Effect of isothermal radiofrequency radiation on cytolytic T lymphocytes.

Previous in vitro studies provide evidence that RF electromagnetic radiation modulates proliferation of human glioma, lymphocytes, and other cell types. The mechanism of RF radiation cell proliferation modulation, as well as mechanisms for effects on other cell physiologic endpoints, are not well understood. To obtain insight regarding interaction mechanisms, we investigated effects of RF radiation exposure on interleukin 2 (IL-2) -dependent proliferation of cytolytic T lymphocytes (CTLL-2). After exposure to RF radiation in the presence or absence of IL-2 cells were cultured at various physiological concentrations of IL-2. Treatment effects on CTLL-2 proliferation were determined by tritiated thymidine incorporation immediately or 24 h after exposure. Exposure to 2450 MHz RIF radiation at specific absorption rates (SARs) of greater than 25 W/kg (induced E-field strength 98.4 V/m) induced a consistent, statistically significant reduction in CTLL-2 proliferation, especially at low IL-2 concentrations. At lower SARs, 2450 MHz exposure increased CTLL-2 proliferation immediately after exposure but reduced 24 h postexposure proliferation. RF radiation effects depended on the mitotic state of the cells at the time of exposure. Comparison of the effects of temperature elevation and RF radiation indicated significant qualitative and quantitative differences.

Effects of the protein kinase C stimulant bryostatin 1 on the proliferation and colony formation of irradiated human T-lymphocytes.

The protein kinase C stimulant bryostatin 1 (Bryo) was used in examining human peripheral blood T-lymphocyte radiosensitivities in proliferation assays. Bryo was similar to PMA in inducing T-cell proliferation by the CD3, CD28 and CD69 pathways. No difference in radiosensitivities was observed in T-cells stimulated by the three independent surface antigen-mediated activation pathways. CD3 was chosen as the second signal for comparing the potencies of the three different first signals Bryo, phorbol 12-myristate, 13-acetate (PMA), and interleukin 2 (IL-2) in stimulating T-cell proliferation and in maintaining this response after radiation. Though there were radioresponse differences among various individuals, the irradiated lymphocytes consistently showed significantly greater proliferation when treated with Bryo or PMA than with IL-2 (p < 0.05- < 0.005). No difference in proliferative responses was observed in T-cells irradiated between 4 h before and 15 h after the addition of stimulants. Colony forming assays showed higher colony survival for irradiated T-cells stimulated with Bryo than with PMA. These results support the important role of protein kinase C in T-cell radiation responses, and suggest a potential role for Bryo in enhancing T-lymphocyte survival during radiation therapy.

Expansion and tumour specific cytokine secretion of bryostatin-activated T-cells from cryopreserved axillary lymph nodes of breast cancer patients.

Current adoptive immunotherapy strategies in cancer patients require large numbers of activated T-cells and are limited by the availability of autologous tumour. We describe a novel method of T-cell activation that produced relatively rapid, high-fold expansion of stored, frozen lymphocytes obtained from the lymph nodes of 20 breast cancer patients during axillary dissection but does not require autologous tumour. In vitro exposure of thawed cells to bryostatin-1 (B), a non-tumour promoting protein kinase C activator and ionomycin (I), a calcium ionophore, at day 0 followed by culture in low dose interleukin-2 (IL-2 20 units ml-1) and restimulation again on day 10 results in 269-28,206 fold (geometric mean = 2254) expansion in cell numbers counted 17 days after initial stimulation. Analysis of cell surface markers revealed that B/I expanded human cells were predominantly T-cells (83-97%) and consisted of a mixture of CD8+ (46-74%) and CD4+ (4-30%) cells. B/I expanded cells did not lyse autologous tumour cells when tested in a 4-h 51Cr release assay, but murine studies reported previously have demonstrated specific and curative in vivo efficacy in MCA-105 tumour-bearing mice despite an inability to lyse autologous tumour in vitro. B/I expanded T-cells from five of six patients secreted the cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in response to co-culture with autologous tumour cells but not with irrelevant tumour. These results are analogous to findings in a murine model, in which non-cytolytic B/I expanded T-cells mediated specific, curative anti-tumour effects in vivo, and lay the groundwork for a clinical trial of this novel strategy for the adoptive immunotherapy of breast cancer patients.

