Impaired Thymic Negative Selection Causes Chronic Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation.
Authors: Sakoda Yukimi , Hashimoto Daigo , Takeuchi Kengo , Asakura Shoji , Akashi Koichi , Tanimoto Mitsune , Harada Mine , Teshima Takanori 
Published: October 2019

Abstract
Chronic GVHD continues to be the most common late complication of allogeneic BMT. In contrast to acute GVHD, the basic pathophysiology of chronic GVHD remains poorly defined. We herein tested the hypothesis that impaired thymic negative selection of the recipients allows for the emergence of donor or host reactive T cells and causes chronic GVHD in a mouse model of allogeneic BMT. C3H/HeN (H-2k) recipients were lethally irradiated and reconstituted with 5 × 106 T cell-depleted bone marrow (TCD BM) cells from wild-type (wt) or MHC II-deficient (II-KO) B6 (H-2b) mice. In recipients of TCD BM from II-KO mice, MHC II molecules were expressed on the radioresistant thymic epithelium that supports positive selection, but not on the radiosensitive hematopoietic elements responsible for negative selection after BMT. Replacement of host thymic dendritic cells (DCs) by donor-derived MHC II-deficient DCs was confirmed by flow cytometric analysis 4 weeks after BMT. Analysis of the thymus showed the emergence of TCRαβCD4 single positive thymocytes after BMT in recipients of TCD BM from II-KO donors, thus confirming that radioresistant thymic epithelial cells mediated positive selection. CD4+ T cells isolated from these mice 6 weeks posttransplant proliferated to both B6 (donor) and C3H/HeN (recipient) stimulators in vitro, demonstrating the emergence of donor and host reactive CD4+ T cells in the absence of thymic negative selection. Although recipients of TCD BM from wt donors did not show any signs of GVHD, those of TCD BM from II-KO donors developed weight loss and alopecia 6 weeks after BMT. Ten weeks after BMT, analysis of the peripheral blood showed pancytopenia and elevation of serum levels of liver enzymes and bilirubin in these mice (Table). Analysis of the thymus and spleen demonstrated severe lymphoid atrophy (Table). Histologic examination of the skin and liver showed standard pathologic features of human chronic GVHD, including sclerodermatous skin changes, such as epidermal atrophy, fat loss, follicular dropout and dermal thickness, as well as bile duct loss and fibrosis in the portal area of the liver. Pathology scores of the skin which sums changes of five pathological parameters were significantly higher in recipients of TCD BM from II-KO donors than in those of wt donors (Table). This GVHD was lethal with only 33% survival at day 80 after BMT in recipients of II-KO TCD BM, whereas all recipients of wt TCD BM survived. Thymectomy prevented the disease, confirming the causal association of the thymus with its development. Adoptive transfer of these CD4+ cells caused similar chronic GVHD in irradiated secondary C3H/HeN recipients. These results demonstrated that impaired thymic negative selction of the recipients allowed for the emergence of donor BM-derived T cells that are both donor and host reactive and caused the disease resembling human chronic GVHD. Our model thus may help to provide insight into the development of chronic GVHD and the similarity of chronic GVHD to autoimmune diseases.
Table Donor Weight WBC PLT AST Spleen Skin Score Survival % 102/μl 104/μl IU/l 106/μl % *P〈 0.05 wt 110±1 130±14 80±8 94.±19 124±29 0±0 100 II-KO 80±7* 44±10* 31±10* 374±9* 14±15* 4.4±0.9 *33*