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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 or EBV-related posttransplantation lymphoproliferative disease (EB-LPD), suggesting PTCy-based immune modulation leads the favorable immune reconstitution after transplant. Recently, we studied the immune reconstitution dynamics of each lymphocyte subset after transplant using PTCy and reported that PTCy-based immune-modulation strongly promoted Treg-dominant T-cell reconstitution and stem cell-derived mature B-cell generation by using murine model of haploidentical HSCT with PTCy. TCR- and BCR- repertoire analysis demonstrated that PTCy resulted in the broader diversity of both T cell and B cell repertoire than non-PTCy controls. However, the mechanism of coordination of increased Tregs and B cells after PTCy-based HSCT has not well studied. To address this issue, we here investgated the impact and role of PTCy on Tregs and B cell recovery and the mutual influence between the two important subsets by using murine BMT model. Irradiated B6D2F1 mice were transplanted with 5x106spleen cells from the CD45.1 B6 mice together with 5x106TCD-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 day21. The chimeric balances among host-residual H-2kd+ cells, donor graft-derived cells and donor BM-derived cells in Tregs and B cells were monitored separately. To evaluate the homeostatic stability, proliferation marker Ki-67 and anti-apoptotic BCL-2 were also quantitatively examined respectively. To examine the detailed phenotype of Tcells, CD4, CD8, CD25, Foxp3, ICOS, CXCR5 were tested. Also, to examine the detailed phenotype of B cells, the expression of B220, CD21, CD23, CD24 were tested. At day21 after HSCT, the number of Treg cell in spleen was significantly higher on PTCy-treated recipients than that of non-PTCy control (P < 0.05). We also checked the emergence of donor-derived follicular helper T cells (Tfh) in lymph node because recent studies have shown that Tfh could contribute B cell differentiation after HSCT. In our analysis at day21 in this PTCy model, Tfh cells were still few in both PTCy group and non-PTCy group, suggesting the early T cell recovery after PTCy did not promote B cell differentiation into pathological plasmablast. The number of B cell was markedly higher in PTCy-treated recipients than that of non-PTCy control (P < 0.01). The increased B cell population was mainly transitional-1 phenotype suggesting these B cells were recent emigrants from bone marrow. In consistent with low recovery of Tfh cells, plasmablast-like phenotype of B cells were not observed. Next, to evaluate the impact of graft-derived Treg recovery on B cell reconstitution, we depleted Treg from spleen cells of B6 donors by using CD25+ magnetic beads. After the depletion, residual Treg cell in the graft was under 1% of total CD4 T cells. At day21, splenic Treg cells is under 3% of total CD4 T cells in Treg-depleted group, that is much lower than Treg in Treg-repleted group. Interestingly, the number of B cells at day21 in PTCy-treated recipients was equivalent to that of non-PTCy recipients when we depleted Treg from donor graft, suggesting that well-balaced B cell recovery promoted by PTCy was completely abolished by Treg-depletion. Taken together, our data suggested that PTCy could induce the expansion of Treg recovery and naïve B cell recovery without urging early emergence of Tfh cells in lymph node, resulting in well-diversed T and B cell reconstitution. Treg appears to contribute to promote naïve B cell emergence from bone marrow, indicating the T and B cell recovery might be mutually influenced. Moreover, slow recovery of Tfh cells in lymph node could be advantage for preventing B cell from forced differentiation into plasmablast causing GVHD. These data provide the important information for understanding the immunological reconstitution after PTCy-based haploidentical HSCT.