sq-23377 and bryostatin-1

Adoptive immunotherapy (AIT) of cancer with T lymphocytes may be limited by the need to activate tumor antigen-sensitized cells in vitro. In murine models, we have shown that AIT with tumor-sensitized T cells that have been pharmacologically activated with bryostatin 1 and ionomycin plus interleukin-2 can induce tumor regression. A Phase I clinical trial was carried out to assess the feasibility and toxicity associated with using tumor- or vaccine-draining lymph node cells, activated pharmacologically and expanded in culture with low-dose interleukin-2 and infused intravenously, followed by IL-2 infusion. Nine patients were entered into the trial, and six were treated as planned. Average expansion of cell numbers over 13 to 27 days in culture was 118-fold. No patient's cells reached the target cell number (2.5 x 10(10)). Infusion of these cells did not result in any unexpected toxicities. The toxicities observed were related to IL-2 infusion, and conformed to the expected range of side-effects. Based on these Phase I results, additional trials, with tumor antigen vaccine-sensitized DLN and technical modifications of the culture technique, are planned.

Topics: Adult; Bryostatins; Cells, Cultured; Female; Humans; Immunotherapy, Adoptive; Interleukin-2; Ionomycin; Lactones; Lymph Nodes; Lymphocyte Activation; Macrolides; Male; Middle Aged; Neoplasms; Pilot Projects; T-Lymphocytes

HIV-1 persists in a latent reservoir despite antiretroviral therapy (ART). This reservoir is the major barrier to HIV-1 eradication. Current approaches to purging the latent reservoir involve pharmacologic induction of HIV-1 transcription and subsequent killing of infected cells by cytolytic T lymphocytes (CTLs) or viral cytopathic effects. Agents that reverse latency without activating T cells have been identified using in vitro models of latency. However, their effects on latently infected cells from infected individuals remain largely unknown. Using a new ex vivo assay, we demonstrate that none of the latency-reversing agents (LRAs) tested induced outgrowth of HIV-1 from the latent reservoir of patients on ART. Using a quantitative reverse transcription PCR assay specific for all HIV-1 mRNAs, we demonstrate that LRAs that do not cause T cell activation do not induce substantial increases in intracellular HIV-1 mRNA in patient cells; only the protein kinase C agonist bryostatin-1 caused significant increases. These findings demonstrate that current in vitro models do not fully recapitulate mechanisms governing HIV-1 latency in vivo. Further, our data indicate that non-activating LRAs are unlikely to drive the elimination of the latent reservoir in vivo when administered individually.

Topics: Anti-HIV Agents; Azepines; Bryostatins; CD4-Positive T-Lymphocytes; Cell Cycle Proteins; Depsipeptides; Disulfiram; Histone Deacetylase Inhibitors; HIV Infections; HIV-1; Humans; Hydroxamic Acids; Indoles; Ionomycin; Lymphocyte Activation; Nuclear Proteins; Panobinostat; Tetradecanoylphorbol Acetate; Transcription Factors; Triazoles; Virus Latency; Vorinostat

Two major barriers in the immunotherapy of breast cancer include tumor-induced immune suppression and the establishment of long-lasting immune responses against the tumor. Recently, we demonstrated in an animal model of breast carcinoma that expanding and reprogramming tumor-sensitized lymphocytes, ex vivo, yielded T memory (Tm) cells as well as activated CD25+ NKT cells and NK cells. The presence of activated CD25+ NKT and NK cells rendered reprogrammed T cells resistant to MDSC-mediated suppression, and adoptive cellular therapy (ACT) of reprogrammed lymphocytes protected the host from tumor development and relapse. Here, we performed a pilot study to determine the clinical applicability of our protocol using peripheral blood mononuclear cells (PBMCs) of breast cancer patients, ex vivo. We show that bryostatin 1 and ionomycin combined with IL-2, IL-7, and IL-15 can expand and reprogram tumor-sensitized PBMCs. Reprogrammed lymphocytes contained activated CD25+ NKT and NK cells as well as Tm cells and displayed enhanced reactivity against HER-2/neu in the presence of MDSCs. The presence of activated NKT cells was highly correlated with the rescue of anti-HER-2/neu immune responses from MDSC suppression. Ex vivo blockade experiments suggest that the NKG2D pathway may play an important role in overcoming MDSC suppression. Our results show the feasibility of reprogramming tumor-sensitized immune cells, ex vivo, and provide rationale for ACT of breast cancer patients.