Modulation of the activity of a human granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein (pIXY 321) by the macrocyclic lactone protein kinase C activator bryostatin 1.

We have examined the effect of the macrocyclic lactone protein kinase C (PK-C) activator bryostatin 1 on the proliferative capacity and lineage commitment of CD34+ human bone marrow cells exposed to the granulocyte-macrophage colony-stimulating factor/interleukin-3 (GM-CSF/IL-3) fusion protein pIXY 321. pIXY 321 administered at a dose of 10 ng/mL was as effective as the combination of plateau concentrations of recombinant (r) IL-3 and rGM-CSF (e.g., 50 ng/mL) in stimulating the growth of day-14 committed myeloid progenitors (colony-forming units granulocyte/macrophage [CFU-GM]). In the large majority of samples tested, coadministration of 0.5 to 100 nM bryostatin 1 with either pIXY 321 or the combination of rIL-3 plus rGM-CSF led to modest but significant increases (e.g., 30 to 75%) in the number of CFU-GM, compared to administration of growth factors alone. The degree of bryostatin 1-induced potentiation, however, was considerably less than that previously observed in the case of cells exposed to either rIL-3 or rGM-CSF, where increases of 100 to 150% were regularly noted. While at least 50% of day-14 CFU-GM stimulated by either pIXY 321 or the combination of rIL-3 plus rGM-CSF were of the pure or mixed eosinophilic variety, coadministration of bryostatin 1 resulted in a dramatic inhibition of eosinophilic colonies and a corresponding increase in pure and mixed neutrophil and macrophage colonies. Although coadministration of recombinant granulocyte colony-stimulating factor (rG-CSF) or recombinant colony-stimulating factor-1 (rCSF-1) mimicked the capacity of bryostatin 1 to increase the total number of pIXY 321-induced day-14 CFU-GM, these growth factors, unlike bryostatin 1, were not capable of inhibiting eosinophilic colony formation. Furthermore, whereas addition of neutralizing antibodies to G-CSF or CSF-1 blocked the capacity of these growth factors to potentiate colony formation in the presence of pIXY 321, it did not abrogate the effect of bryostatin 1 on progenitor cell growth or lineage commitment. Finally, in contrast to its effects on committed myeloid progenitors, bryostatin 1 did not increase the growth of erythroid (burst-forming units-erythroid [BFU-E]) and multipotent (multipotent colony-forming units [CFU-GEMM]) progenitors stimulated by pIXY 321, but instead inhibited colony formation at higher concentrations (e.g., 10 to 100 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

gamma-Interferon plays a key role in T-cell-induced tumor regression.

Recent studies have demonstrated that noncytolytic T-cells can mediate regression of murine tumors. In this report, we demonstrate that MCA-105 tumor-draining lymph node cells (DLN) activated with the protein kinase C activator, bryostatin 1, plus a calcium ionophore are capable of inducing specific tumor regression in vivo when adoptively transferred to mice with established metastases. However, these activated DLN cells lack in vitro cytotoxicity against autologous tumor. Antibody against gamma-interferon (IFN-gamma) markedly inhibited the therapeutic efficacy of these activated DLN cells. Anti-tumor necrosis factor produced a statistically significant but weaker inhibition of tumor regression. IFN-gamma, but not tumor necrosis factor alpha, could be shown to be secreted by activated DLN cells in vitro in response to specific tumor. Secretion of IFN-gamma was primarily a function of CD8+ T-cells. IFN-gamma was not directly cytotoxic to sarcoma cells in vitro. Moreover, tumor cells incubated with IFN-gamma were not more susceptible to lysis by activated DLN cells. However, recombinant murine IFN-gamma had a significant antiproliferative effect against MCA-105 tumor cells when tested in a [3H]thymidine uptake assay. Similarly, supernatants obtained from DLN/autologous tumor cocultures markedly inhibited MCA-105 proliferation; this antiproliferative effect was abrogated by the addition of anti-IFN-gamma antibody to the cultures. These results suggest that secretion of IFN-gamma by adoptively transferred DLN cells plays an essential role in tumor rejection. The dominant effect of IFN-gamma may be its demonstrated antiproliferative activity.

