Cladribine

Five days post-immunization, cladribine (10 mg/kg; Merck) was administered daily via oral gavage in 0.5% aqueous carboxymethylcellulose for 5 days (further referred to as ‘cladribine-treated EAE group’). The vehicle group received 0.5% aqueous carboxymethylcellulose for 5 days at the same period (further referred to as ‘vehicle-treated EAE group’). Moreover, an additional control group of naïve mice (without EAE or any oral treatment), further referred to as ‘controls’, was used for ex vivo electrophysiological analysis. Prior to the EAE experiments, titration experiments of cladribine were conducted, indicating 10 mg/kg as the appropriate dose to reach an adequate brain concentration of approximately 80–100 ng/g over a period of 2.5 h (Additional file 2: Fig. S2). This time interval was chosen in advance, as cladribine is rapidly available following oral administration and maximum plasma levels are found within 1 to 2 h of ingestion [8 (link)].

TCR-activated cells were incubated for 48 hours with 10^-7 M cladribine (Sigma Aldrich, cat. no. C4438) or in the absence of cladribine, as control samples. After the incubation period, the cytotoxic effect of cladribine was immediately assessed by absolute CBC using Sysmex XS 800i Hematology Analyzer, followed by a viability assay using the FACSAria III flow cytometer and Becton Dickinson Horizon™ Fixable Viability Stain 780 (FVS 780, BD cat. no. 565388). Necrotic cells were determined by higher levels of FVS 780, with a 10-20-fold increase in intensity over viable cells.
TCR-activated lymphocytes cultured for 48h with or without cladribine were transferred to a cladribine-free medium with rh-IL2 for up to 7 and 14 days. Lymphocytes were analyzed for viability at day 0 (before culture initiation) and at day 7 or 14, as described above.
The changes in absolute lymphocyte number reflected cell proliferation or depletion in response to the cytotoxic action of cladribine. Survival indexes were established as a ratio between absolute viable cell number without or after cladribine exposure, and initial absolute viable cell number. Proliferation indexes were calculated as a ratio of absolute viable cell number before and after 7 days and 14 days of culture with rh-IL2.