Topics: Breast Neoplasms; Bryostatins; Dendritic Cells; Female; Humans; Immunotherapy, Adoptive; Interleukin-15; Interleukin-2; Interleukin-7; Ionomycin; Leukocytes, Mononuclear; Lymphocyte Activation; Lymphocyte Subsets; Myeloid Cells; Natural Killer T-Cells; Neoplasm Staging; Receptor, ErbB-2; Tumor Burden

It was reported that breast cancer patients have pre-existing immune responses against their tumors(1,2). However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses(3). However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively(4). This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7+IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Topics: Animals; Bryostatins; Female; Immunotherapy, Adoptive; Ionomycin; Lymphocyte Activation; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; T-Lymphocytes

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). Tumor antigen-sensitized DLN lymphocytes from BALB/c mice with 10-day 4T1 mammary carcinomas were harvested, activated with B/I, and expanded in culture with either interleukin-2 (IL-2) or IL-7 + IL-15. Cell yields, proliferation, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for antitumor activity against established 4T1 mammary carcinomas after inoculation of tumor cells subcutaneously (s.c.). IL-7/15 resulted in much faster and more prolonged proliferation of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately 5-10-fold greater yields of viable cells. Culture in IL-7/15 yielded higher proportions of CD8(+) T cells and a higher proportion of cells with a central memory phenotype. T cells grown in IL-2 had higher interferon-gamma (IFN-gamma) release responses to tumor antigen than cells grown in IL-7/15. Adoptive transfer of B/I-activated T cells grown in IL-7/15 demonstrated much greater efficacy against 4T1 tumors in vivo. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in AIT of cancer.

Topics: Animals; Antigens, Neoplasm; Bryostatins; Carcinoma; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Female; Immunologic Memory; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-15; Interleukin-2; Interleukin-7; Ionomycin; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Phenotype; Recombinant Proteins; T-Lymphocyte Subsets; Time Factors; Tumor Burden

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apoptosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-gamma release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a "per-cell" basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.

Topics: Adjuvants, Immunologic; Animals; Apoptosis; Bryostatins; Cell Proliferation; Flow Cytometry; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-15; Interleukin-2; Interleukin-7; Interleukins; Ionomycin; Lung Neoplasms; Lymph Nodes; Lymphocyte Activation; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Tumor Cells, Cultured

