CD4⁺CD25^-CD69⁺ t Cells Is a Novel Subset of Regulatory T Cells Involved In the Prevention of Acute Graft-Versus-Host Disease In Human

Acute graft-versus-host disease (aGVHD) remains the major obstacle to a more favorable therapeutic outcome of allogeneic hematopoietic stem cell transplantation (HSCT). Regulatory T cells (Tregs) play an important role in aGVHD prevention and a variety of Treg populations with distinct suppressive mechanism have been described in mice or human so far. In this study, we sought to identify Tregs associated with aGVHD in unmanipulated allogeneic HSCT in human by examining the frequency of immunophenotypes of three subsets of Tregs in periphery blood including CD4⁺CD25⁺Foxp3⁺, CD4⁺CD25⁺CD62L⁺ and CD4⁺CD25^-CD69⁺ Tregs in three clinical patterns: First, we monitored 17 aGVHD patients along the course of disease and all of them got complete response (CR) to aGVHD finally. All samples were divided into three groups according to clinical status of aGVHD: before aGVHD-specefic treatment; partial response (PR); and CR, only CD4⁺CD25^-CD69⁺ subset of Tregs showed significant changes, and the frequency decreases with the onset of aGVHD and increases after aGVHD recovery in peripheral blood, (Fig.1) furthermore, decreased frequency is associated with severity of aGVHD. Compared with healthy donor (HD), frequency of CD4⁺CD25^-CD69⁺ Treg increased significantly even at the onset of aGVHD (Fig. 1), while frequency of CD4⁺CD25⁺Foxp3⁺ Treg decreased significantly after HSCT. Second, we compared the frequency of Tregs at +30day-post-HSCT between 16 post-HSCT patients who developed aGVHD and 31 patients who did not develop, only CD4⁺CD25^-CD69⁺Treg had significantly lower percentages in patients with aGVHD (p=0.042), the other two subsets showed no significant difference; Third, we detected the association between Tregs in allografts and the incidence of aGVHD in 50 post-HSCT patients in an unbiased fashion, there were no differences between the aGVHD-group and No-aGVHD-group in the aspect of patients’ age and sex, disease status, stem cell and donor source, while there were significant difference between the two in the aspect of donors’ age, conditoning regimen and patient/donor HLA compatibility. We compared both the frequency and absolute number of the allograft components of patients with aGVHD and those without aGVHD, and absolute number of CD3⁺ T cell and CD4⁺CD25^-CD69⁺Treg were statistically different, however, only CD4⁺CD25^-CD69⁺Treg showed significant difference in multivariate Cox proportional hazards models including patients’ factors with significant difference and other factors closely associated with aGVHD. Then, we chose the median of frequency and absolute number of CD4⁺CD25^-CD69⁺Treg as the borderline (1.08% and 1.34*10⁶/kg, respectively) to identify the association between this subset in allografts and aGVHD. Both higher frequency and higher absolute number of CD4⁺CD25^-CD69⁺ Tregs in allografts correlate with lower incidence of aGVHD by Kaplan-Meier, and log-rank test (Fig. 2). Moreover, our results show that CD4⁺CD25^-CD69⁺Tregs expand significantly early after HSCT, the frequency at +30day reach the summit and then decrease gradually, and a low percentage of CD4⁺CD25^-CD69⁺Tregs at +30day-post-HSCT is associated with aGVHD. The reconstitution of this Tregs early after HSCT is strongly associated with its component in allograft by spearman correlation analysis (r=0.449, p=0.003). Taken together, our findings suggest that CD4⁺CD25^-CD69⁺T cell is a novel subset of regulatory T cell providing protection against aGVHD.