A Calcineurin Inhibitor Does Not Affect the Post-Transplantation Cyclophosphamide -Induced Regulatory T Cell Expansion after Bone Marrow Transplantation in a Murine Chronic Gvhd Model
Authors: Saeki Kyosuke , Maeda Yoshinobu , Seike Keisuke , Tani Katsuma , Kuroi Taiga , Yoshida Shohei , Nishimori Hisakazu , Tanimoto Mitsune 
Published: October 2019

Abstract
Chronic graft-versus-host disease (GVHD) remains a major late complication of allogeneic hematopoietic stem cell transplantation (HSCT). Prolonged administration of a calcineurin inhibitor, such as cyclosporin A (CsA), does not decrease the risk of chronic GVHD. Indeed, we and others have reported that long-term use of CsA impairs reconstitution of bone marrow-derived regulatory T cells (Tregs) and increases susceptibility to chronic GVHD. Recently, the administration of post-transplantation cyclophosphamide (PTCY) has been used widely in GVHD prophylaxis and lower incidences of acute and chronic GVHD have been reported. Here, we evaluated the effects of PTCY and CsA on Tregs and cytokine production in effector T cells using the well-defined chronic GVHD mouse model of B10.D2 (H2d) cells into BALB/c (H2d) mice. Sublethally-irradiated recipient BALB/c mice were transplanted with 8.0 × 106 T cell-depleted bone marrow cells and 2.0 × 106 T cell-replete spleen cells from donor B10.D2 mice. PTCY was administered by intraperitoneal injection at days 3 and 4 post bone marrow transplantation (BMT). CsA was injected peritoneally once daily, starting on day 0 of BMT until the day of analysis. Administration of PTCY, with or without CsA, improved the clinical chronic GVHD score significantly at day 50 versus the controls and the CsA-alone group (controls: 2.8 ± 0.34, PTCY: 0.5 ± 0.0, p < 0.01; CsA alone: 1.8 ± 0.73, PTCY-CsA: 0.5 ± 0.0, p < 0.01). There was no significant difference in the GVHD score between the PTCY and PTCY-CsA groups. Histopathological examinations of the skin at day 50 after BMT also showed significantly reduced chronic GVHD damage in the PTCY group versus the controls and the CsA-alone group (controls vs. PTCY, p < 0.001; CsA alone vs. PTCY, p < 0.01). Flow cytometry analysis of the cells isolated from the peripheral lymph nodes 28 days after BMT revealed that the proportions of Tregs to CD4+ T cells in the PTCY (25.8 ± 2.1%) and PTCY-CsA (29.5 ± 2.2%) groups were remarkably higher than the controls (8.3 ± 2.7%) and CsA-alone (6.7 ± 1.6%) groups (p < 0.05). There was no significant difference in the Treg ratio between the PTCY and PTCY-CsA groups. Intracellular staining showed that IFN-γ single-positive and IFN-γ/IL-17 double-positive CD4 T cells were well suppressed in the PTCY and PTCY-CsA groups versus the controls and CsA-alone group (controls: 38.2 ± 3.5%, PTCY: 11.9 ± 3.1%, p < 0.01; CsA alone: 46.9 ± 2.2%, PTCY-CsA: 17.9%, p < 0.01). Our data suggest that PTCY treatment attenuated chronic GVHD via enhanced reconstitution of donor Tregs and suppression of IFN-γ/IL-17-producing effector T cells. Moreover, CsA does not appear to hinder this suppressive effect of PTCY. This result seems helpful for understanding how PTCY prevents chronic GVHD in clinical settings.

Disclosures
Maeda: Mundipharma KK: Research Funding.