We have shown that tumor vaccine-sensitized draining lymph node (vDLN) cells activated ex vivo with bryostatin and ionomycin (B/I) were capable of inducing antigen-specific regression of a murine mammary tumor, 4T07. vDLN cells not activated with B/I were ineffective. We hypothesized that B/I selectively activates tumor-sensitized (CD62Llow) lymphocytes, to account for the highly potent and tumor-specific activity. We hypothesized that CD8+ CD62Llow cells may be preferentially activated by B/I treatment, infiltrate the tumors and mediate tumor regression in mice. 4T07-IL2 tumor cells were injected into one hind footpad of BALB/c mice. Ten days later, vDLN were harvested and separated based on CD62L expression. After separation, cells were activated with B/I, expanded with IL2 (40 IU/ml) for 10 days, and adoptively transferred to 4T07 tumor bearing mice. Naive mice were also treated with different subsets of T cells and later were challenged with 4T07 tumor cells. To test in vitro responses to antigen, expanded lymphocytes were cultured either alone or with irradiated 4T07 tumor cells. Supernatants were harvested after 24 h and tested by ELISA for IFN-gamma. The importance of the host immune response was tested by AIT into 4T07-bearing nude athymic mice. Host mice were depleted in vivo of CD4 or CD8 T cells after vDLN AIT to ascertain the mediators of tumor regression. In order to track B/I activated vDLN cells, they were prestained with CFSE prior to adoptive transfer into tumor-bearing hosts. At various time points, tumors, spleens and lymph nodes of host mice were harvested, dual stained for activation marker expression and analyzed by flow cytometry. CD62Llow cells expanded 12-fold more than CD62Lhigh lymphocytes during the 10 day culture period. Supernatant from CD62Llow cells + 4T07 cultures contained 33-fold more IFN-gamma than supernatant from CD62Lhigh cells + 4T07 cultures (843.9 pg/ml +/- 135.8 vs 25.89 pg/ml +/- 0.01). Adoptive transfer of CD62Llow lymphocytes induced complete tumor regressions in all mice, while tumors regressed in only 17% of mice treated with CD62Lhigh lymphocytes. Naive mice that received B/I-activated CD62Llow cells were protected from future tumor challenges, while mice given CD62Lhigh cells did not exhibit the same resistance to tumor growth. Tumors in nude host mice regressed after AIT treatment. In vivo depletion of CD4 T cells after AIT did not inhibit tumor regression, but CD8 T cell depletion abrogated tumor regress

Topics: Animals; Bryostatins; Cytokines; Immunologic Memory; Immunotherapy, Adoptive; Ionomycin; L-Selectin; Lactones; Lymphocyte Activation; Macrolides; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; T-Lymphocytes

Tumor cell vaccines have been successful at inducing immunity in naïve mice, but only in a few reports has vaccination alone induced regression of established tumors and, generally, only when they are very small. Clinically, vaccinations alone may not be able to cause regression of established human cancers, which tend to be weakly immunogenic. We hypothesized that pharmacologic ex vivo amplification of a vaccination-induced immune response with subsequent adoptive immunotherapy (AIT) to tumor-bearing animals would be more effective in treatment of these animals than vaccination alone. The 4T1 and 4T07 mammary carcinomas are derived from the same parental cell line, but 4T1 is much less immunogenic and more aggressive than 4T07. Vaccination with either 4T1, 4T1-IL-2, or 4T07-IL-2 was not effective as treatment for established 4T1 tumors. However, 4T1 or 4T07-IL-2-vaccine-sensitized draining lymph node (DLN) cells, activated ex vivo with bryostatin 1 and ionomycin and expanded in culture, induced complete tumor regressions when adoptively transferred to 4T1 tumor-bearing animals. This was effective against small tumors as well as more advanced tumors, 10 days after tumor cell inoculation. Furthermore, as would be required for this approach to be used clinically, vaccine-DLN cells obtained from mice with established progressive 4T1 tumors (inoculated 10 days before vaccination) also induced regression of 4T1 tumors in an adoptive host. In none of these experiments was exogenous IL-2 required to induce tumor regression. The response to tumor cell vaccine can be amplified by ex vivo pharmacologic activation of sensitized T cells, which can then cure an established, weakly immunogenic and highly aggressive tumor that was resistant to vaccination alone.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Alkylating; Bryostatins; Cancer Vaccines; Cyclophosphamide; Enzyme Activation; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-2; Ionomycin; Lactones; Macrolides; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; T-Lymphocytes; Tumor Cells, Cultured; Tumor Stem Cell Assay; Vaccination

