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.

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.

Precursor frequency analysis of bryostatin activated lymphocytes.

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)

Immunomodulatory effects of transforming growth factor-beta on T lymphocytes. Induction of CD8 expression in the CTLL-2 cell line and in normal thymocytes.

We investigated the role of transforming growth factor-beta 1 (TGF-beta) in regulation of T cell growth and differentiation. Treatment of CTLL-2 cells with TGF-beta inhibited IL-2-dependent proliferation and caused morphologic changes as well as increased adherence. A major change of phenotype in TGF-beta-treated cells was the de novo expression of CD8 alpha chain in 35% of cells, which required the continuous presence of TGF-beta. Of the CD8 alpha+ cells, 20 to 30% co-expressed CD8 beta chain. Increased CD8 expression occurred even in the total absence of cell growth, was not a consequence of growth inhibition, and was not a result of selective growth or survival of CD8+ cells. New RNA synthesis was required for TGF beta-induced CD8 alpha surface expression, inasmuch as this was prevented by treatment with actinomycin D. Northern blot analysis demonstrated that cells treated with IL-2 + TGF-beta rapidly accumulated mRNA encoding both chains of the CD8 dimer, to a level fourfold greater than control by 6 to 12 h. In contrast, the IL-2-dependent increases in IL-2R alpha, IL-2R beta, and Granzyme B mRNA levels in these cultures were profoundly inhibited by TGF-beta. When unfractionated murine thymocytes were stimulated with phorbol dibutyrate plus ionomycin and cultured with IL-2 + TGF-beta, an increase in CD8 alpha mRNA was seen and greater numbers of CD8+ cells with higher levels of CD8 alpha and CD8 beta surface expression resulted, as compared to controls treated with IL-2 alone. Furthermore, similar treatment of CD4-CD8-(double negative) thymocytes with TGF-beta induced de novo CD8 alpha expression by a substantial number of cells, and the majority of these CD8+ cells lacked TCR/CD3. These data suggest that TGF-beta has both positive and negative regulatory effects on the expression of gene products important for T lymphocyte differentiation and function.

Inhibition of tumor-specific cytotoxic T-lymphocyte responses by transforming growth factor beta 1.

Transforming growth factor beta (TGF-beta) is a potent immunosuppressive cytokine that is produced by neoplastic and normal cells. It has not been demonstrated directly, however, that TGF-beta can inhibit antigen-specific T-cell responses to tumor cells in vitro. We show here that generation of antitumor cytotoxic T-lymphocyte (CTL) activity in mixed-lymphocyte tumor cultures of splenocytes from DBA/2 mice immunized with the syngeneic P815 mastocytoma + Corynebacterium parvum was consistently and profoundly inhibited when 0.675 to 10 ng/ml of TGF-beta were added on Day 0 of culture. TGF-beta added on Day 1 or later had little or no effect. In contrast to the results with P815 immune mice, mixed-lymphocyte tumor cultures established with splenocytes from P815 tumor-bearing hosts showed variable degrees of inhibition by TGF-beta, depending on the stage of the ongoing in vivo immune response. Addition of recombinant murine tumor necrosis factor alpha (1,000 or 10,000 units/ml) partially reversed inhibition of CTL responses by TGF-beta, while recombinant interleukin 2 nearly completely reversed the suppression. These data indicate that one level at which TGF-beta may act to inhibit mixed-lymphocyte tumor cultures is that of cytokine production. To determine whether TGF-beta also has any direct effect on CTL, P815-specific CTL clones derived from tumor-bearing host mice were utilized. We found that proliferation of rested CTL clones in response to tumor cells + interleukin 2 was inhibited by 5 ng/ml of TGF-beta, while the interleukin 2-dependent reactivation of cytolytic activity was not affected by TGF-beta. In contrast to rested CTL, when TGF-beta was added to cultures of previously activated CTL, proliferation was not inhibited. These data demonstrate that TGF-beta has profound inhibitory effects on the in vitro generation of effector CTL from tumor-specific murine splenocytes, and this inhibition may be an indirect result of suppressed cytokine production as well as a direct antiproliferative effect on CTL.

Cyclophosphamide and abrogation of tumor-induced suppressor T cell activity.

Previously we have demonstrated that the in vitro generation of P815-specific anti-tumor cytotoxic T lymphocytes (CTL) was suppressed by splenic suppressor T cells from late tumor-bearing hosts (TBH). Suppression is not caused by in vitro growth of P815 from splenic metastases, since suppression was also seen with spleen cells from late TBH mice bearing a hypoxanthine/aminopterin/thymidine-sensitive subline (PHS-5) of P815 in the presence of HAT. Cyclophosphamide has been shown to inhibit the induction of suppressor cells selectively in a number of immune responses, but evidence that it can inhibit active tumor-induced suppressor T cells is limited. We have found that suppressor T cells already induced by P815 in syngeneic late TBH are sensitive to low doses of cyclophosphamide (50 mg/kg) given 1 day before spleen harvest, but the in vitro CTL response of late TBH spleen cells could not be restored by pretreating the mice with cyclophosphamide, even when exogenous interleukin-2 was added to the cultures. Although 50 mg/kg cyclophosphamide did not inhibit the CTL response of spleen cells from mice immunized with P815 + Corynebacterium parvum, the same dose of cyclophosphamide eliminated the CTL response of spleen cells from early TBH. Interleukin-2 (IL-2) did not overcome this effect of cyclophosphamide, suggesting a direct effect on CTL. "Ultra-low" -dose cyclophosphamide (10 mg/kg) did not adversely effect early TBH CTL but was still able to eliminate suppressor T cell activity from late TBH. Nevertheless, late TBH CTL remained unresponsive after pretreatment of mice with ultra-low-dose cyclophosphamide, even when exogenous IL-2 was added in vitro. CTL precursor frequency analyses demonstrated that cyclophosphamide pretreatment had little or no effect on the numbers of CTL precursors from early TBH. Late TBH CTL precursor cells were not detectable in these studies, with or without suppressor T cell inhibition by cyclophosphamide pretreatment. Thus, it appears that most CTL precursor cells may be lost or irretrievably inactivated in the spleens of late TBH mice.

Phenotype of syngeneic tumor-specific cytotoxic T-lymphocytes and requirements for their in vitro generation from tumor-bearing host and immune spleens.

Cells required for the in vitro generation of syngeneic cytotoxic T-lymphocytes (CTL) against the P815 mastocytoma in the DBA/2 mouse strain were investigated. For both immune and tumor-bearing host spleen cells, CTL effector cells were eliminated by treatment with anti-Thy1.2, anti-Lyt1.1, or anti-Lyt2.1 and C', but were resistant to anti-L3T4 (GK1.5). Thus, CTL effectors (and their precursors) were Lyt1+2+, L3T4-. However, P815-specific CTL could not be generated in the absence of L3T4+ cells, whose function could be replaced with exogenous interleukin-2 (IL-2). When monoclonal antibodies against L3T4 were added to mixed leukocyte tumor cultures, CTL generation was markedly inhibited. Depletion of accessory cells also led to a marked reduction in CTL generation, which could be restored to control levels by adding adherent cells from normal spleens or with exogenous IL-2, but not with IL-1. Thus, accessory cells are apparently required to present the tumor antigens of this Ia-negative tumor to T-helper cells.