IntroductionDespite improved molecular characterization of malignancies and development of targeted therapies, 1 acute leukemia is not curable and few patients survive more than 10 years after diagnosis. 2,3 To further improve outcome, we studied the potential efficacy of boosting the patient's immune response. Therapeutic vaccination aimed at promoting T-cell immunity requires: activation of the innate system, optimal presentation of major histocompatibility complex (MHC) class I-binding peptides, and provision of CD4 ϩ T-cell help. DNA vaccines have the potential to supply all of these. In an animal model of acute promyelocytic leukemia (APL), 4 we developed a promyelocytic leukemia-RAR␣ (PML/RAR␣)-targeted DNA-based vaccine 5 and show that DNA combined with all-trans retinoic acid (ATRA) has a pronounced survival advantage, concomitant with time-dependent antibody production, 5,6 and an increase in interferon-␥ (IFN-␥). 5 A similar approach confirmed these findings. 7 The role of ATRA as an immunomodulator is well documented. [8][9][10][11] Westervelt et al 12 show that ATRA responses are influenced by the presence of an intact adaptive immune response.The present study is aimed at investigating the immune responses involved in the antileukemic effect of the combined ATRA ϩ DNA therapy, particularly those mediated by CD4 ϩ and CD8 ϩ T cells. MethodsWe extended our previous study using the same protocol illustrated in supplemental Figure 1A (available on the Blood website; see the Supplemental Materials link at the top of the online article) where we combined ATRA (5 mg) and a PML-RAR␣FrC DNA construct in an APL mouse model. 5 Peripheral blood (PB) was collected approximately every 20 days from day 19 after APL engraftment to follow the mice clinically. At specific days, mice were killed to evaluate responses. Methods are detailed in the figure legends. Animal studies were undertaken according to the guidelines of the institutional animal care committee of Hôpital Saint-Louis. Results and discussion DNA vaccination combined with ATRA induces long-term survivalOur finding of ATRA ϩ DNA treatment significantly extending survival compared with ATRA alone was confirmed in additional protocols (Figure 1A-B; supplemental Figure 1B-C). We have previously reported that DNA alone elicited a modest survival advantage 5 and that FrC alone failed to give long-term survival 13 (supplemental Figure 1D).ATRA ϩ DNA-treated mice had increased white blood cell counts on day 21 after leukemia engraftment, possibly resulting from the For personal use only. on May 11, 2018. by guest www.bloodjournal.org From increases in CD4 ϩ and CD8 ϩ cells (supplemental Figure 2A). ATRAand nearly half of the ATRA ϩ DNA-treated mice had reduced platelets by day 60 (supplemental Figure 2A). Long-term survivors (LTSs) from the ATRA ϩ DNA-treated group (Ͼ 120 days) had significantly reduced bone marrow blast counts (2%-12%; supplemental Figure 2B). ATRA-alone-treated mice died by day 90.PB absolute counts of CD3 ϩ CD4 ϩ and CD3 ϩ CD8 ϩ subsets, as...

Polyplex micelles have demonstrated biocompatibility and achieve efficient gene transfection in vivo. Here, we investigated a polyplex micelle encapsulating genes encoding the tumor-associated antigen squamous cell carcinoma antigen recognized by T cells-3 (SART3), adjuvant CD40L, and granulocyte macrophage colony-stimulating factor (GM-CSF) as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Intraperitoneally administrated polyplex micelles were predominantly found in the lymph nodes, spleen, and liver. Compared with mock controls, the triple gene vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer cells, of which long-term surviving mice showed complete rejection when re-challenged with CT26 tumors. Moreover, the DNA vaccine inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung tumors in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c+ DCs and CD4+/CD8a+ T cells into tumors. Depletion of CD4+ or CD8a+ T cells by neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. In conclusion, a SART3/CD40L+GM-CSF gene-loaded polyplex micelle can be applied as a novel vaccine platform to elicit tumor rejection immunity regardless of the recipient MHC haplotype.

Esophageal rupture is a rare entity. Delay in the diagnosis and treatment may threaten the patient's life. The decision for surgical or nonsurgical treatment, however, remains controversial because advocates of both treatments have reported comparable results. To quantify the decision making, we suggest the systemic inflammatory response syndrome (SIRS) score for triage of an esophageal rupture. Using this criterion for 12 patients resulted in the survival of all of them. Therefore, we advocate use of the SIRS score for triage of an esophageal rupture.

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 H2q haplotyes, blocking anti-H2q 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-H2q 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.