We have shown that adoptive transfer of tumor-sensitized lymphocytes activated in vitro with bryostatin-1 and ionomycin (B/I), and expanded in culture, can induce regression of small established tumors. We set out to determine whether similar treatment would be effective against larger tumors and what cells mediate this effect. We also attempted to shorten the ex vivo culture period with the ultimate aim of developing a more clinically useful protocol. BALB/c mice were injected in one footpad with IL-2-transfected 4T07 mammary tumor cells. Ten days later, popliteal draining lymph nodes (DLN) were harvested and activated with B/I for 18 h. Mice with either 3-day or 10-day 4T07 flank tumors were treated with cyclophosphamide (100 mg/ kg ip, CYP) alone or CYP followed the next day by infusion of either B/I-activated lymphocytes transferred immediately or activated cells that had been expanded in vitro for 3 or 10 days. In some experiments, mice were also treated with rat anti-mouse CD4 monoclonal antibody (GK1.5) or anti-CD8 antibody (2.43). All mice receiving CYP alone or CYP + sensitized, nonactivated DLN cells demonstrated progressive tumor growth. One hundred percent (6/6) of mice treated with CYP + AIT with B/I-activated,10-day expanded cells had complete regression of 3-day flank tumors. Treatment with activated, nonexpanded cells, induced tumor regression in a majority of mice, but was not as reliable as AIT with expanded cells. We developed a protocol with a shortened expansion period (3-day) that was efficacious for treatment of 4T07 when adoptively transferred to either 3 or 10 day tumor-bearing mice. In vivo depletion of CD4(+) cells had no effect on regression of 3-day tumors, but treatment with anti-CD8 antibody abrogated the effect of immunotherapy. Adoptive transfer of B/I-activated cells, with or without long-term expansion, induced regression of early and late stage 4T07 tumors and is dependent on CD8(+) but not CD4(+) T cells.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Antineoplastic Agents, Alkylating; Bryostatins; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cyclophosphamide; Female; Immunotherapy, Adoptive; Ionomycin; Ionophores; Lactones; Macrolides; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C

Because the requirement for long-term cell culture can make adoptive cellular immunotherapy cumbersome, experiments were designed to determine whether smaller numbers of tumor-sensitized T cells activated briefly with bryostatin 1 and ionomycin (B/I) could be returned immediately to recipient mice without in vitro expansion and still have an anti-tumor effect in vivo. Popliteal tumor-draining lymph nodes (DLNs) from mice bearing progressive MCA-105 and MCA-203 footpad sarcomas were harvested and treated for 18 h with B/I. These cells were then washed and transferred immediately to naive C57B1/6 mice. In some experiments, these mice were irradiated (500 rads) before adoptive transfer and were given interleukin-2 (IL-2, 7,500 IU i.p., b.i.d. for 3 days) after receiving the activated lymphocytes. Recipient mice were challenged with sarcoma cells (4 x 10(5) i.v.) 6 to 32 days after receiving the activated lymphocytes. Mice receiving 10(6) B/I-activated lymphocytes before tumor challenge had significantly fewer metastases than did controls. This protective effect did not require exogenous IL-2 or host irradiation. Using Thy-1 congenic donors, it was shown that B/I-activated T cells expanded in recipients when IL-2 was also given, and these cells were a prominent component (15% of total cells) in the infiltrates found in the lungs of mice 7 days after i.v. tumor challenge. Combining these B/I-"pulsed" cells with cyclophosphamide (CYP) and IL-2 to treat mice with established (3-day) metastases resulted in significant reduction in pulmonary nodules, with complete regression in many of the treated mice, which was rarely seen with CYP alone or with CYP + IL-2. Thus, adoptive transfer of tumor-sensitized, B/I-activated DLN cells confers protection against i.v. tumor challenge, without prior in vitro expansion of the effector cells. Phenotyping studies demonstrate that donor cells activated with B/I do expand in recipient mice after adoptive transfer and can move to sites of tumor. Moreover, these cells can mediate a therapeutic effect on established tumor metastases, when combined with chemotherapy.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Animals; Bryostatins; Cell Division; Cell Movement; Cyclophosphamide; In Vitro Techniques; Interleukin-2; Ionomycin; Ionophores; Lactones; Lung; Lymphocyte Activation; Lymphocytes; Macrolides; Mice; Mice, Inbred C57BL; Sarcoma, Experimental; Spleen