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the curative treatment of many hematological malignancies, but the relapse is one of the major causes of treatment failure and mortality. Previous clinical studies have shown that low-dose IL-2 was well tolerated and could lower the relapse rate in human T cell depleted allogeneic BMT. On the other hand, we also demonstrated that low-dose IL-2 could preferentially enhance regulatory T cell (Treg) and improve the symptoms of chronic GVHD (NEJM 2011,Sci Trans Med 2013). In murine bone marrow transplantation (BMT) model showed that Treg can protect from lethal GVHD while preserving graft-versus-leukemia (GVL) effect. Especially, CD62L+ Tregs has a higher capacity in suppressing GVHD than their CD62L- counterpart. Our recent study reported that exogenous low-dose IL-2 induced the expression of PD-1 on Treg preferentially and it was most evident in CD44+CD62L+central-memory Treg (Blood 2016). However, the impact of CD62L+ Treg enhanced by low-dose IL-2 on GVHD and GVL has not been well investigated. In this study, we studied the effect of exogenous IL-2 on the lymphocyte phenotypic profiles including naïve- versus memory-type balance and also examined donor-derived immunity, especially GVHD and GVL, by using leukemia-bearing BMT model. Lethally irradiated B6D2F1 recipients were transplanted spleen cells and BM cells from B6 donors and 5000 IU Teceleukin (recombinant IL-2) was injected once a day for 14 days. The number of CD4+CD25+Foxp3+ Treg, CD4+CD25-Foxp3- conventional T cell (Tcon) and CD8+ T cell was compared between IL2- and vehicle-treated groups. The expressions of CD44, CD62L, CCR4, CCR7, Ki-67, PD-1, CTLA-4, LAG3, GITR, ICOS and TIM-3 in each subset were also examined. Lymphocytes profiling analysis showed that IL-2 treatment in the first 7 days resulted in the dominant expansion of effector T cells without Treg increase (Treg 0.10 vs 0.16,p=0.35, Tcon 2.83 vs 4.20, p=0.01, CD8 T cell 5.52 vs 10.4, p=0.01, million). On the contrary, the extended IL-2 treatment in the next 7 days increased CD62L+ Treg (21.5 vs 48.2 % of total Treg, p<0.001, 0.02 vs 0.07 million, p=0.006) without increasing effector T cells. PD-1 on Treg was very high in BMT-recipients even without receiving IL-2, so further enhancement of PD-1 expression by IL-2 was not observed. However, the expression of other inhibitory molecules including CTLA-4 (MFI, 6.61 vs 9.92, p=<0.001), LAG-3 (1.73 vs 2.17, p=0.04) and ICOS (2.41 vs 3.84, P<0.001) was enhanced by the IL-2 treatment. To elucidate the mechanisms of the increase in CD62L+ Treg, the in vivo violet dye dilution assay was performed. We first sorted out CD62L+ T cells by micro-beads from spleen cells, and then adaptively transferred into recipients. Five days treatment of low-dose IL-2 resulted in the enhancement of the division of CD62L+ Treg (% divided cells; 19.5 vs 29.8 % of Treg, p=0.005, 62.3 vs 108 in number, p=0.002). Interestingly, when CD62L- T cells were transferred, IL-2 treatment also induced the division of the cells and, of note, induced the phenotypic change of CD62L- into CD62L+ Treg (7.63 vs 19.8 % of Treg, p=0.009, 91.3 vs 361, p=0.01). These results suggested that IL-2 have the possibility to enhance the proliferative activity of Treg with the up-regulation of CD62L expression. Lastly, we transplanted lucipherase+P815 leukemia cells into this BMT model and evaluated the clinical impact of low-dose IL-2 on GVL by using in vivo bioluminescence system. To assesse the GVL and GVHD independently, we used two different tumor burden models. In the high tumor burden model, IL-2 injection decreased bioluminescence intensity (BLI) of P815 (BLI at week3, 8.3x10E7 vs 8.1x10E6 photon/second, p<0.001) and significantly extends overall survival. On the other hand, in the low tumor burden model, both treatment groups did not die from leukemia but vehicle-treated control group showed a significant higher clinical GVHD score and died from GVHD. These data demonstrated the clinical benefits on anti-tumor effect of IL-2, which could enhance the GVL activity without the detrimental effect on GVHD. In conclusion, our data indicate multiple effects of IL-2 on post-transplant immunity can coordinate to intensify GVL separately from GVHD on the mechanism of sequential enhancement of effector T cell and CD62L+ Treg in the different phases. These data provide the important information for developing Treg-targeted therapy.