Expansion of Polyclonal CD4⁺CD25^high Foxp3⁺ Regulatory T-Cells in High Risk Myelodysplastic Syndromes (MDS).

Foxp3⁺ regulatory T-cells(Tregs) play a central role in maintaining tolerance. A reduction in Tregs activity is a key feature of autoimmune diseases, whereas their expansion in malignant diseases leads to suppression of the host anti-tumour responses. Expansion of oligoclonal cytotoxic T cells, inhibition of MDS CD34⁺ progenitors proliferation and response to immunosuppressive therapy suggest that autoimmunity contributes to the pathophysiology of low risk MDS. However, this is not generally a feature of high risk MDS. We hypothesise that in high risk MDS patients an increased number of Tregs may suppress immune responses against the dysplastic clone. In early MDS, these cells may be reduced and or be associated with coexistent autoimmune diseases, an uncommon but recognised association of low risk MDS. We therefore studied CD4⁺ CD25^high Foxp3⁺ and CD8⁺ CD25⁺Foxp3⁺ Tregs in peripheral blood of MDS patients and examined correlation with bone marrow blasts, cytogenetic Status, IPSS score and progression to AML. Clonality of CD4⁺CD25⁺ Tregs was assessed by TCR spectratype analysis of CDR3 size distribution and by CDR3 sequence analysis.

52 patients with MDS (30 male, 22 female) with a mean age of 62 years (range 40 to 82 years) were studied. According to WHO classification, 5/52 (10%) had a diagnosis of 5q⁻ syndrome, 9/52(17%) refractory anaemia (RA), 18/52 (35%) refractory cytopenia with multilineage dysplasia (RCMD), 16/52 (31%) refractory anaemia with excess blasts (RAEB) and 4/52 (7%) patients MDS/MPD (one with JAK2 V617F Mutation). Cytogenetic study shows normal pattern in 57%, 5q- in 22%, stable cytogenetic in 10% and complex findings in 11%. All samples were taken at diagnosis prior to any treatment. In addition 5 samples were analyzed pre and post 5-Azacytidine therapy. The absolute number of CD4⁺CD25^highFoxp3⁺ regulatory T cells in 5q- syndrome was 0.5±0.28×10⁷/l, RA0.6±0.56×10⁷/l, RCMD1.42±0.97×10⁷/l, RAEB2.8±2.2×10⁷/l and MDS/MPD 2.9±2×10⁷/l. In cases with <5% bone marrow blasts (RA, 5q⁻ & RCMD)absolute number and percentage of Tregs was significantly lower than those with ≥5% BM blasts (p=0.001). The mean number was also significantly lower at 0.73±0.57×10⁷/l in low risk cases(IPSS 0) compared with 2±1.5×10⁷/l in intermediate and high risk groups (p=0.008). CD8⁺ Tregs were not significantly different between the subtypes of MDS and between low and high risk IPSS subgroups. Tregs number did not differ significantly between various cytogenetic subgroups. The spectratype of CD4⁺CD25⁺ TCR amplicons, showed a polyclonal pattern and the overall complexity of Vβ subfamilies was not different between low risk and high risk MDS, suggesting that the expanded Tregs in high risk MDS are not clonal and likely to arise by peripheral expansion rather than an antigen-driven response. CD4⁺ CD25^high Foxp3⁺ Tregs in five patients were studied pre and post 5-Azacytidine. The numbers were significantly decreased after treatment in the one patient who responded to treatment (p=0.001), whereas Treg numbers were unchanged or increased in non responsive cases. In 5 RCMD cases with concomitant autoimmune diseases the percentage of Tregs was lower than other patients within the same subgroup of MDS, however, this did not achieve statistical significance.

The findings indicate Tregs are altered in MDS and may be important in the pathophysiology of MDS. Monitoring of Tregs numbers can be a useful indicator for disease progression and response to immunosuppressive therapy.