Sensitizing T-lymphocytes for adoptive immunotherapy by vaccination with wild-type or cytokine gene-transduced melanoma.

BACKGROUND: For the relatively nonimmunogenic B16-F10 murine melanoma, it has been found that genetically engineered expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin (IL)-2, IL-4, or interferon-gamma (IFN-gamma) resulted in a vaccine that could induce resistance to rechallenge. Because T cells from lymph nodes draining the sites of some progressive tumors can mediate tumor regression after in vitro activation, it seemed possible that even apparently nonimmunogenic melanoma cells might induce similar preeffector cells in the vaccine-draining lymph nodes (DLNs). METHODS: C57BL/6 mice were vaccinated with B16-F10 cells that were either unmodified or genetically modified to produce IL-2, IL-4, GM-CSF, or IFN-gamma. DLNs were harvested 10 days after vaccination for adoptive immunotherapy (AIT). The DLN cells were activated with bryostatin 1 and ionomycin (B/I), expanded for 10 days in culture, and transferred to mice with 3-day pulmonary metastases. Pulmonary nodules were counted 14 days after AIT. RESULTS: Adoptive transfer of expanded DLN lymphocytes sensitized by inoculation of WT B16-F10, or IL-4, GM-CSF, or IFN-gamma expressing cells significantly reduced pulmonary metastases. Despite the spontaneous regression of IL-2-transduced B16-F10 tumors, DLN from mice inoculated with IL-2 producing B16 cells had little or no antitumor activity. CONCLUSIONS: B16-F10 vaccination strategies that apparently do not induce systemic immunity can effectively sensitize DLN preeffector cells.

Biologic therapy of melanoma with cytokines and lymphocytes.

Melanoma has a somewhat unpredictable behavior, and spontaneous regressions do occasionally occur. Many have surmised that these are the result of immunologic attack by the host. Immunologic treatment has been more successful for melanoma than for most other neoplasms, even with relatively crude therapies, such as bacterial products. With the availability of recombinant cytokines, immunotherapy for melanoma has entered a new era. Interleukin-2 (IL-2), which acts entirely through immunologic mechanisms, has been tested extensively, either alone, in combination with other cytokines, or with adoptive cellular therapy. Alone, it has only modest antitumor activity, even at high doses. Its utility may be greater when combined with immunocompetent cells, especially tumor-sensitized T lymphocytes, in adoptive immunotherapy. On the other hand, nonspecific lymphokine-activated killer (LAK) cells do not appear to add significantly to the efficacy of IL-2 alone. Interferon-alpha (IFN-alpha) [corrected] also has had fairly limited activity in the advanced disease setting, but, on the basis of a recently completed randomized trial, has arguably become the "standard of care" in the adjuvant setting for patients with high-risk melanoma, particularly node-positive patients. A number of regimens combining IL-2, IFN-alpha, and chemotherapeutic agents have yielded striking response rates in small trials and await confirmation in larger studies. With better delineation of the host immune response and definition of relevant tumor antigens, we can look forward to exciting results with combinations of vaccines, cytokines, and adoptive cellular approaches, particularly in patients with micrometastatic disease.

Bryostatin 1 activates splenic lymphocytes and induces sustained depletion of splenocyte protein kinase C activity in vivo after a single intravenous administration.

Bryostatin 1 activates and subsequently down-regulates protein kinase C (PKC) in vitro and has potential use as an immunomodulator and as an anti-cancer agent. Despite extensive examination of its activities in vitro and anti-tumor effects in vivo, previous studies have failed to document that bryostatin 1 modulates total cellular PKC activity in tumor or normal tissues when administered in vivo. After a single bolus injection of bryostatin 1 (1.0 microgram) in normal C57BI/6 mice, blood was drawn at various intervals and assayed for bryostatin 1 levels. In addition, spleens from bryostatin-treated mice were harvested 10 min to 10 days after treatment, weighed and analyzed for cell numbers, PKC activity and cell surface phenotypes. Bryostatin 1 levels in plasma rose rapidly, reaching peak levels of 56.5 nM less than 1 min after injection, and then declined to undetectable levels by 1 h. A similar pattern was observed when bryostatin 1 was incubated with leukemia cells in vitro, raising the possibility that the rapid fall in plasma levels results from intracellular uptake and binding. Bryostatin 1 induced marked depletion of total splenocyte PKC activity (as much as 69% relative to control values) at 24-96 h after drug administration, but not at earlier times (i.e. 1 h). A single injection of bryostatin 1 also induced expression of the T cell activation marker CD69, leading to positivity in 53% of cells at 3-24 h versus 11% in control mice, and resulted in marked splenomegaly, associated with increased numbers of nucleated cells at 48-96 h. Together, these studies demonstrate that despite rapid disappearance of the drug from plasma, a single i.v. dose of bryostatin 1 exhibits significant and sustained effects on normal murine spleen cells, including early lymphocyte activation, prolonged depletion of PKC activity, splenocyte proliferation and splenomegaly. These findings may have implications for attempts to understand the in vivo effects of bryostatin 1 in normal host tissues.

Adoptive transfer of bryostatin-activated tumor-sensitized lymphocytes prevents or destroys tumor metastases without expansion in vitro.

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.