Dysregulated Distribution of Naïve and Memory FoxP3 Positive Regulatory T-Cells Associated with Anemia in Myelodysplastic Syndrome (MDS)

Background: An increase in CD4+FoxP3+ regulatory T cells (Tregs) has been associated with disease progression in myelodysplastic syndrome (MDS). Tregs provide peripheral tolerance through naturally occurring negative regulatory mechanism that control self-reactivity and autoimmunity. MDS is a heterogeneous hematopoietic disease linked to several distinct pathophysiologic mechanisms that occurs primarily in older individuals. Through the work of our laboratory and others, the balance of CD4 and CD8 T cells has been shown to be disturbed in MDS patients that respond to immunosuppressive therapy. IST offers hematologic benefit and improved survival in a subpopulation of MDS patients with evidence of a T-cell autodominant immune process. It is critical to clarify the correlation between dysregulated naïve and memory CD4+ T cells and dysfunction of Tregs since it has been reported that the supply and function of naïve Tregs is important for protection against autoimmune diseases mediated by T-cells.

Methods: We analyzed the direct correlation among FoxP3, CD25 and CD127 expression, and that of CD45RA and CD27 to define Tregs with naïve, central memory, and effector memory phenotypes in 45 MDS patients and 17 healthy controls. We developed an 8-color flow cytometry staining approach that included the following phenotypic markers to define the cell populations: antibodies to CD3, CD4, CD25, CD127 (IL-7Rα), CD45RA, CD27, FoxP3, and aqua fluorescent dye for live:dead cell discrimination.

Results: A total of 23 men and 22 women were enrolled from the Bone Marrow Failure Rare Disease Research Network with a diagnosis of MDS with a median age of 69 years old. Based on International Prognostic Scoring System classification, 90% were in a lower risk category (low and intermediate −1 risk). The controls were 17 healthy individuals that donated blood to the Southwest Florida Blood Services (SFBS) and consisted of 8 men and 9 women (median age 52 years old). We found that bright CD25 expression and dim expression of CD127 were most closely associated with FoxP3 expression in healthy controls. The transcription factor FoxP3, which is the most specific phenotypic marker of Tregs, displayed a distinct association with CD25 and CD127 in naïve and memory cells. Therefore, naïve and memory Tregs were defined by FoxP3 expression and the following phenotypes: naïve Tregs (CD45RA+/CD27+/FoxP3+), central memory Tregs (CD45RA−/CD27+/FoxP3+), and effector memory Tregs (CD45RA−/CD27−/FoxP3+). The mean percentage of FoxP3+ Tregs in MDS patients did not differ from healthy controls (5.24% and 4.83%, respectively P=0.94). Younger age, < 61 years of age, has been strongly associated with responsiveness to IST and T-cell-mediated autoimmunity. When the Tregs were examined in patients divided into two groups based on an age of 60 years old, a higher percentage of FoxP3 positive cells was observed in the older age group compared to the younger group (mean, 7.46% and 4.98%, respectively, P=0.12) of MDS patients. Interestingly, the percentage of FoxP3+ Tregs tended to be inversely correlated with the lymphocyte count (P=0.08) and the CD4/CD8 ratio (P=0.1) in patients. When naïve and memory Tregs were compared between cases and controls, these Treg populations demonstrated distinct skewing with effector memory Tregs dramatically higher in some patients. MDS patients were then divided into two groups based on the percentage of effector memory Tregs. Using a cutpoint equal to the mean + 2 S.D of controls, the patients were grouped by the percentage of effector memory (EM) Tregs into two groups: higher EM-Tregs (mean 39.3% ± 12.5%) and lower EM-Tregs (mean 6.39% ± 5.16%). The patient group with higher EM-Tregs was significantly associated with the presence of anemia (P=0.01) compared to the group with lower effector memory Tregs

Conclusions: Memory Tregs have been reported to express higher levels of integrin alpha_E, display differential tissue distribution, and a higher turnover rate compared to naïve Tregs. Memory Tregs differentiate from naïve CD25−FoxP3− non-Tregs in the peripheral blood after antigenic challenge under tolerogenic conditions. Our findings suggest that the conversion of effector memory Tregs may be important during development of anemia in MDS patients with autoimmune-mediated bone marrow failure.