Influences of Mycophenolate Mofetil and Cyclosporine-a on the Ex Vivo-Expanded Vδ2 T Cells

Abstract

Background. γδ T cells are increasingly recognized as the important players in host defense against infections and malignancies. The potent ability of γδ T cells to kill hematopoietic and solid tumors has attracted great interest for using them as an adoptive cell therapy strategy to treat cancer. It has been extensively reported that Vδ2 cells, the dominant subpopulation of peripheral γδ T cells in human, are easily expanded by aminobisphosphonates (e.g pamidronate) ex vivo. Several clinical trials of Vδ2 cells-based immunotherapy have been conducted with promising results. Given that γδ T cells do not cause graft versus host disease (GvHD), but can exert strong graft-versus leukemia (GvL) effect, this special T-cell population becomes popular for cellular immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (alloHSCT). Immunosuppressive drugs are commonly used for conditioning and GvHD prophylaxis and treatment, which could affect the efficacy of adoptive Vδ2 cells. However, whether the phenotype and function of ex vivo-expanded Vδ2 cells are influenced by immunosuppressive drugs remains unknown. Our unpublished data implicated that mycophenolate mofetil (MMF) impaired the recovery of Vδ2 cells after alloHSCT. We next explored the impacts of MMF and cyclosporine A (CsA) on the pamidronate-expanded Vδ2 T cells.

Methods. PBMCs from healthy donors were stimulated with pamidronate in the presence of IL-2 for 10-14 days. The proliferation, differentiation maker, functional receptor expression, and cytotoxic cytokines-production capacity of pamidronate-expanded Vδ2 cells were determined by Flow Cytometry following 1, 3, and 6 days treatments with mycophenolic acid (MPA, the active form of MMF) and CsA respectively. These experiments were performed in the primary Vδ2 cells at the same time. Furthermore, the influences of MMF and CsA on the cytotoxic activity of pamidronate-expanded Vδ2 cells on leukemia cells were investigated using calcein AM pre-staining of target cells.

Results. Compared to primary Vδ2 T cells in PBMCs, pamidronate-expanded Vδ2 cells exhibited markedly increased expressions of effector cell marker (CD45RO), functional receptor (NKG2D), and cytotoxic cytokines (IFN-γ and TNF-α). Upon treatment with MPA and CsA, we observed that the differentiation status and functional receptor expression of expanded Vδ2 cells were not significantly changed. Notably, their proliferation and cytokine-production capacity were attenuated differentially by MPA and CsA treatment. It seemed that pamidronate-expanded Vδ2 T cells were more tolerant to MPA and CsA than primary Vδ2 T cells, in comparing the reduction degrees of cytotoxic cytokines produced after 1-day of CsA treatment (TNF-α 13.8% Vs 37.0% , P=0.02; IFN-γ 28.4% Vs 77.2% , P=0.00003), and after 3-day of MPA treatment (IFN-γ 34.1% Vs 89.7% , P=0.02). In addition, the cytotoxic elect of pamidronate-expanded Vδ2 T cells against K562 cells was moderately impaired after exposure to MPA and CsA.

Conclusions. We demonstrated for the first time the influences of MPA and CsA on pamidronate-expanded Vδ2 T cells. These results indicate that MPA and CsA each has distinct effect on the phenotypes and cytotoxic function of pamidronate-expanded Vδ2 T cells. It is worth emphasizing that pamidronate-expanded Vδ2 T cells were more tolerant to MPA and CsA than the primary Vδ2 T cells, which may have important implications for Vδ2 cells-based immunotherapy after alloHSCT.

Acknowledgments. This study is supported by the National Natural Science Foundation of China (No. 81770191), and Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 81621001).