DNA Vaccination and All-Trans Retinoic Acid Treatment-Induced Long Term Survival Requires CD4+ and CD8+ T-Cell Activation in An APL Mouse Model.

OBJECTIVES: DNA vaccines can be effective in the acquisition of humoral and cellmediated immune responses. Our studies on an acute promyelocytic leukemia (APL) mouse model show that DNA vaccination combined with all-trans retinoic acid (ATRA) results in a survival advantage with a significant increase in the Th1 cytokine IFNg. ATRA alone can act as an adjuvant to induce immune responses as measured by an increase in anti-RARa antibody production, which correlated with improved survival in mice. Similar increases in antibody production have been observed in our patients after maintenance therapy. The aim of this study is to use immunomonitoring and functional assays to evaluate the presence of activated T-cells and to demonstrate APL-specific killing and to determine if the protective effect of DNA vaccination is CD4+ and CD8+ mediated.

METHODS: Using an APL transplant model in FVB/N mice, CD107a, expressed on the surface of lytic granules of activated T-cells, was measured by flow cytometry. A flow based CFSE assay was used to measure APL specific cell killing by cytotoxic T-cells (CTLs). As FVB/N mice have H2^q haplotyes, blocking anti-H2^q antibodies were used to determine if the cytotoxic activity was MHC restricted. Immunophenotyping by measuring CD4+ and CD8+ absolute counts were conducted. Mice injected with APL cells were depleted of CD4+ or CD8+ cells with anti-CD4 or anti-CD8+ antibody treatment initiated the day after DNA vaccination (early) and continued every 5 days and assayed for efficacy of the DNA+ATRA combined therapy or CD4+ or CD8+ cells were depleted in long term survivors (>100 days, late).

RESULTS: Th1 cytokines TNFa and IFNg were increased indicative of DNA effects and specific activated CD3/CD8 T cells were detected and observed to release cytotoxic granules in the presence of APL cells in long term survivors. A dose dependent decrease in CFSE positive cells was observed assaying effectors from spleens of ATRA alone, ATRA+DNA treated mice and CD107a+ sorted cells from the latter using APL cells as targets. This effect was MHC restricted as anti-H2^q antibodies reduced the specific cytotoxic activity. CD4+ absolute numbers measured on day 38 significantly correlated with survival (p=0.005). Although not significant a similar trend was observed for CD8+ counts. The CD4+ or CD8+ depleted mice treated with DNA + ATRA died earlier compared with the undepleted animals. When DNA + ATRA treated long term survivors were depleted of CD4+ or CD8+ cells, the CD4+ depleted mice relapsed and died in 3 months whereas the CD8+ depleted mice survived for a further 3 months when the experiment was terminated. These data are consistent with an increase in anti-RARa antibody production previously measured in other protocols. Interestingly the late depletions show that CD4+ cells are required for the maintenance of the remissions and show that memory T-cells are required.

CONCLUSION: Therefore we have been able to detect protective cellular and humoral responses in mice with the combined treatment of DNA+ATRA, which correlates with outcome.