mice of different strains expressing low and high levels of NK cell activity were equally highly cytotoxic. However, A-LAK cells obtained from nude or beige mice had relatively lower levels of cytotoxicity. Stimulation of NK cell activity by poly I:C or inhibition by in vivo or in vitro treatment with anti-asialo GM1 serum did not affect the generation of A-LAK cells. A-LAK cells derived from spleen or bone marrow of C57BL/6 or nude mice treated with anti-asialo GM1 serum were found to be asialo GM1+ suggesting that A-LAK cell could be generated from the asialo GM1- precursor cells. Expansion of plastic adherent A-LAK cells in the presence of IL-2 could provide large numbers of highly purified cytotoxic A-LAK cells suitable for cancer immunotherapy.     

High expression of NKR-P1 is not an absolute requirement for natural killer activity in BDIX rats

 NKR-P1 has been identified as a triggering structure selectively expressed on rat natural killer (NK) cells and adherent lymphokine-activated killer (A-LAK) cells. In vivo treatment with anti-NKR-P1 monoclonal antibody (mAb 3.2.3) was shown to induce complete inhibition of NK cytotoxicity and elimination of LAK cell precursors in Lewis and Fisher rat strains. We investigated the effects of mAb 3.2.3 in a colon tumor model in BDIX rats. Inoculation of animals with mAb 3.2.3 even at very high doses induced a strong but incomplete inhibition of NK cytotoxicity in nylon-wool-non-adherent spleen and peripheral blood cells. Generation of adherent A-LAK cells from their spleen precursors was also strongly but not fully inhibited. We also investigated the effect of treatment with mAb 3.2.3 on the tumorigenicity of the NK-sensitive REGb cell line. When subcutaneously inoculated in syngeneic animals, REGb cells induce tumors that first grow for 2 weeks, then spontaneously regress and disappear. In contrast with previous results using anti-asialoGM1, no significant difference in tumor growth was observed between rats treated with mAb 3.2.3 and control animals, even with a long-term treatment. In vitro, mAb 3.2.3 exhibited the same incomplete efficiency. Nylon-wool-non-adherent spleen cells treated with mAb 3.2.3 plus complement were completely free of 3.2.3^bright cells, but retained a substantial NK activity and generated LAK cells after culture with IL-2. After an overnight incubation in standard medium of 3.2.3-depleted spleen cells, 3.2.3^bright cells were partially recovered and the NK cytotoxic activity, as well as the generation of LAK cells, was significantly enhanced. These results suggest that a strong expression of NKR-P1 is not required for BDIX mononuclear cells to exhibit NK function and generate LAK cells under IL-2 activation. Received: 11 July 1995 / Accepted: 16 November 1995     

Factors,including transforming growth factor β, released in the glioblastoma residual cavity,impair activity of adherent lymphokine-activated killer cells

Adherent lymphokine-activated killer (A-LAK) cells were obtained from peripheral blood lymphocytes of patients with recurrent glioblastoma. In vitro features of A-LAK cultures were assessed in comparison to those of non-adherent lymphokine-activated killer (NA-LAK) cells of the same patients with regard to cytotoxic activity, proliferation and surface markers. Only in a minority of cases did A-LAK cells show a markedly higher cytotoxicity on K562, Daudi and allogeneic glioblastoma cells. Nevertheless, A-LAK cells proliferated significantly better than NA-LAK and contained higher percentages of CD16⁺, CD56⁺ and CD25⁺ cells, indicating that A-LAK cells from these patients represent a subpopulation of lymphocytes enriched for activated natural killer cells. We also investigated whether immunosuppressive factor(s) were present in the tumour bed of recurrent gliomas. To this end, samples of glioblastoma cavity fluid (GCF), which accumulates in the cavity of subtotally removed tumour, were recovered and tested for the presence of immunosuppressive activity. All GCF samples analysed were shown to inhibit in vitro proliferation and antitumour cytotoxicity of 1-week-cultured A-LAK cells in a dose-dependent manner. Such GCF activity was effectively antagonized by a transforming growth factor (TGF) neutralizing antibody, indicating the involvement of TGF in lymphocyte inhibition. These results show that in the tumour cavity remaining after subtotal glioblastoma resection a marked immunosuppressive activity, probably due to local release of TGF, is present; such activity may negatively influence the therapeutic effectiveness of local cellular immunotherapy.     