Lymphocytes obtained from tumor-draining lymph nodes (DLN) can have potent in vivo antitumor activity after in vitro activation with bryostatin 1 and ionomycin. However, the presence of visceral metastases in the donor can inhibit the effectiveness of such lymphocytes. In the present study, we tested the ability of low-dose cyclophosphamide to overcome metastasis-induced immunosuppression in a murine model.. Mice were injected with MCA-105 sarcoma cells in the footpad alone or in the footpad and the tail vein to establish lung metastases. Cyclophosphamide was given i.p. 1 day before harvesting the draining popliteal lymph nodes. For all donor groups, DLN cells were activated with 5 nM bryostatin 1 and 1 microM ionomycin and cultured for 7 days in 20 U/ml IL-2. Activated DLN cells were then adoptively transferred to syngeneic mice with 3-day lung metastases.. The adoptive transfer of DLN cells from mice with footpad tumors only significantly reduced the number of lung metastases compared to untreated mice. However, activated DLN cells obtained from mice with both footpad and lung tumors were significantly less effective. Treatment of similar donor mice with 10 mg/kg cyclophosphamide significantly improved the anti-tumor activity of adoptively transferred cells. This dose of cyclophosphamide did not reduce the number of cells obtained from each lymph node or the expansion of cell numbers in vitro.. These results suggest that the administration of low-dose cyclophosphamide prior to harvesting DLN cells may improve the success of adoptive immunotherapy in cancer patients.

Topics: Animals; Bryostatins; Cyclophosphamide; Female; Immune Tolerance; Immunotherapy, Adoptive; Ionomycin; Lactones; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Macrolides; Mice; Mice, Inbred C57BL; Mitogens; Neoplasm Metastasis; Neoplasm Transplantation; Sarcoma, Experimental

Bryostatin 1 (Bryo) is a potent activator of protein kinase C. When T cells are stimulated with Bryo and the calcium ionophore ionomycin (Io), they expand rapidly in low-dose IL-2 (20 U/ml). We have shown that Bryo/Io-activated T-cells from murine tumor-draining lymph nodes have striking antigen-specific antitumor efficacy in vivo. To account for the specificity despite using a nonspecific T cell activation method, it was postulated that the Bryo/Io combination might preferentially activate antigen-sensitized T cells. To test this hypothesis, an allogeneic response model was used. C57BL/6 mice were primed by intraperitoneal (ip) injection with DBA/2 mouse splenocytes. After 9 days, the C57BL/6 spleens were harvested and H-2d-specific cytolytic T lymphocyte (CTL) precursor frequency (PF) was determined by limiting dilution analysis (LDA). A portion of the same spleen cells was treated for 18 hr with Bryo/Io and expanded for 7 days in IL-2 (20 U/ml); the LDA was then repeated to analyze PF after expansion. The entire experiment was also done with responder and stimulator strains reversed (DBA/2 mice immunized with C57BL/6 cells). The resulting PF values were [table: see text] Normal spleen cells treated with Bryo/Io exhibited < 10% release vs the same targets at any effector:target ratio, ruling out nonspecific cytolysis after Bryo/Io. T lymphocytes or CD8+ T cells were selected from primed splenocytes and the PF values before and after Bryo/Io were analyzed. These data showed that the increase in PF after expansion could not be attributed to T cell or CD8+ cell enrichment alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bryostatins; Cell Transplantation; Flow Cytometry; Germ-Free Life; Immunization; Interleukin-2; Ionomycin; Lactones; Lymphocyte Activation; Macrolides; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Recombinant Proteins; Spleen; Stem Cells; T-Lymphocytes; T-Lymphocytes, Cytotoxic

