Peripheral Blood Mononuclear Cells (PBMCs) were collected from at least 3-month untreated RRMS patients, stimulated by anti-CD28 and anti-CD3 and incubated with 1*10^-7M of cladribine. After 72h, cladribine washed out and the survived PBMCs were cultured in presence of IL2 for 10 days. Thereafter, CD4+, CD8+, CD14+ and CD19+ subpopulations were separated and subjected to RNA-Seq transcriptional analysis. Differentially expressed genes (DEGs, qvalue<0.05, Fold Change≥1.5) between cells incubated in absence or presence of cladribine were analyzed.     

PBMCs were obtained from 10 female RRMS patients mean age 48.6±4.3 years, Expanded Disability Status Scale (EDSS) 1.7±1.3 and disease duration of 15.1±3.3 years. Incubation with cladribine reduced the number of live PBMCs by 44±5% (p=0.04). After 10 days of culture the total number of surviving cells recovered and the number of CD4+, CD8+, CD19+ and CD14+ cells were comparable in samples incubated in absence or presence of cladribine. In recovered cells the significant transcriptional changes of 52 DEGs were observed in CD4+ T-cells  This transcriptional profile was enriched by genes involved in various Th2 cells activation mechanisms including  movement of Th2 cells (7.0E-06), recruitment of Th2 cells (p=7.2E-05), chemotaxis of Th2 cells (p=9.7E-05) and migration of Th2 cells (p=6.7E-0.4).  Of note, CD8+ T-cells characterized by 19 DEGs, while no transcriptional changes in CD14+ and CD19+ subpopulations were observed.

CD4+ T-cells recovered after incubation with cladribine showing Th2 associated transcriptional profile.