Serum IL-1beta, IL-2, and IL-6 in insulin-dependent diabetic children

Insulin-dependent diabetes mellitus (IDDM) is a chronic disease characterized by T-cell-dependent autoimmune destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans, resulting in an absolute lack of insulin. T cells are activated in response to islet-dominant autoantigens, the result being the development of IDDM. Insulin is one of the islet autoantigens responsible for the activation of T-lymphocyte functions, inflammatory cytokine production, and development of IDDM. The aim of this study was to investigate serum concentrations of interleukin (IL)-1beta, IL-2, IL-6, and tumor necrosis factor (TNF)-alpha in children IDDM. The study population consisted of 27 children with IDDM and 25 healthy controls. Children with IDDM were divided into three subgroups: (1) previously diagnosed patients (long standing IDDM) (n : 15), (2) newly diagnosed patients with diabetic ketoacidosis (before treatment) (n : 12), and (3) newly diagnosed patients with diabetic ketoacidosis (after treatment for two weeks) (n : 12). In all stages of diabetes higher levels of IL-1beta and TNF-alpha and lower levels of IL-2 and IL-6 were detected. Our data about elevated serum IL-1beta, TNF-alpha and decreased IL-2, IL-6 levels in newly diagnosed IDDM patients in comparison with longer standing cases supports an activation of systemic inflammatory process during early phases of IDDM which may be indicative of an ongoing beta-cell destruction. Persistence of significant difference between the cases with IDDM monitored for a long time and controls in terms of IL-1beta, IL-2, IL-6, and TNF-alpha supports continuous activation during the late stages of diabetes.     

Effect of virus-modified tumor cell extracts,autologous mononuclear cell infusions and interleukin-2 on oncolytic activity of effector cells of patients with advanced ovarian cancer

Summary We studied the biological responses of six ovarian cancer patients after intraperitoneal (i.p.) injections of virus-modified tumor cell extracts (VMTE) and autologous peripheral blood mononuclear cells, collected by leukapheresis after two injections of VMTE. VMTE was prepared from allogeneic ovarian cell lines, OV2774 and CaOV3, modified by influenza virus, A/PR8/34. A dose of 9 mg VMTE was given i.p. in total of 2–4 injections, and (1–9) × 10⁸ autologous mononuclear cells were infused i.p., 24 h after the second VMTE injection, and 24 h and 72 h after the third VMTE injection. Both peripheral blood (PB) and peritoneal cavity (PC) effector cell cytotoxicity was significantly enhanced against the K562 cell line in the majority of patients, 24–48 h after the second and third VMTE injections. This was accompanied by a dramatic influx of neutrophils into PC (57-550-fold), increase in absolute numbers of lymphocytes, (including large granular lymphocytes) and monocytes, and resulted also in a significant decrease in the number of ascitic tumor cells (98% reduction). The infusion of autologous mononuclear cells did not appear to influence either cytotoxicity or cell infiltration of the peritoneal cavity. We also investigated the in vitro effect of recombinant interleukin-2 (IL-2) on effector cells from PB and PC from patients before and after VMTE treatment. Cytotoxicity of both of these compartments was significantly potentiated after culture with IL-2. In three out of five VMTE-treated patients, PC cytotoxicity was significantly higher after activation with IL-2 than that of patients before VMTE treatment. These data suggest that VMTE induces regional cellular immunity, which could be further potentiated by culture of PC effector cells with IL-2. Thus, combination of VMTE and IL-2 after regional administration could represent the effective therapy for patients with advanced ovarian cancer.     

