All-Trans Retinoic Acid Enhances the Cytotoxic Effect of T Cells with Anti-CD38 Chimeric Receptor in Acute Myeloid Leukemia
Authors: Mihara Keichiro , Bhattacharyya Joyeeta , Takihara Yoshihiro , Kimura Akiro 
Published: October 2019

Abstract
Abstract 1901
Majority of AML blasts express CD38, while human hematopoietic stem cells express CD34 but not CD38. Accordingly, we focused on CD38 for a therapeutic target and developed a cellular immunotherapy by using T cells bearing anti-CD38-chimeric antigen receptor (CAR). We recently reported T cells with anti-CD38-CAR efficiently eliminated B-cell lymphoma cells expressing CD38 in vitro and in vivo. However, intensity of CD38 in lymphoma cells is much higher than that in AML cells. Additionally, leukemic stem cells(LSCs) phenotypically express CD34+CD38−. Thus, to fully employ anti-CD38-CAR against AML blasts including LSCs, intensity of CD38 expression need to be raised for the clinical application. All-trans retinoic acid (ATRA) is widely used for treatment of patients with acute promyelocytic leukemia (APL). Interestingly, it has been reported that ATRA enhances CD38 expression on the surface of AML cells. In this study, we investigated whether human peripheral T cells retrovirally transduced with anti-CD38-CAR kills AML cells and furthermore, whether the cytotoxicity is enhanced in the presence of ATRA. First of all, we confirmed that T cells bearing anti-CD38-CAR expressed GFP as well as the anti-goat mouse IgG-PerCP, whose expression is consistent with the expression of anti-CD38-CAR on the T cells. Next, we evaluated the cytotoxic effect of CD38-specific T cells against two AML cell lines (THP-1 and CMK), which highly express CD38 (>99%). Lactate dehydrogenase (LDH) releasing assay and flow cytometry were performed for cytotoxicity. We co-incubated THP-1 or CMK cells with T cells transduced at a variety of effector (E): target (T) ratios for 3 consecutive days in vitro. Interestingly, these assays showed that T cells bearing anti-CD38-CAR were cytotoxic against THP-1 and CMK cells in dose- and time-dependent manners. However, in the cases of KG1a, U937 and HL60 cells which partially express CD38, killing effect was restricted to CD38+ cells by T cells bearing anti-CD38-CAR, and CD38− AML cells remained alive. These results suggested that augmentation of CD38 expression is essential for the sufficient cytotoxicity against AML cells not expressing CD38 by T cells bearing anti-CD38-CAR. We, then, investigated whether ATRA augmented CD38 expression and resultantly enhanced the cytotoxicity against AML cell lines. Interestingly, ATRA augmented CD38 expression in HL60 cells as well as even in KG1a and U937. However, ATRA by itself exerted no effect on cytotoxicity or proliferating activity in KG1, U937 and even in HL60 cells. These results showed that ATRA contributed to enhancement of CD38 expression, but not to cytotoxicity and proliferation on AML cell lines. Next, we attempted to examine cytotoxic effect of T cells with anti-CD38-CAR on KG1, U937 or HL60 cells following ATRA treatment. AML cell lines were co-cultured with the effector T cells transduced for 3 days in the presence of ATRA. The killing effect of T cells bearing anti-CD38-CAR against AML cell lines was limited in the absence of ATRA. Intriguingly, T cells with anti-CD38-CAR exerted enhanced cytotoxic effect on AML cells in the presence of ATRA. Next, we applied our settings above to AML cells freshly isolated from AML patients. Firstly, we confirmed that CD38 expression was enhanced by ATRA in AML cells from patients as observed in AML cell lines. Furthermore, 3-day incubation of patients' AML cells with T cells bearing anti-CD38-CAR abrogated AML cells in the presence of ATRA. These results indicated that T cells expressing anti-CD38-CAR efficiently eliminated AML cells from patients as well as AML cell line cells through the enhancement of CD38 expression by ATRA. Here, we propose that pre-treatment of ATRA in patient with AML enhances CD38 expression on the leukemic cell surface, which augments the cytotoxic effect of T cells bearing CAR to eradicate leukemic cells including LSCs.

Disclosures:
No relevant conflicts of interest to declare.