Multiple sclerosis (MS) represents the leading cause of neurological deficit among young adults, affecting women more frequently than men. In MS, the extent of central nervous system lesions is determined by the net balance between self-reactive and regulatory T-cells at any given time, among other factors, as well as by the effect of inflammatory response. Here, we studied both CD4+ and CD8+ T-Reg in parallel in blood and CSF during MS relapse. A recruitment of both regulatory CD4+ and CD8+ T cells (T-Reg) within the cerebrospinal fluid (CSF) takes place during MS relapse. Not previously described, the presence of CD4+ T-Reg in CSF was higher in women than in men, which could account for the sexual dimorphism in the incidence of MS. A direct correlation between plasma oestradiol (E2) and IL-2 levels was observed, in line with a putative circuit of E2 and perforin expression by CD4+ T-Reg playing a role in MS. Also, serum IFN-alpha was higher in females, with direct correlation with serum E2 levels. This is the first study to analyze perforin expression by CD4+ T-Reg in MS, which was greatly enhanced in CSF, what points out a relevant role of this molecule in the suppressive effects of the CD4+ T-Reg in MS, and contributes to the understanding of MS pathophysiology.