Effects of granulocyte-colony-stimulating factor and interleukin-2 on ascites formation and the survival time of nude mice bearing human ovarian cancer cells

 The aim of this study was to elucidate the effect of intraperitoneal (i.p.) instillations of granulocyte-colony-stimulating factor (G-CSF) and/or interleukin-2 (IL-2) on ascites formation and the survival time of nude mice with malignant ascites, produced by i.p. inoculation of human ovarian cancer cells. When the nude mice were treated with medium alone, ascites was observed in all mice 28 days after tumor inoculation. When the mice were treated with cis-diamminedichloroplatinum(II) (cisplatin) alone, G-CSF alone or IL-2 alone, it took 35 days for the ascites to form in all mice. When cisplatin was combined with G-CSF or IL-2, one of ten mice did not form ascites during the observation period. Surprisingly, when G-CSF and IL-2 were simultaneously administered, ascites formation was not observed in any mice. Although i.p. treatment with cisplatin alone significantly prolonged the survival time, compared to medium alone, the lytic activity of spleen cells against HRA cells was significantly suppressed. When G-CSF or IL-2 was combined with cisplatin, the suppression by cisplatin was eliminated and subsequently resulted in a prolongation of the survival time. When G-CSF was combined with IL-2, both the peritoneal and splenic macrophages/monocytes were stimulated and the splenic lytic activity was about double that following treatment with G-CSF alone on IL-2 alone, suggesting that complete inhibition of ascites formation results not only from a significant increase of the peritoneal macrophages but also from enhancement of the lytic activity. Two mice, died from dissemination of tumor in the abdominal cavity, but eight mice survived without tumor for more than 90 days. As confirmed by monitoring body weight and hematocrit, G-CSF and IL-2 seemed to have no adverse effect. From these results, we conclude that a combination therapy with G-CSF and IL-2 might be of clinical use for inhibiting large amounts of ascites, which may inhibit therapeutic effects for ovarian cancer patients. Received: 20 May 1996 / Accepted: 19 September 1996     

Generation of tumor cell lysate-loaded dendritic cells preprogrammed for IL-12 production and augmented T cell response

Dendritic cells (DC) loaded with tumor associated antigens (TAA) are often used for the vaccination of cancer patients; however methodologies for the vaccine preparation have not yet been standardized. The purpose of this work was to optimize the ex-vivo production of functional TAA-loaded DC that would produce interleukin-2 (IL-12) and enhance the T cell response. We generated ex-vivo DC from human monocytes with granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4, and whole necrotic tumor cells (cell lysates) of cancer cell lines were used as model TAA. DC were loaded with lysates without or with additional tumor necrosis factor-alpha (TNF-alpha), or cytokine combination treatments and tested for functional ability in vitro. Tumor cell lysates alone did not fully mature DC either phenotypically or functionally. After antigen uptake additional maturation signals were necessary. TNF-alpha matured DC phenotypically, but additional interferon-gamma (IFN-gamma) treatment was necessary to achieve functional maturation, the production of significant amounts of IL-12. Since IL-12 production by DC increased during the first 24 h of maturation and declined by 48 h, proper timing of the ex-vivo DC treatment was crucial for the generation of functionally mature antigen-loaded DC. Our results suggest that after allowing 4 h of tumor lysate uptake by immature DC, further treatment with TNF-alpha and IFN-gamma for 24 h provides the optimal conditions to obtain functional TAA-loaded DC. These TAA-loaded cytokine pretreated DC then prime na?ve T cells, and enhance both T helper 1 (Th1), Th2 and cytotoxic T lymphocyte (CTL) responses, that are necessary to achieve an effective, specific anti-tumor response.     

Pilot study of treatment of biochemotherapy-refractory stage IV melanoma patients with autologous dendritic cells pulsed with a heterologous melanoma cell line lysate

