Reconstitution of Regulatory T Cells Involves in the Development of Acute Graft-Versus-Host Disease after Hematopoietic Stem Cell Transplantation.
Authors: Karakawa Shuhei , Nakamura Kazuhiro , Hara Keiichi , Mizoguchi Yoko , Miki Mizuka , Kawaguchi Hiroshi , Sato Takashi , Nishimura Shin-ichiro , Kobayashi Masao 
Published: April 2020

Abstract
Graft-versus-host disease (GVHD) is a significant complication in hematopoietic stem cell transplantation (SCT). On the other hand, graft-versus-tumor (GVT) effect has been known to be concerned with the prevention of relapses in various hematological or non-hematological malignant disorders. The regulation of GVHD without suppressing GVT effect is a pivotal role in the success of hematopoietic SCT. Recently CD4+CD25+regulatory T cells have been recognized to regulate the maintenance of self-tolerance, and associate with several autoimmune diseases. In transplant immunity, CD4+CD25+regulatory T cells have been reported to regulate GVHD without suppressing GVT effect in several animal studies. Natural killer T (NKT) like cells also have been recognized to associated with the maintenance of self-tolerance by inducing CD4+CD25+regulatory T cells through the production of IL-2. In this study, we examined the roles of CD4+CD25+regulatory T cells and NKT like cells in cases underwent hematopoietic SCT. Blood samples from patients underwent SCT in our institution during past 3 years (7 months through 26 years of age, n =19) were obtained every two weeks until day 90 after SCT. Primary disorders of patients were non-malignant hematological disease such as aplastic anemia (n=3), chronic granulomatous disease (n=8) and malignant disease such as leukemia (n=5), and solid tumors (n=3). Pre-conditioning regimens used in this study were myeloablative regimens in 10 cases and reduced intensity regimens in 9 cases, respectively. The frequencies of CD4+CD25+regulatory T cells were assessed by the expression of CD4 and CD25, and those of NKT like cells were assessed by the expression of CD3, CD16, and CD56 using flow cytometry. The mRNA expression of FOXP3 in purified CD4+CD25+regulatory T cells were determined by quantitative real-time PCR method. In 13 patients who had none or Grade 1 acute GVHD, the frequency of CD4+CD25+regulatory T cells in CD4+ T cells was increased up to 25–90% at early period after SCT and normalized below 20% after day 45 post SCT. On the other hand, four of 6 patients who had acute GVHD (more than Grade 2) showed that the frequency of CD4+CD25+regulatory T cells in CD4+ T cells persisted below 20%. In other one patient, the development of acute GVHD (Grade 2) was associated with decreasing the frequency of CD4+CD25+regulatory T cells (30 to 10%) and the recovery of GVHD was found with increasing CD4+CD25+regulatory T cells (10 to 30%). The mean frequency of CD4+CD25+regulatory T cells in CD4+ T cells on day 15 after SCT was 44% in patients without GVHD and 21% in patients with GVHD (p=0.07). No difference in the expressions of FOXP3 mRNA in purified CD4+CD25+regulatory T cells was noted between patients with GVHD and those without GVHD. The reconstitution pattern of CD3+CD16+CD56+ NKT like cells after SCT was not associated with the development of GVHD. These results suggest that the development of acute GVHD may be strongly associated with the reconstitution of donor derived CD4+CD25+regulatory T cells in CD4+ T cells. The measurement of CD4+CD25+regulatory T cells in CD4+ T cells might lead to the early diagnosis and the prevention of acute GVHD. (Future studies will be needed to examine the association between the frequency of regulatory T cells and GVT effect.)