Protein kinase C (PKC)-activating tumor promoters, such as PMA, are mitogenic to human T lymphocytes but, in contrast to many physiological mitogenic agonists, PMA does not operate via a receptor-coupled, IL-2-dependent signaling pathway. Bryostatin, another PKC activator, is not mitogenic to human T cells and, moreover, it inhibits the PMA-induced mitogenic response. Inhibition of the PMA-induced human T cell proliferation by bryostatin correlates with the bryostatin-induced increased degradation rate of conventional PKC (cPKC) isoenzymes. Nevertheless, bryostatin synergizes with suboptimal doses of various T cell mitogens operating via a Ca(2+)-dependent signaling pathway to induce a strong IL-2-dependent proliferative response. Our findings indicate that Ca2+ signals do not abolish the bryostatin-induced enhanced degradation rate of cPKC observed in human T cells. The results suggest that transient activation of cPKC by bryostatin induces some irreversible biochemical events that, by themselves, are not sufficient for the induction of cell proliferation, but that can be complemented by a Ca(2+)-mediated signal(s) leading to IL-2-dependent T cell proliferation. In contrast, PMA-induced IL-2-independent proliferation of human T cells appears to be dependent on the continuous presence of cPKC (that may mediate some critical events at late stages of the response) and, therefore, is inhibited by bryostatin, that induces a rapid degradation of potentially active cPKC.

Topics: Biological Transport; Bryostatins; Calcium; Cell Membrane; Cells, Cultured; Drug Synergism; Humans; Interleukin-2; Ionomycin; Lactones; Lymphocyte Activation; Macrolides; Mitogens; Protein Kinase C; Signal Transduction; T-Lymphocytes; Tetradecanoylphorbol Acetate

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.

Topics: Adjuvants, Immunologic; Axilla; Breast Neoplasms; Bryostatins; Cryopreservation; Female; Humans; Immunotherapy; Interferon-gamma; Ionomycin; Lactones; Lymph Nodes; Lymphocyte Activation; Lymphocyte Subsets; Lymphocytes, Tumor-Infiltrating; Macrolides; Phenotype; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

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.

Topics: Adjuvants, Immunologic; Animals; Antibodies; Bryostatins; Female; Immunoglobulin G; Immunotherapy, Adoptive; Interferon-gamma; Ionomycin; Lactones; Lung Neoplasms; Lymph Nodes; Lymphocyte Activation; Macrolides; Methylcholanthrene; Mice; Mice, Inbred C57BL; Sarcoma, Experimental; T-Lymphocytes; Tumor Necrosis Factor-alpha

Treatment of human cancer with tumour-specific T lymphocytes is limited by the frequent unavailability of autologous tumour to stimulate T-cell growth and by the toxicity associated with high-dose interleukin-2 (IL-2) treatment. In the present study we demonstrate that Bryostatin 1 (B) plus ionomycin (I) can substitute for tumour antigen and activate tumour-bearing hosts' T-cells which provide long-term protection against tumour challenge after adoptive transfer. Lymphocytes obtained from the popliteal lymph nodes (DLN) draining an MCA-105 footpad sarcoma were stimulated with B/I, and then cultured for 7 days with 20 U ml-1 IL-2. This in vitro stimulation protocol consistently expanded cell numbers greater than 20-fold during 7 days. Mice given B/I-stimulated draining lymph node (DLN) cells were protected from specific i.v. tumour challenge for at least 15 weeks after adoptive transfer, even in the absence of IL-2 treatment. Tumour immunity conferred by B/I-activated DLN cells was systemic and independent of host T-cells. However, resistance to tumour challenge was lost when either CD4+ or CD8+ T-cells were depleted in vivo. These studies indicate that DLN cells activated with bryostatin 1 and ionomycin persist long-term in vivo as functional memory cells after adoptive transfer.

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents; Bryostatins; Evaluation Studies as Topic; Female; Immunotherapy, Adoptive; Ionomycin; Lactones; Lymphocyte Activation; Macrolides; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Transplantation; Sarcoma, Experimental; T-Lymphocytes; Time Factors

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.