Eleven AJCC stage IV melanoma patients with progressive disease after treatment with biochemotherapy were treated with autologous dendritic cells pulsed with heterologous tumor cell lysates. The vaccine used mature DCs (CD1a⁺⁺⁺, CD40⁺⁺, CD80⁺⁺, CD83⁺, and CD86⁺⁺⁺) generated from peripheral blood monocytes in the presence of GM-CSF and IL-4. After 7 days, DCs were matured with a defined cocktail of cytokines (IL-1+IL-6+TNF-+PGE₂) and simultaneously pulsed with lysates of heterologous melanoma cell lines, for 2 days. A total of 4×10⁶ DCs was injected monthly under ultrasound control in an inguinal lymph node of normal appearance. The study was closed when all patients died as a consequence of tumor progression. No sign of toxicity was observed during the study. One patient experienced a partial response lasting 5 months, and two patients showed a mixed response which lasted 3 months. The median survival of the whole group was 7.3 months (range 3–14 months). This vaccination program had specific antitumoral activity in highly pretreated and large tumor burden stage IV melanoma patients and was well tolerated. The clinical responses and the median survival of the group of patients, together with the low toxicity of our DC vaccine, suggest that this approach could be applied to earlier AJCC stage IV melanoma patients.     

Alteration in interactions between tumor-infiltrating lymphocytes and tumor cells in human melanomas after chemotherapy or immunotherapy

Summary Alteration in interactions between tumor-infiltrating lymphocytes (TILs) and tumor cells after chemotherapy or immunotherapy was studied in metastatic melanoma patients. Tumors were harvested from surgical specimens 17 days after the end of chemotherapy with cisplatin, vinblastine, and dacarbazine (CVD). Tumors of nonlymph-node metastases from two responders yielded neither TILs nor tumor cells, whereas those from all four nonresponders had both TILs [(1.1–13.8) × 10⁶ cells/g tumor] and tumor cells [(2.8–30.8) × 10⁶ cells/g tumor). Tumors of lymph node metastases from nine patients yielded substantial numbers both of TILs and tumor cells, regardless of different clinical responses, except with one complete responder, whose tumor did not contain tumor cells. The mean increase of TILs from these tumors (n = 14) 3–4 weeks after incubation with 200 U/ml recombinant interleukin-2 (rIL-2) was 2.5-fold, whereas there was a 56-fold increase in TILs from untreated tumors (n = 3). CD3⁺ T cells predominated in TILs before and after expansion with IL-2. IL-2-activated TILs from five of six tumors tested displayed higher cytotoxicity against autologous tumor cells than against cells from any of three allogeneic tumors. Mean tumor cell numbers (10⁶ cells/trial) obtained by serial needle biopsies for the same tumor in five patients decreased from 1.2 before therapy to 0.25 at day 4 of therapy (interferon alone), and to 0.02 at day 8 (interferon and IL-2). This decrease did not correlate with clinical responses. Yields (× 10⁶ cells/g tumor) of TILs and tumor cells in subcutaneous melanomas obtained by excisional biopsies in one nonresponder under IL-2 therapy were respectively 0.2 and 1.1 before therapy (day 0), 0.1 and <0.01 during (day 7), 0.2 and <0.01 at the end of therapy (day 21), and 0.5 and 0.5 at the time of tumor progression (day 66). Yields of TILs and tumor cells in the other nonresponder were respectively 3 and 26 before (day 0), 16 and 3 during (day 7), and 0.4 and <0.01 at the end of IL-2 therapy (day 17), and 2.5 and 6 at the time of progression (day 62). TILs in these two patients before therapy proliferated well in culture with IL-2 (570-and 720-fold, respectively), and showed higher cytotoxicity against autologous tumor cells, whereas none of those from the five tumors biopsied during or at the end of IL-2 therapy proliferated. TILs at the time of progression showed modest proliferation (54- and 76-fold, respectively) and showed major-histocompatibility-complexnonrestricted cytotoxicity. In summary, a decrease in the number of live tumor cells did not always correlate with clinical response in either therapy. CVD chemotherapy may simply impair IL-2-induced proliferation of TILs. IL-2 therapy may induce transient unresponsiveness of TILs to IL-2.This work was supported in part by grant CA 47 891 from the National Institutes of Health and a grant from the University Cancer Foundation, and Mr Richard Hunton Melanoma Found.     

