Characterization Of T Follicular Helper Cells In Patients With Low Risk Myelodysplastic Syndromes

A subpopulation of CD4+helper T cells, T-follicular helper cells (TFH), are characterized by the surface expression of CXCR5, ICOS, and PD1, the transcription factor Bcl-6, and produce mainly IL-21, but also IL-17, IL-4, and IFN-γ. They represent the major population that helps B cells to turn into plasma cells and they are implicated in the pathogenesis of different autoimmune diseases. Peripheral CXCR5+CD4+ T helper cells (p-TFH) are the circulating component of TFH. p-TFH cells have been extensively studied in the context of inflammation and autoimmunity. Patients with systemic lupus erythematosus and rheumatoid arthritis have increased p-TFH. Immune dysregulation characterizes low risk myelodysplastic syndromes (MDS). The expression of p-TFH in patients in low risk MDS has not been previously evaluated. To examine the expression of p-TFH in this disease we isolated peripheral blood mononuclear cells (PBMCs) from patients with low risk MDS (n=20, Refractory Anemia,RA; and Refractory Cytopenias with Multilineage Dysplasia, RCMD) and ten healthy, age- matched controls. Written informed consent was obtained from all study subjects. PBMCs were left untreated or activated with PMA and Ionomycin, in the presence of Brefeldin A, for 5 hours. Subsequently, cells were stained with the surface markers CXCR5, CD4, CD45RO, ICOS and intracellular IL-21, and analyzed by flow cytometry.

Patients with MDS show decreased CD4+ICOS+ cells compared to healthy controls (5,34±1,44% vs 8,69±4,08% respectively, p=0.028). The median percentages of CXCR5+CD4+ cells (estimated on CD4+ cells) before stimulation were lower in MDS patients although not statistically significant different from the control subjects. The expansion of the CXCR5+CD4+ population after stimulation was higher in MDS patients compared to healthy individuals (ratio of stimulated to unstimulated CXCR5+CD4+ cells of MDS patients: 1.44±0.47 vs control: 1.23± 0.32, p=0.027). MDS patients at diagnosis showed increased median levels of IL-21 producing CXCR5+CD4+ cells compared to patients previously treated with erythropoietin (EPO), (21,34±10,14% vs 17,55±12,91, respectively). Additionally, patients with RA showed increased CXCR5+CD4+IL-21+ cells compared to the RCMD patients (22,39±11,76% vs 17,84±10,70%, respectively). Collectively, the percentage of IL-21 producing CXCR5+CD4+ cells did not differ significantly between the MDS and control groups (20,57± 10,12% vs 19,86± 11,03%, respectively). The presence of marrow fibrosis did not affect the differences observed and described above. The p-TFH cell subset was identified in every case as a memory component of CD4+ T helper cells, as previously described. Although the number of patients analyzed is limited, our results suggest that low risk MDS patients show a trend of lower CXCR5+CD4+ cells compared to age–matched control subjects. EPO treatment eliminates IL-21 production from CXCR5+CD4+ cells compared to treatment-naïve patients. Further analysis of a larger pool of subjects along with the examination of the specific transcription factor Bcl-6 will reveal the role of p-TFH cells in low risk MDS.