OBJECTIVES: To assess potential roles of effector cells and immunologic markers in demyelinating CNS lesion formation, and their modulation by interferon β-1a (IFN-β-1a). METHODS: Twenty-three patients with relapsing-remitting multiple sclerosis (RRMS) received IFN-β-1a for 6 months. Immunologic marker results were correlated with brain MRI lesion volumes, and volumes of normal-appearing brain tissue (NABT) with decreasing or increasing voxel-wise magnetization transfer ratio (VW-MTR), suggestive of demyelination and remyelination, respectively. RESULTS: Baseline expression of Th22 cell transcription factor aryl hydrocarbon receptor (AHR) and interleukin (IL)-17F, and percentages of IL-22-expressing CD4(+) and CD8(+) cells, were significantly higher in patients vs 15 healthy controls; IL-4 in CD4(+) cells was lower. Baseline percentage of IL-22-producing CD8(+) cells positively correlated with T2 lesion volumes, while percentage of IL-17A-producing CD8(+) cells positively correlated with T2 and T1 lesion volumes. IFN-β-1a induced reductions in transcription factor AHR, T-bet, and retinoic acid-related orphan nuclear hormone receptor C (RORc) gene expression, while it increased GATA3's expression in CD4(+) cells. Percentages of IL-22-, IL-17A-, and IL-17F-expressing T cells significantly decreased following treatment. Increased percentages of IL-10-expressing CD4(+) and CD8(+) cells correlated with greater NABT volume with increasing VW-MTR, while decreased percentage of IL-17F-expressing CD4(+) cells positively correlated with decreased NABT volume with decreasing VW-MTR. CONCLUSIONS: Findings indicate that IFN-β-1a suppresses Th22 and Th17 cell responses, which were associated with decreased MRI-detectable demyelination. CLASSIFICATION OF EVIDENCE: This pilot study provides Class III evidence that reduced Th22 and Th17 responses are associated with decreased demyelination following IFN-β-1a treatment in patients with RRMS.