Possible Involvement of γδ-T Cells in the Development of Chronic Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Koh Ki-Ryang , Nakamae Hirohisa , Ohta Kensuke , Li Hongzhang , Koh Hideo , Nakane Takahiko , Takeoka Yasunobu , Hirose Asao , Hagihara Kiyoyuki , Sakamoto Erina , Nakamae Mika , Terada Yoshiki , Yamane Takahisa , Hino Masayuki 
Published: April 2020

Abstract
Background: Chronic graft-versus-host disease (cGVHD) remains the major cause of late morbidity and non relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the pathogenesis of cGVHD is poorly understood. This study was designed to explore the immunological mechanism of cGVHD with comprehensive analysis of peripheral T cell subsets (CD4, CD8, Th1, Th2, γδ-T and NKT), dendritic cell (DC) subsets and serum 17 different cytokines.
Methods: 20 patients (pts) with complete donor cell engraftment over 100 days after allo-HSCT were enrolled, including 6 pts without cGVHD (control) and 14 pts with cGVHD (5 limited/ 9 extensive). All of the pts with extensive cGVHD were under systemic immunosuppressive therapy, and the pts with limited cGVHD or without cGVHD were free from immunosuppressant. T cell subsets including CD4 (CD3+CD4+CD8−), CD8 (CD3+CD4−CD8+), Th1 (CD4+CXCR3+CCR4−), Th2 (CD4+CXCR3−CCR4+), γδ-T (CD3+TCRVδ2+), and NKT (CD3+CD161+) and DC subsets (CD11c+DC and CD123+DC) were determined with a flow cytometer. Serum cytokines, including IL–1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF-α, IFN-γ, G-CSF, GM-CSF, MIP-1β, and MCP-1) were analyzed by Bio-Plex Cytokine Assay. Differences among extensive cGVHD, limited cGVHD and control were evaluated by one-way ANOVA followed by Bonferroni method as a post hoc test.
Results: A significant difference was found only in γδ-T cells, but not in any of the other cells, including CD4, CD8, Th1, Th2, NKT, and DC subsets among extensive cGVHD, limited cGVHD and control. There was a significant decrease in the mean absolute number of γδ-T cells in extensive cGVHD (4.0 /μl, n=9, p&lt;0.001) and limited cGVHD (16.0 /μl, n=5, p=0.01), compared to that in control (56.7 /μl, n=5). This marked reduction of γδ-T cells in cGVHD was found also in the mean percentage of γδ-T cells in CD3 T cells (%-γδ-T) as follows: extensive cGVHD (0.53%, n=9, p&lt;0.0001), limited cGVHD (1.00%, n=5, p&lt;0.001) in contrast with control (4.74%, n=5). On the other hand, dramatical increase in IL-6 was observed in extensive cGVHD (18.4pg/ml, n=5), in contrast with limited cGVHD (2.5pg/ml, n=3, p=0.02) or control (0.8pg/ml, n=5, p=0.004), demonstrating IL-6 involvement in the severity of cGVHD. Moreover, IL-10 as a kind of Th2 cytokines was likely to increase in extensive cGVHD (3.8pg/ml, n=5, p=0.02), compared to that in control (0.2pg/ml, n=5), suggesting Th2 involvement in extensive cGVHD. The other cytokines including IL-4, TNF-α, IFN-γ did not show any significant changes between cGVHD and control. Interestingly, a positive correlation was observed between IL-6 and the reciprocal of the percentage of γδ-T cells in CD3 T cells (1/%-γδ-T) (n=12, r=0.79, p=0.002) with all samples put together, suggesting γδ-T cells as a negative regulator of IL-6.
Discussion: γδ-T cells have been reported to control the innate immune system and participate in anti-tumor or anti-infection immunity in a major histocompatibility complex-unrestricted manner. Our data suggest the possible involvement of the reduction of γδ-T cells in the pathogenesis of cGVHD, and the usefulness of monitoring γδ-T cells in predicting the development of cGVHD after allo-HSCT.