Hematopoietic stem cell–derived Tregs are essential for maintaining favorable B cell lymphopoiesis following posttransplant cyclophosphamide
Yuichi Sumii, Takumi Kondo, Shuntaro Ikegawa, Takuya Fukumi, Miki Iwamoto, Midori Filiz Nishimura, Hiroyuki Sugiura, Yasuhisa Sando, Makoto Nakamura, Yusuke Meguri, Takashi Matsushita, Naoki Tanimine, Maiko Kimura, Noboru Asada, Daisuke Ennishi, Yoshinobu Maeda, Ken-ichi Matsuoka
Yuichi Sumii, Takumi Kondo, Shuntaro Ikegawa, Takuya Fukumi, Miki Iwamoto, Midori Filiz Nishimura, Hiroyuki Sugiura, Yasuhisa Sando, Makoto Nakamura, Yusuke Meguri, Takashi Matsushita, Naoki Tanimine, Maiko Kimura, Noboru Asada, Daisuke Ennishi, Yoshinobu Maeda, Ken-ichi Matsuoka
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Research Article Hematology Transplantation

Hematopoietic stem cell–derived Tregs are essential for maintaining favorable B cell lymphopoiesis following posttransplant cyclophosphamide

Abstract

Posttransplant cyclophosphamide (PTCy) is associated with a low incidence of chronic graft-versus-host disease (cGVHD) following hematopoietic stem cell (HSC) transplantation. Previous studies have shown the important roles of B cell immunity in cGVHD development. Here, we investigated the long-term reconstitution of B lymphopoiesis after PTCy using murine models. We first demonstrated that the immune homeostatic abnormality leading to cGVHD is characterized by an initial increase in effector T cells in the bone marrow and subsequent B and Treg cytopenia. PTCy, but not cyclosporine A or rapamycin, inhibits the initial alloreactive T cell response, which restores intra-bone marrow B lymphogenesis with a concomitant vigorous increase in Tregs. This leads to profound changes in posttransplant B cell homeostasis, including decreased B cell activating factors, increased transitional and regulatory B cells, and decreased germinal center B cells. To identify the cells responsible for PTCy-induced B cell tolerance, we selectively depleted Treg populations that were graft or HSC derived using DEREG mice. Deletion of either Treg population without PTCy resulted in critical B cytopenia. PTCy rescued B lymphopoiesis from graft-derived Treg deletion. In contrast, the negative effect of HSC-derived Treg deletion could not be overcome by PTCy, indicating that HSC-derived Tregs are essential for maintaining favorable B lymphopoiesis following PTCy. These findings define the mechanisms by which PTCy restores homeostasis of the B cell lineage and reestablishes immune tolerance.

Authors

Yuichi Sumii, Takumi Kondo, Shuntaro Ikegawa, Takuya Fukumi, Miki Iwamoto, Midori Filiz Nishimura, Hiroyuki Sugiura, Yasuhisa Sando, Makoto Nakamura, Yusuke Meguri, Takashi Matsushita, Naoki Tanimine, Maiko Kimura, Noboru Asada, Daisuke Ennishi, Yoshinobu Maeda, Ken-ichi Matsuoka

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Figure 2

PTCy is associated with a decrease in graft-derived effector T cells in the bone marrow soon after BMT and increase in HSC-derived mature T cells in the later period.

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PTCy is associated with a decrease in graft-derived effector T cells in ...
(A) Lethally irradiated (10 Gy) BDF1 recipients (H2Kb/dCD45.2+) received transplants of 5 × 106 Ly 5.1 B6 (H2Kb/bCD45.1+) splenocytes with 5 × 106 B6 (H2Kb/bCD45.2+) TCD-BM cells. All recipient mice were injected intraperitoneally with 50 mg/kg of cyclophosphamide or vehicle on day 3 after allogeneic BMT (vehicle-treated, n = 30; and PTCy-treated, n = 32). Animals were euthanized on days 7, 14, 21, and 28 (early phase) and days 56 and 84 (late phase) after allogeneic BMT to harvest bone marrow, spleens, and thymi. (B) The kinetics of total nucleated cell recovery in the bone marrow after allogeneic BMT. (C and D) Representative flow cytometry plots identifying CD4+ and CD8+ T cell subsets (C) and chimerism (D) in the bone marrow and spleen on day 7 after allogeneic BMT. (E) Kinetics of graft- and HSC-derived CD8+ T cell, CD4+ Tcon, and CD4+ Treg recovery in the bone marrow and spleen after allogeneic BMT. * and † (E) indicate the comparison between graft-derived T cells in vehicle-treated group versus those in PTCy-treated group and HSC-derived T cells in vehicle-treated group versus those in PTCy-treated group, respectively. Gray bars (B and E) indicate mean reference values ± SEM of NC (n = 3). (F) Representative flow cytometry plots identifying CLP (Lin−c-KitintSca-1intIL-7Rα+Flt3+) cells and the total number of CLP cells in the bone marrow on day 56 after allogeneic BMT (vehicle-treated, n = 4, and PTCy-treated, n = 5). (G) Representative flow cytometry plots identifying DP (CD4+CD8+) cells and number of DP cells in the thymus on day 56 after allogeneic BMT (vehicle-treated, n = 5, and PTCy-treated, n = 5). Graft- and HSC-derived cells were defined as H2Kd−CD45.1+ and H2Kd−CD45.1− gated cells by flow cytometry, respectively. Data from 2 independent experiments were combined and expressed as the mean ± SEM. P values were determined using the Mann-Whitney U test. *,†P < 0.05, **,††P < 0.01, ***P < 0.001. CY, cyclophosphamide; CLP, common lymphoid progenitor; DP, double-positive; Lin, lineage.