Comprehensive Analyses of Early Lymphocyte Reconstitution after Haploidentical HSCT with Posttransplant Cyclophosphamide: Coordinated Treg-Dominant T-Cell Reconstitution and Stem Cell-Derived Mature B-Cell with Broad BCR-Repertoir Diversity
Authors: Iwamoto Miki , Matsuoka Ken-ichi , Meguri Yusuke , Asano Takeru , Yoshioka Takanori , Kishi Yuriko , Nakamura Makoto , Sando Yasuhisa , Suzuki Ryuji , Kitaura Kazutaka , Maeda Yoshinobu , Tanimoto Mitsune 
Published: October 2019

Abstract
Posttransplant cyclophosphamide (PTCy) is an effective prophylaxis for both acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Recent studies reported that PTCy has been associated with low incidence of viral infections and EB-LPD, suggesting PTCy-based immune modulation leads the favorable immune reconstitution after transplant. However, the immune reconstitution dynamics of each subset after HSCT using PTCy remains poorly understood. To address this issue, we explored the impact and role of PTCy on the early lymphocyte reconstitution by using murine BMT model. Irradiated B6D2F1 mice were transplanted with 5x106 spleen cells from the CD45.1 B6 mice together with 5x106 TCD-BM from CD45.2 B6 donors. Cyclophosphamide 100mg/kg or control vehicle was administered at day 3 after transplant. Peripheral blood mononuclear cells (PBMCs) and splenic cells were sequentially obtained at day7, 14 and 21.The chimeric balances among host-residual H-2kd+ cells, donor graft-derived cells and donor BM-derived cells in CD8+ T cells, CD4+ Tcons, Tregs, B cells and NK cells were monitored separately. To evaluate the homeostatic stability of each lymphocyte subset at various time points, proliferation marker Ki-67 and anti-apoptotic BCL-2 were also quantitatively examined in each subset. To evaluate the clonal diversity of T and B cells, we performed the TCR- and BCR- repertoire analysis at day 21. Between day 0, transplanted recipients were developed severe acute GVHD, however, recipients received PTCy at day 3 promptly showed the recovery of the weight and improvement of the clinical GVHD score after day 5, whereas control continue to lose weight, suggesting the effect of PTCy to ameliorate acute GVHD. At day 7, all T cell subsets were critically depleted from both peripheral blood and spleen. The number of T cells was markedly lower in PTCy group than in control group (CD8 T+ cells; 5.1 vs 155.1/mm2, P<0.01: CD4 Tcons; 5.2 vs 61.2/mm2, P<0.01: Treg; 0.02 vs 0.52/mm2, P<0.01, respectively). Especially, Ki-67+ proliferating cells, including Tcons and Tregs, were completely depleted, indicating these activated cells are very sensitive to cyclophosphamide intervention. However, interestingly, surviving T cells in recipients just after cyclophosphamide intervention showed significantly high-levels of BCL-2 expression than control recipients (MFI: CD8 T cells; 2.8 vs 10.0: CD4 Tcns; 2.9 vs 9.9: Treg; 1.8 vs 5.3, respectively). Based on the elevated anti-apoptotic elements, T cell in PTCy-treated recipients undergo aggressive homeostatic proliferation and the number of CD4 T cell subset, especially Tregs, took over that of control recipient by day 14. CD8+ T cell proliferation after PTCy was less aggressive than CD4 T cells, resulting Treg ratio to CD8 T cells in PTCy recipents was greatly higher than in control (Treg/CD8: 0.061 vs 0.031, P<0.05). During 3 weeks, T cell recovery was basically maintained by donor graft-derived cell, though PTCy recipents involved averagely 10% of host-residual T cells. In comparison to T cells, main reconstitution of B cells was maintained by donor stem cell-derived cell. In PTCy recipients, CD23+CD24+ Transitional-2 naïve B cell and CD21-CD24+ mature follicular B cell overwhelmingly increased by Day 21(Follicular B cells in PTCy group and control; 1.22e6 vs 1.31e5, P<0.0001). BCR-repertoire diversity analysis demostrated that PTCy resulted in the broad diversity of B cell repertoire (Inverse Simpson Index; 40.5 vs 13.9). Our data clearly indicated that PTCy contributes the favorable immune reconstitution by modulating coordinate T and B cell recovery. These findings might provide important information to promote immune tolerance after PTCy-based transplant.

Disclosures
Maeda: Mundipharma KK: Research Funding.