Immunotherapy of intraperitoneal cancer with interleukin 2 and lymphokine-activated killer cells reduces tumor load and prolongs survival in murine models

The control of malignancy disseminated within the peritoneal cavity is an important problem in the management of low-grade gastrointestinal and ovarian neoplasms. A model of peritoneal carcinomatosis in the mouse was used to investigate the potential of lymphokine-activated killer (LAK) cells and exogenous interleukin 2 (IL-2) to control intraperitoneal tumor. LAK cells are splenocytes activated in vitro by IL-2. C57BL/6 mice were injected intraperitoneally with a lethal inoculum of syngeneic MCA-105 tumor. Three days later, the established tumor was treated with adoptively transferred LAK cells and/or exogenous IL-2 administration. LAK cells alone were ineffective in reducing intraperitoneal tumor. Administration of IL-2 alone resulted in limited tumor reduction. Treatment with exogenous IL-2 in conjunction with LAK cells resulted in the greatest reduction of intraperitoneal tumor. The larger the number of LAK cells given, the greater the reduction in tumor. Frequent intraperitoneal bolus administration of IL-2 was more effective than a single daily intraperitoneal injection and intraperitoneal administration of IL-2 and LAK was more effective than systemic treatments. Marked prolongation of life was seen in mice treated with LAK cells plus exogenous IL-2. We conclude that intraperitoneal LAK cells plus exogenous IL-2 is an effective treatment regimen for reducing intraperitoneal tumor in this murine model.     

Suppression of tumor necrosis factor receptor associated factor (TRAF)-2 attenuates the proinflammatory and proliferative effect of aggregated IgG on rat renal mesangial cells

Immune-complex (IC) mediated glomerulonephritis (GN) is a common cause of chronic kidney disease associated with increased levels of tumor necrosis factor (TNF)-α in renal cells. TNF-α signaling pathways involve complicated interactions between multiple proteins including TNF-receptor-associated factor (TRAF)-2. We have previously found markedly up-regulated expression of TRAF-2 in renal tissues from IC mediated lupus nephritis patients. Here we investigated the effect of TRAF-2 on inflammatory response in rat mesangial cells (MCs). The results showed that treatment with soluble aggregated IgG (AIgG) resulted in a time- and dose-dependent increase in the expression of interleukin (IL)-1β and IL-6. Significant cell proliferation was also observed after the treatment with soluble AIgG. Knockdown TRAF-2 by siRNA significantly suppressed soluble AIgG induced up-regulation of TRAF-2, IL-1β, and IL-6. Meanwhile the cell proliferation was inhibited and apoptotic cells were increased. It was concluded that TRAF-2 could induce the proinflammatory and proliferative effects of soluble AIgG on rat MCs. Thus, TRAF-2 may represent a future target for therapy of IC mediated GN.     

Interleukin-15 modulates interferon-gamma and beta-chemokine production in patients with HIV infection: implications for immune-based therapy

Interleukin (IL)-15 is a cytokine that has lymphocyte stimulatory activity similar to that of IL-2, and plays important immunoregulatory functions during HIV disease. To evaluate the role of IL-15 in HIV infection the following patients were studied: 18 antiretroviral-naive patients with advanced disease; 19 patients with continuous viral suppression and immunological response after 48-120 weeks of highly active antiretroviral therapy (HAART); and 12 patients with evidence of virological and immunological HAART treatment failure. Nineteen healthy blood donors were included as controls. The production of IL-15 by human peripheral blood monocytes stimulated with lipopolysaccharide and Mycobacterium avium complex, the priming effect of IL-15 on IFN-gamma production from purified CD4(+) and CD8(+) T cells, and the ability of IL-15 to stimulate the beta-chemokine release from purified CD4(+) and CD8(+) T cells were analyzed. In the present work IL-15 production by human peripheral blood monocytes was significantly increased in HIV-infected patients with long-term virological and immunological response to HAART. IL-15 enhanced the in vitro priming of CD4(+) and CD8(+) T cells for IFN-gamma production, also in patients receiving HAART. Finally, IL-15 had positive effects on RANTES, MIP-1alpha, and MIP-1beta release by CD4(+) and CD8(+) T cells. In conclusion IL-15 could affect the immune response of HIV-infected patients by augmenting and/or modulating IFN-gamma production and beta-chemokine release. These data about functional properties of IL-15 could provide new implications for immune-based therapies in HIV infection.     

Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells.

The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen-stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity.     

Interleukin-2 increases the antibody response in patients receiving autologous intralymphatic tumor cell vaccine immunotherapy.

The production of tumor-binding antibodies was studied in a group of cancer patients undergoing active specific immunotherapy with irradiated, cholesterol-treated, cell culture-derived autologous tumor cells injected by the intralymphatic route. Fifteen patients were analyzed: nine patients (four melanoma, one breast, one sarcoma, one colon, and one undifferentiated cancer) received three injections of 10 to 15 x 10(6) tumor cells, spaced 2 weeks apart, and six patients (two melanoma, two renal, one breast, and one colon cancer) received tumor cells admixed with 3 x 10(6) U recombinant interleukin-2 (IL-2) (Proleukin, Cetus, Emeryville, CA, USA) plus a 10-day intravenous infusion of 15 x 10(6) U/kg/day IL-2 after each immunization. Serum antibody binding to autologous tumor cells was measured at 2 and 4 weeks after initiation of therapy using an enzyme-linked immunosorbent assay with patient serum being added to adherent tumor cells bound to 96-well microtiter plates. After 4 weeks, we found a significant difference (0.02 less than P less than 0.04) in serum titer in the group receiving IL-2 (33% mean increase) compared with the non-IL-2 group (8% mean increase). Although neither group showed clinical improvement in response to the therapy, the results clearly demonstrated the efficacy of IL-2 in augmenting patient antibody response to autologous intralymphatic tumor cell immunization.     

IL-12 rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo

Tumor-associated macrophages (TAMs) play a major role in promoting tumor growth and metastasis and in suppressing the antitumor immune response. Despite the immunosuppressive environment created by the tumor and enforced by tumor-associated macrophages, treatment of tumor-bearing mice with IL-12 induces tumor regression associated with appearance of activated NK cells and activated tumor-specific CTLs. We therefore tested the hypothesis that IL-12 treatment could alter the function of these tumor-associated suppressor macrophages. Analysis of tumor-infiltrating macrophages and distal TAMs revealed that IL-12, both in vivo and in vitro, induced a rapid (<90 min) reduction of tumor supportive macrophage activities (IL-10, MCP-1, migration inhibitory factor, and TGFbeta production) and a concomitant increase in proinflammatory and proimmunogenic activities (TNF-alpha, IL-15, and IL-18 production). Similar shifts in functional phenotype were induced by IL-12 in tumor-infiltrating macrophages isolated from the primary tumor mass and in TAMs isolated from lung containing metastases, spleen, and peritoneal cavity. Therefore, although TAMs display a strongly polarized immunosuppressive functional profile, they retain the ability to change their functional profile to proinflammatory activities given the appropriate stimulus. The ability of IL-12 to initiate this functional conversion may contribute to early amplification of the subsequent destructive antitumor immune response.     

Priming of the antitumor response promotes efficacy of interleukin-2 therapy

 We have studied the effect of active specific immunization (ASI) on the antitumor response induced by locoregional, low-dose interleukin-2 (IL-2) therapy. On day 0, mice were injected i.p. with viable, syngeneic tumor cells and with irradiated tumor cells (ASI). Low-dose IL-2 treatment was given i.p. for 5 consecutive days. ASI led to extended survival in two out of seven models tested. In these two models, enhanced efficacy was observed when both ASI and IL-2 were administered. In the five models in which ASI had no therapeutic value, ASI+IL-2 treatment was no more effective than IL-2 therapy. In the SL2 lymphoma model, use of ASI prior to IL-2 therapy given as early as days 1–5 led to at least 60% cure, whereas IL-2 therapy without ASI was only effective when administered after day 9. In the P815 mastocytoma model, however, ASI, IL-2, and the combination caused negative (suppressive) effects when administered on days 6–10. IL-2 administered on days 6–10 was therapeutically effective in this model when mice were treated with cyclophosphamide on day 6. In both the SL2 and the P815 tumor models, cured mice were specifically immune. The positive and negative effects observed were not due to the increased number of cells injected (non-specific inflammation) nor to possible antigenic alteration of the ASI cells by irradiation, as ASI with fragmented tumor cells was also effective in inducing synergy. Investigations into the underlying mechanism indicated that CD4⁺ cells play an important role. In total, the results indicate that ASI may be a good supplement to locoregional IL-2 treatment if care is taken to alleviate immunosuppressive activities. Received: 6 February 1997 / Accepted: 6 March 1997     