Bryostatin 1 modulates the proliferation and lineage commitment of human myeloid progenitor cells exposed to recombinant interleukin-3 and recombinant granulocyte-macrophage colony-stimulating factor.

The activity of protein kinase C (PK-C) has been implicated in the regulation of the growth and differentiation of both normal and neoplastic hematopoietic cells. We have examined the effects of the PK-C-activating agents phorbol 12,13-dibutyrate (PDBu), mezerein, and bryostatin 1 on the proliferation and lineage commitment of CD34+ human myeloid progenitor cells stimulated by recombinant interleukin-3 (rIL-3) and/or recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Although each of the PK-C activators administered alone induced no colony formation, coadministration of these agents with plateau concentrations of each cytokine (eg, 50 ng/mL) increased the number of day 14 granulocyte-macrophage colony-forming units by 100% to 150%. The number of pure and mixed neutrophil and macrophage colonies was substantially enhanced in the presence of PK-C activators, whereas the percentage and, in most cases, the absolute number of eosinophilic colonies was significantly reduced. The inhibition of eosinophilic colony formation was not overcome by the addition of rIL-5. Although addition of bryostatin 1 24 hours before rIL-3 abrogated the increase in total colony formation observed with simultaneous administration of factors, the inhibition of eosinophilic colonies and the increase in neutrophil/macrophage colonies persisted under these conditions. The addition of bryostatin 1 for up to 144 hours after rIL-3 continued to potentiate total colony formation, whereas the inhibition of eosinophilic commitment was lost after 120 hours. Together, these results suggest that pharmacologic interventions at the level of PK-C may regulate both the proliferation as well as the lineage commitment of human hematopoietic progenitors exposed to rGM-CSF and rIL-3.

Bryostatin 1 activates T cells that have antitumor activity.

Several strategies have been used to stimulate the growth of tumor-specific T cells in place of tumor antigen. One approach is to use pharmacologic agents to activate the second messenger pathways of T-cell activation. In the present study, we examined the ability of the protein kinase C activator bryostatin 1 (B) plus the calcium ionophore ionomycin (I) to stimulate the growth of lymphocytes obtained from the axillary lymph nodes (DLN) draining a progressively growing intradermal plasmacytoma tumor. Draining lymph node cells were initially cultured with autologous tumor cells and 20 U/ml of interleukin-2 (IL-2) for 7 days. The lymphocytes were then incubated with various concentrations of bryostatin 1 plus 1 microM ionomycin and cultured for an additional 14 days in IL-2. DLN cells initially cultured with autologous tumor and then restimulated with 5 nM bryostatin 1 and 1 microM ionomycin exhibited marked in vitro proliferation and 15-fold expansion of cell numbers over 2 weeks. The cells expanded with B/I were predominantly CD8+ T cells and retained specific in vitro cytotoxicity against autologous tumor. When adoptively transferred to mice with established liver metastases, DLN cells restimulated with B/I-mediated specific tumor regression.

Activation of CD8+ murine T cells from tumor-draining lymph nodes by phorbol dibutyrate plus calcium ionophore.

