Absence of LTB4/BLT1 Axis Promotes Generation of Long-Lasting Antitumor Memory Responses Induced by Administration of GM-CSF Gene-Transduced Tumor Cells, in a CD4⁺ T Cell-Dependent Manner,

Abstract 3246

GM-CSF (Granulocyte macrophage-colony stimulating factor) gene-transduced leukemia cell vaccine has been reported to be promising approach to enhance antitumor immune response in leukemia patients. The enhancement of this specific antitumor immunity is considered to be clinically beneficial to maintain complete remission for long time after chemotherapy without stem cell transplantion. Leukotriene B4 (LTB4) is an extremely potent lipid inflammatory mediator derived from membrane phospholipids, known to recruit and activate leukocytes including neutrophils, mediated by the same class of receptors, the GPCR (G-protein coupled receptor) superfamily, BLT1 and BLT2. So far, the role of LTB4 in tumor immunology is not well known. Previously, we demonstrated that the GM-CSF gene transduction into murine monocytic leukemia cell line of WEHI3B (WGM) eliminated the tumorigenicity in subcutaneous challenge model using wild type (WT) BALB/c mice. At day 50 after the challenge, we rechallenged WEHI3B cells into the opposite flank of both WT and BLT1-KO mice which had rejected WGM cells (WT/WGM, KO/WGM, respectively). Intriguingly, more KO/WGM mice re-rejected the rechallenged WEHI3B cells and showed significantly prolonged survival compared with WT/WGM mice.

To clarify this unique mechanism of the long-lasting antitumor effects observed in KO/WGM mice, we performed following immunological assays. Our in vivo immune cell depletion assays (NK, CD4⁺ T, CD8⁺ T cell) showed that KO/WGM mice treated with anti-CD4 antibody displayed rather enhanced tumor growth compared with WT/WGM mice. Thus, we next compared the rates of different subsets of memory T cells, so called memory stem T cell subsets (T_SCM), central memory T cell subsets (T_CM) and effector memory T cell subsets (T_EM) in TDLNs (Tumor draining lymph nodes) between WT/WGM mice and KO/WGM mice at the day 46 after tumor challenge. Expectedly, results showed that TDLNs harvested from KO/WGM mice exhibited higher subset ratios of CD44⁺CD62L⁺ (T_CM) cell to CD4⁺ T cells as well as CD44⁺CD62L⁻ or (T_EM) cell to CD4⁺ T cells than those from WT/WGM mice. In the case of use of CD122 for memory marker, similar results were obtained. Of note, TDLNs from KO/WGM mice exhibited increased ratios of CD44⁺CD122⁺CD62L⁻ (T_SCM) cell. In addition, the cell numbers of immunosuppressive CD3⁺ CD4⁺ PD-1⁺, CD3⁺ CD4⁺ GITR⁺ and CD3⁺ CD4⁺ CTLA4⁺ cells were reduced in both TDLNs and spleen derived from KO/WGM mice compared with those from WT/WGM mice. Furthermore, our results of CBA (Cytometric Bead Array) assay using splenocytes harvested from day 2 to day 15 showed that the Th2 cytokine production level of IL-4 and IL-5 from splenocyte harvested from KO/WGM mice were higher than those from WT/WGM mice. In regard to IL-2 and IFN-g (Th1 cytokine), the production level at both day 7 and day10 from splenocytes harvested from KO/WGM mice were also higher than those from WT/WGM mice, implicating that loss of LTB4/BLT1 signaling promotes systemic activation of both tumor antigens specific Th1 and Th2 CD4⁺ T cell subsets. Finally, our flow cytometric analyses demonstrated that exogenously GM-CSF-driven upregulated MFI of CD40⁺, CD80⁺ and CD86⁺ DCs in TDLNs from KO/WGM mice were further significantly increased compared with those from WT/WGM mice (p<0.05), and more numerous number of CD86⁺ DCs that had phagocytosed TAAs of WEHI3B cells was detected in TDLNs harvested from KO/WGM mice than that from WT/WGM mice.

In conclusion, we for the first time demonstrate that loss of LTB4/BLT1 axis can sustain long-lasting memory CD4⁺ T cell-dependent antitumor immunity against syngeneic tumor cells long after GM-CSF triggering tumor rejection, allowing us to expect the possibility that the strategy of blocking LTB4/BLT1signaling may be useful to maintain antitumor immunity induced by GM-CSF gene-transduced leukemia cell vaccine in clinical settings.