Topics: Animals; Bryostatins; Enzyme Activation; Immunotherapy, Adoptive; Ionomycin; Lactones; Lymph Nodes; Lymphocyte Activation; Macrolides; Mast-Cell Sarcoma; Mice; Mice, Inbred DBA; Protein Kinase C; T-Lymphocytes, Cytotoxic

Although there is evidence that corticosteroids inhibit receptor-ligand-induced phospholipid hydrolysis, the immunosuppressive effects of these agents downstream of protein kinase C (PK-C) activation and cytosolic Ca2+ mobilization is unclear. Previous studies indicated that T cell proliferative activation could be achieved with simultaneous short-term (e.g., 15-120 min) exposure to agents activating PK-C and elevating cytosolic Ca2+. In the studies reported here, similar procedures were utilized for determining whether corticosteroids alter T cell activation signals downstream of second messenger events. Dexamethasone interfered with T cell activation induced by short-term exposure to phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore, ionomycin. The inhibitory effect was evident with as little as 15 min of exposure to dexamethasone and T cell activating agents, making mechanisms involving de novo protein synthesis unlikely. Dexamethasone's effects in this system were blocked by the steroid receptor antagonist RU-486, indicating that the inhibition was mediated through the glucocorticoid receptor. The inclusion of recombinant interleukin-2 (IL-2) only partially overcame the dexamethasone inhibitory effect. Long-term (i.e., 48 hr) direct stimulation of PK-C with either PDBu or the non-tumor-promoting PK-C activator, bryostatin 1, also substantially overcame dexamethasone's effects, resulting in a recovery of IL-2 production and significant restoration of the T-cell proliferative response. These observations suggest that treatment with a PK-C-activating agent such as bryostatin 1 could reduce glucocorticosteroid-induced immunosuppression.

Topics: Adrenal Cortex Hormones; Bryostatins; Calcium; Cell Division; Dexamethasone; Drug Interactions; Enzyme Activation; Humans; Interleukin-2; Ionomycin; Lactones; Lymphocyte Activation; Macrolides; Mifepristone; Monocytes; Phorbol 12,13-Dibutyrate; Protein Kinase C; Second Messenger Systems; T-Lymphocytes; Time Factors