When lymphocytes from the lymph nodes draining the site of a progressively growing MCA-105 sarcoma are stimulated in vitro with autologous tumor and low-dose interleukin-2 (IL-2), they will grow and develop the ability to lyse autologous tumor cells in vitro; these lymphocytes can also eradicate tumor metastases in vivo. Phorbol esters and calcium ionophores activate signal transduction pathways in T cells and mimic the events triggered by antigen binding. We therefore sought to determine whether large numbers of MCA-105 tumor-specific, therapeutically active T cells could be obtained from MCA-105 draining lymph nodes (DLNs) following a brief exposure to phorbol dibutyrate (PDBu) and ionomycin (Io). DLN cells primarily stimulated with autologous tumor, followed by a secondary stimulation with PDBu-Io and cultured in 20 U/ml IL-2, demonstrated marked expansion of cell numbers during 3 weeks in culture, had moderate cytolytic activity [37% at effector:target ratio (E:T) = 80:1], and were all CD8+ T cells. In contrast, DLN cells stimulated primarily with PDBu-Io and cultured in 20 U/ml IL-2 demonstrated at least 8-10-fold greater growth than antigen-stimulated DLN cells during 3 weeks, were moderately cytolytic (31% at E:T = 80:1), and were a mixed population of CD8+ and CD4+ T lymphocytes. DLN cells that were expanded by either protocol, like cells stimulated repeatedly in vitro with tumor cells, could eliminate MCA-105 pulmonary metastases when given with IL-2 in an adoptive immunotherapy model. DLN cells stimulated primarily with PDBu-Io completely eradicated MCA-105 metastases but had no in vivo antitumor activity against the syngeneic B16 melanoma or MCA-203 sarcoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Bryostatin 1-activated T cells can traffic and mediate tumor regression.

Adoptive immunotherapy in humans may be limited by the lack of autologous tumor cells to activate and expand tumor-specific T cells. Pharmacologic manipulation of protein kinase C (PKC) and intracellular calcium may substitute for tumor antigen and stimulate T cells for adoptive immunotherapy. In the present study, we evaluated the ability of the PKC activator Bryostatin 1 (B) plus the calcium ionophore ionomycin (I) to activate lymphocytes obtained from popliteal lymph nodes (DLN) draining an MCA-105 footpad tumor. The adoptive transfer of B/I-stimulated DLN cells eradicated MCA-105 pulmonary metastases. These lymphocytes do not require concomitant IL-2 administration to mediate regression of lung metastases. Three days after intrasplenic injection of tumor cells and splenectomy, mice were given iv injections of B/I-stimulated DLN cells. Adoptive immunotherapy with these cells induced regression of established liver metastases. In an intradermal tumor model, the adoptive transfer of B/I-stimulated MCA-105 DLN cells cured mice of MCA-105 intradermal (id) tumors, but did not induce regression of MCA-206 tumors. Mice cured of MCA-105 id tumors were protected against MCA-105, but not MCA-203, tumor challenge in the footpad 7 weeks after adoptive immunotherapy.

Effect of a combined exposure to cytosine arabinoside, bryostatin 1, and recombinant granulocyte-macrophage colony-stimulating factor on the clonogenic growth in vitro of normal and leukemic human hematopoietic progenitor cells.

We have examined the effect of a combined 24 h exposure to cytosine arabinoside (ara-C) and the protein kinase C activator bryostatin 1, either alone or in conjunction with recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), on the clonogenic growth of 14 primary samples from acute myelogenous leukemia (AML) patients, as well as normal human committed and early hematopoietic progenitors. Incubation of blasts with 1 microM ara-C and 12.5 nM bryostatin 1(+/- 1.25 ng/ml rGM-CSF) resulted in a heterogeneous pattern of inhibitory effects toward primary leukemic colonies, ranging from 32-98%, and subadditive to synergistic drug interactions. However, exposure of blasts to ara-C and bryostatin 1, either with or without rGM-CSF, eliminated leukemic cell self-renewal in 80-93% of samples, and very substantially reduced growth in the remainder. Exposure of normal human bone marrow mononuclear cells to identical concentrations of ara-C and byostatin 1 permitted the survival of 23% of committed myeloid progenitors (granulocyte-macrophage colony-forming units), and greater than 50% when rGM-CSF was included. Finally, exposure of bone marrow populations highly enriched for progenitor cells (CD34+, DR-, CD71-) to ara-C and bryostatin 1 +/- rGM-CSF for 24 h led to minimal reductions (e.g. 10-15%) in the survival of early hematopoietic progenitors (high proliferative potential colony-forming cells). Together, these findings indicate that combined exposure in vitro to ara-C and bryostatin 1, both with and without rGM-CSF, effectively inhibits the growth of leukemic cells with self-renewal capacity, while sparing a significant fraction of normal committed and primitive hematopoietic progenitors.