P711: the prevalence of foxp3-e2 isoform in regulatory t cells correlates with the degree of dysplasia in myelodysplastic syndromes

Background: An altered immune-tolerance control, mediated Regulatory T cells (Tregs), has been largely described in MDS. Indeed, in early MDS stage, reduced Tregs levels might favor pro-inflammatory and autoimmune mechanisms, likely able to damage polyclonal hematopoiesis, while in advanced stages, increased Treg-dependent suppression of immune effectors has been observed to foster leukemia progression of the dysplastic clone/s. The expression of the master regulator gene FoxP3 has been largely recognized to be responsible for the immune modulating properties of the Tregs. Alternative splicing mechanisms generate two main isoforms of Foxp3, one containing the exon 2 (Foxp3-E2) and another, shorter, lacking the exon 2 (Foxp3-Δ2). The expression of Foxp3-E2 has been recently demonstrated to be essential for Tregs suppressive activity; indeed, Foxp3-Δ2 has been associated with the generation of unstable Tregs and with an autoimmune phenotype in a murine model. Aims: To investigate whether the expression of Foxp3-E2 versus Foxp3-Δ2 isoform by the Tregs, resident in the bone marrow (BM), might improve the evaluation of immune tolerance control in MDS. The possibility that such analysis might add a new valuable prognostic biomarker to the evaluation of MDS patients has been also considered. Methods: 27 newly diagnosed MDS patients, according to WHO 2022 and 8 healthy donors were recruited in the study, approved by the Local Ethical committee. BM samples obtained from consenting healthy donors were part of their marrow donation. Patients were categorized according to IPSS-M. Immune profile, Foxp3-E2, overall Foxp3 and ki67 expression by Treg have been evaluated by immune fluorescence and multi-parametric flow cytometry. Mann-Whitney test and Spearman correlation test have been used for statistical analysis. Results: According to IPSS-M, 21 patients were low risk (9 very low, 10 low, 2 moderate low) and 6 were high risk (4 high, 1 moderate high, 1 very high). In high risk MDS patients, Foxp3-E2 expression was significantly increased, as compared to low risk and controls. In addition, in high risk, both Foxp3-E2 and overall Foxp3 CD4+ T cells showed an increased proliferative capability, as assessed by ki67 expression, compared to controls and low risk patients. Foxp3-E2/overall Foxp3 ratio, was significantly decreased in all MDS patients, both low risk and high risk patients, as compared to controls. A significative direct correlation between the BM percentage of CD16-CD11b- dysplastic neutrophils and Foxp3-E2/overall Foxp3 ratio has been identified in the MDS cohort (Figure 1, A). Moreover, the proliferation level of Treg, as evaluated by their ki67 expression, has been observed to directly correlate with the percentage of BM blasts (Figure 1, B). In the end, in low risk, Foxp3-E2/overall Foxp3 ratio has been observed to inversely correlate with the activation status of CD8 T cells, as measured trough CD54 expression. Summary/Conclusion: Here we show that MDS high risk subjects are characterized by an increased number of Tregs expressing the Foxp3-E2 isoform, thus with a highly suppressive phenotype. Moreover, the degree of BM dysplasia directly correlate with the prevalence of actively suppressive Tregs and their proliferative status. Understanding how the immune system contributes to MDS physiopathology opens future possibility to identify new therapeutic targets, ameliorating MDS clinical management.Keywords: T regulatory cells, Tumor immunity, Myelodysplastic syndrome