Lymphocyte Dynamics during the Treatment of SR-aGVHD with Mesenchymal Stem Cells Derived from Human Umbilical Cord Blood - a Phase III, Multi-Center, Prospective, Single-Arm Pivotal Study

Background: Acute graft versus host disease (aGVHD) is one of the most common and severe complications after allo-HSCT treatment. Systemic corticosteroid has been considered as the standard initial treatment for aGVHD. However, steroid refractoriness occurs in more than 40% of patients and is associated with dismal prognosis. Especially for severe (grade III-IV) aGVHD with failed corticosteroid treatment, the prognosis remains extremely poor, with a 5-year survival rate of only 5%. In the treatment of aGVHD, mesenchymal stem cells (MSCs) have garnered significant attention due to their immunomodulatory and tissue repair capabilities. Our center led a phase III, multicenter, prospective, single-arm, pivotal study to verify the efficacy and safety of MSC therapy for SR-aGVHD. This study explored the changes in lymphocytes during the treatment of SR-aGVHD with MSCs.

Methods: 1. Peripheral blood samples were collected from patients before mesenchymal stem cells (MSCs) infusion and on days 15, 28, and 56 post-infusion to detect lymphocyte subsets. The extent of immune reconstitution varies among different patients. Mathematical transformations were applied to the data at the four time points, focusing on the changes at days 15, 28, and 56 post-MSCs infusion compared to pre-infusion, to evaluate the changes brought by MSCs infusion. 2. Twenty-seven patients with SR-aGVHD who received conventional second-line therapy without MSCs infusion were used as a historical control group to compare the differences in lymphocyte subset changes between the two groups.

Results:

Part I: MSCs Treatment Group vs. Historical Control Group

T lymphocytes (CD3⁺) showed an increasing trend in both the MSCs treatment group and the historical control group, but the overall increase was less pronounced in the treatment group compared to the control group, with no statistical difference (D15 P=0.565; D28 P=0.905). The CD4⁺ lymphocyte ratio in the MSCs treatment group reversed the rapid decrease observed in the historical control group (D15, P=0.016; D28, P=0.041). Compared to the control group, the increase in CD8⁺ lymphocytes was smaller in the MSC treatment group (D28, P=0.055). Unlike the control group, which showed a continuous increase, B lymphocytes in the MSC treatment group increased initially and then decreased, with a statistically significant difference between the two groups at D28 (P=0.025).NK cells: Both the MSC treatment group and the historical control group showed a decreasing trend, but the decrease was more moderate in the MSCs treatment group (D15 P=0.035, D28 P=0.005). Compared to the control group, Treg cells reversed the trend of continuous decline after MSC infusion, with a statistically significant difference between the two groups at D28 (P=0.009).

Part II: Effective Treatment Group vs. Treatment Failure Group

T lymphocytes (CD3⁺) showed an increase in the effective treatment group and a decrease in the failure group, with a statistically significant difference in the change at D28 between the two groups (P=0.042). CD8⁺ lymphocytes demonstrated a continuous decrease in the treatment failure group and an opposite trend in the effective group (D28 P=0.003). NK cells decreased in both the effective and failure groups early after infusion, but the failure group reversed this trend at D28 compared to the effective group (P=0.048).Th1 cells exhibited distinct trends in the effective and failure groups, with an initial increase followed by a decrease in the effective group and an initial decrease followed by an increase in the failure group (D15 P=0.052, D28 P=0.096). Th2 cells in the effective treatment group experienced an initial decrease followed by an increase, while Th2 cells in the treatment failure group showed an initial increase followed by a decrease (D15 P=0.001, D28 P=0.325).

Conclusion:

1.Human umbilical cord blood-derived MSCs therapy for SR-GVHD demonstrates encouraging efficacy. Additionally, various lymphocyte subpopulations exhibit changes post-infusion compared to the control group, providing direct evidence of the immunomodulatory effects of mesenchymal stem cells.2.Differences in cellular trends are observed between the effective and failure groups after mesenchymal stem cell infusion, indicating a need for further mechanistic studies to enhance the therapeutic outcomes of MSCs therapy.