Maturation of human myeloid cells is associated with quantitative and qualitative changes in protein kinase C (PKC) and increases in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors, actin, and actin regulatory proteins. We have studied the actin responses and cell shape changes caused by FMLP and its second messenger pathways in HL60 cells undergoing neutrophilic maturation. In uninduced cells, the PKC activators 12-O-tetradecanoyl phorbol-13-acetate (TPA), bryostatin, and 1-oleyl-2-acetylglycerol (OAG) resulted in 15% to 30% decreases in F-actin, whereas FMLP had no effect. Ionomycin had no effect on actin but did cause a 10-fold increase in intracellular calcium. Cells grown for 24 hours in 1% dimethyl sulfoxide (DMSO) acquired the ability to polymerize actin in response to FMLP and ionomycin. TPA continued to cause a decrease in F-actin at 24 hours, but caused an increase in F-actin at 48 to 72 hours of maturation. The PKC inhibitor 1-5-isoquinolinesulfonyl 2-methylpiperazine (H7) partially blocked the F-actin increase caused by TPA in induced cells, but had no effect on the decrease in F-actin caused by TPA in uninduced cells or the increase in F-actin seen in FMLP-treated neutrophils. F-actin rich pseudopods developed following TPA or FMLP stimulation of induced HL60 cells; in uninduced cells neither agent caused pseudopod formation but TPA caused a dramatic loss of surface ruffles. The ability of FMLP and ionomycin to elicit a neutrophil-like actin response in HL60 cells within 24 hours after DMSO treatment shows that the actin regulatory mechanism is mature by that time. The inability of ionomycin to increase F-actin in uninduced cells supports the view that calcium increases alone are insufficient for actin polymerization. The longer maturation time required for HL60 cells to develop an actin polymerization response to TPA compared with FMLP, coupled with the inability of H7 to block the FMLP-mediated F-actin increase in neutrophils, suggests that the F-actin increase caused by FMLP is not mediated solely by PKC. Lastly, the TPA-induced F-actin decrease and shape changes in uninduced HL60 cells, and the longer time required for a "mature" response to TPA, may reflect immaturity in the PKC isoenzyme pattern rather than immaturity of the actin regulatory mechanism.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Bryostatins; Calcium; Cell Differentiation; Dimethyl Sulfoxide; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Ionomycin; Isoquinolines; Lactones; Macrolides; Microscopy, Electron, Scanning; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Piperazines; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The aim of this study was to clarify the relationship between the stimulatory effects of protein kinase C activators, including phorbol 12-myristate 13-acetate (PMA) and bryostatin, on the hydrolysis of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) and on PtdCho synthesis. The cell lines used were selected because of their differential responses to protein kinase C activators and included rat-1 fibroblasts, untransformed and A-raf-transformed NIH 3T3 fibroblasts and human HL60 leukaemia cells. Exposure of rat-1 and NIH 3T3 fibroblasts to 100 nM-PMA stimulated phospholipase D-mediated hydrolysis of phospholipids about 2- and 6-fold respectively. In contrast, 100 nM-PMA had similar (2.5-3.0-fold) stimulatory effects on PtdCho synthesis in these cell lines. In the untransformed NIH 3T3 cells, both PMA and bryostatin stimulated both phospholipid hydrolysis and PtdCho synthesis, with 100 nM-bryostatin being somewhat less potent than 100 nM-TPA. In contrast, in A-raf-transformed NIH 3T3 cells or in HL60 cells, only TPA, but not bryostatin, stimulated PtdCho synthesis. In these transformed cells, bryostatin had 3-fold, or higher, stimulatory effects on phospholipid hydrolysis. Addition of ionomycin, a Ca2(+)-elevating agent, partially restored the stimulatory effect of bryostatin on PtdCho synthesis, but it failed to modify the effect of bryostatin on phospholipid hydrolysis. These data indicate that increased phospholipid hydrolysis is not necessarily associated with increased PtdCho synthesis.

Topics: Animals; Bryostatins; Cells, Cultured; Enzyme Activation; Fibroblasts; Hydrolysis; Ionomycin; Lactones; Leukemia, Experimental; Macrolides; Phosphatidylcholines; Phospholipids; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate

In order to examine the role of phosphatidylinositol bisphosphate (PIP2) hydrolysis in B cell activation, we studied the effect of various classes of protein kinase C (PKC) activators on anti-Ig-mediated B cell stimulation. Anti-Ig-stimulated PIP2 hydrolysis, elevations in [Ca2+]i, and induction of DNA synthesis were inhibited by PMA (a phorbol ester) as previously reported. In contrast, indolactam (an alkaloid PKC activator) inhibited PIP2 hydrolysis and elevations in [Ca2+]i, but stimulated rather than inhibited cellular proliferation. In order to examine whether the binding avidity of the PKC activators to PKC played a role in determining their activity to stimulate or inhibit B cell activation, we studied two other PKC activators, bryostatin and mezerein. Again, both inhibited anti-Ig mediated PIP2 hydrolysis and elevations in [Ca2+]i, whereas only the former inhibited induction of DNA synthesis. These data suggest that a) high levels of PIP2 hydrolysis and elevations in [Ca2+]i are not essential for anti-Ig-mediated induction of B cell DNA synthesis and b) activation of PKC may induce both stimulatory and inhibitory pathways of B cell activation, and whether stimulation or inhibition of cell activation is observed may depend on the combined intensity of these two signals.

Topics: Animals; Antibodies, Anti-Idiotypic; B-Lymphocytes; Bryostatins; Calcium; Diterpenes; Enzyme Activation; Immunoglobulin delta-Chains; In Vitro Techniques; Indoles; Ionomycin; Lactams; Lactones; Lymphocyte Activation; Macrolides; Mice; Mice, Inbred DBA; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Protein Kinase C; Receptors, Antigen, B-Cell; Signal Transduction; Terpenes