Generation of therapeutic T lymphocytes from tumor-bearing mice by in vitro sensitization. Culture requirements and characterization of immunologic specificity

We have previously established an in vitro sensitization (IVS) procedure with which lymphocytes from tumor-bearing mice could be expanded and sensitized to acquire antitumor reactivity capable of mediating the regression of established pulmonary metastases from the weakly immunogenic MCA 105 murine sarcoma. Culture conditions required for the optimal generation of therapeutic effector cells were evaluated in the current study. Generation of effector cells by IVS required stimulation by intact tumor cells. Tumor cells killed by heat or disrupted by sonication were ineffective, but the antigenicity of tumor cells was not affected by gamma-irradiation. Long term established tumor cell lines could also serve as antigenic stimulator cells albeit with lower efficiency than fresh tumor cells. IL-2 was essential for cellular proliferation during IVS. The concentration of 1000 U/ml of IL-2 also induced nonspecific lymphokine-activated killer (LAK) activity. However, cytotoxic cells were generated during IVS in response to a broad range of IL-2 concentrations. At low IL-2 concentrations (2 to 10 U/ml), IVS cells were generated which displayed little or no LAK activity, had a greater therapeutic efficacy than those generated with high concentrations of IL-2 (100 to 1000 U/ml). Despite having high LAK activity, IVS cells, from cultures where IL-2 was added 3 or more days after initiation, had no therapeutic effect. Thus, the generation of therapeutic cells occurred independently of LAK cell production. Adoptive immunotherapy with IVS cells from MCA 105 tumor-bearing mice demonstrated cross-reactivity with the immunologically distinct MCA 106 but not the nonimmunogenic MCA 102 tumor. In contrast, IVS cells from MCA 106 tumor-bearing mice exhibited specific in vivo reactivity. In vitro cytotoxicity analyses revealed that IVS cells from MCA 105 and MCA 106 tumor-bearing mice were able to lyse both MCA 105 and MCA 106 target cells, but the reactivity toward inoculating tumors was highest. Considering previous findings that the MCA 105 and MCA 106 sarcomas possessed distinct tumor-specific transplantation Ag, the cross-reactivity observed in this study suggests that the immune response during progressive tumor growth may be different from that elicited in response to active immunization.     

IL-2 immunotoxin therapy modulates tumor-associated regulatory T cells and leads to lasting immune-mediated rejection of breast cancers in neu-transgenic mice

Studies in cancer patients have suggested that breast tumors recruit regulatory T cells (Tregs) into the tumor microenvironment. The extent to which local Tregs suppress antitumor immunity in breast cancer is unknown. We questioned whether inhibiting systemic Tregs with an IL-2 immunotoxin in a model of neu-mediated breast cancer, the neu-transgenic mouse, could impact disease progression and survival. As in human breast cancer, cancers that develop in these mice attract Tregs into the tumor microenvironment to levels of approximately 10-25% of the total CD4+ T cells. To examine the role of Tregs in blocking immune-mediated rejection of tumor, we depleted CD4+CD25+ T cells with an IL-2 immunotoxin. The treatment depleted Tregs without concomitant lymphopenia and markedly inhibited tumor growth. Depletion of Tregs resulted in a persistent antitumor response that was maintained over a month after the last treatment. The clinical response was immune-mediated because adoptive transfer of Tregs led to a complete abrogation of the therapeutic effects of immunotoxin treatment. Further, Treg down-modulation was accompanied by increased Ag-specific immunity against the neu protein, a self Ag. These results suggest that Tregs play a major role in preventing an effective endogenous immune response against breast cancer and that depletion of Tregs, without any additional immunotherapy, may mediate a significant antitumor response.