Abstract
Background: The combination of antigen recognition, costimulatory ligands, and dendritic cells (DC)-derived cytokines (IL-12 or type I IFNs) stimulate T cells upon antigen presentation of DC. Chimeric antigen receptor (CAR) T cells induce anti-tumor cytotoxicity independent of DC by employing antigen recognition portion (single chain variable fragment)/CD3zeta and costimulatory signaling domain. However, the clinically available CARs are not engineered to provide DC-derived cytokine stimulation to T cells. This deficiency may prevent the CAR T cells from developing optimal effector functions, surviving, and forming a responsive memory T cell population. DC can enhance CAR T cell functionality by producing T cell stimulating cytokines. Intratumoral DC, marked by the expression of CD141/CLEC9A, play a critical role in recruiting T cells into the intratumoral area and inducing T cell cytotoxicity against the tumor. IRF8 is an essential transcription factor in developing intratumoral DCs. A co-stimulatory protein, 4-1BB is expressed on activated T cells and is a part of a CAR construct. 4-1BB has been suggested to stimulate IRF8 through the NF-kB signaling and could participate in the generation of intratumoral DC. Therefore, we hypothesized that autologous DCs transduced with 4-1BB CAR would enhance the efficacy of anti-CD33 CAR T cell therapy against acute myelogenous leukemia (AML) by providing DC-derived cytokines and recruiting CAR T cells in bone marrow microenvironment.
Methods: We sorted bone marrow CD34+ progenitors and T cells. Cells were transduced with an anti-CD33 41BBz CAR lentivector (pCCL-HP67.6-4-1BB-CD3z). We sorted transduced T (CAR T), and CD34+ progenitors three days after transduction. While expanding transduced CAR T cells further, we induced the differentiation of transduced CD34+ cells to DC (CAR-DC) in vitro by incubating cells with Flt3L/GM-CSF/IL-4 and AML cell lysate. After an additional four days of culture, we analyzed CAR-DC using flow cytometry. We co-cultured a human AML cell line, Kasumi-1 cells with CAR T +/- CAR-DC (E/T ratio=1), or mock control, and quantified cell death in different CAR T to Kasumi-1 ratios (10, 5, and 2) using CytoTox 96 NonRadioactive Cytotoxicity Assay and Annexin V. We also utilized multiplex cytokine immunoassays to quantify cytokine production. For in vivo studies, we injected luciferase-GFP tagged Kasumi-1 cells (10X106) into NSG mice, followed by injection of CAR T (5X105) +/- CAR-DC (1.5X105) or control T cells (5X105). We monitored the NSG mice using serial bioluminescence imaging and compared the survival of each group.
Results: On phenotypic analysis using flow cytometry, we found that frequencies of cells expressing CD141/CLEC9A+ were significantly higher in CAR-DC vs. control DC (35.2 +/- 4.1 % vs. 9.0 +/- 1.7 % of HLA-DR+ cells), which suggest 4-1BB activation induce CD34+ progenitors to intratumoral DCs. The cytotoxicity assay showed 63.2 +/- 0.6 % Kasumi-1 death with CAR T/CAR-DC compared to 46.5 +/- 3.5 % with CAR T cells alone. CAR T/CAR-DC also demonstrated more Annexin V positive Kasumi-1 cells compared to CAR T and control T cells (78.4 +/- 5.1 % vs 39.9 +/- 7.7 % vs 17.6 +/- 2.2 %). These cytotoxicity assays demonstrated that CAR-DC enhanced the anti-Kasumi-1 cytotoxicity of anti-CD33 CAR T cells. CAR T cells co-cultured with CAR-DC produced a two-fold higher IFN-gamma and TNF-alpha than CAR T cells alone (p<0.01). The IFN-gamma and TNF-alpha production increases in correlation with the counts of CAR T cells. However, CAR T/CAR-DC group produced a four-fold higher IL-12 throughout different E/T ratios compared to CAR T alone group (p<0.01), which suggest DCs are the major source of IL-12 production and CAR T cells produce a higher level of IFN-gamma and TNF-alpha in response to DCs. In vivo NSG mice experiments demonstrated that CAR T/CAR-DC group had increased survival (p<0.01) and decreased AML burden than CAR T alone group.
Conclusions: Our data show that 1) in vitro differentiation of DCs with 4-1BB stimulation increases intratumoral CD141/CLEC9A+ DCs, 2) interaction between CAR-DC and CAR T cells enhances cytotoxic cytokine production in response to DC-derived IL-12. These combined effects resulted in improved anti-CD33 CAR T cytotoxicity in vitro and in vivo NSG AML mice model. Our findings implicate the development of a new strategy of CAR T therapy combined to CAR-DC to increase the efficacy of cancer immunotherapy.
Disclosures
No relevant conflicts of interest to declare.
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