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. 2020 Apr 16;18(1):169.
doi: 10.1186/s12967-020-02329-5.

Simultaneous quantification of natural and inducible regulatory T-cell subsets during interferon-β therapy of multiple sclerosis patients

Marco Chiarini  1   2 , Ruggero Capra  3 , Federico Serana  1   2 , Diego Bertoli  4   2 , Alessandra Sottini  2 , Viviana Giustini  2 , Cristina Scarpazza  3   5 , Marco Rovaris  6 , Valentina Torri Clerici  7 , Diana Ferraro  8 , Simonetta Galgani  9 , Claudio Solaro  10 , Marta Zaffira Conti  11 , Andrea Visconti  12 , Luisa Imberti  13 ; SURROGATE Study Group
Affiliations

Simultaneous quantification of natural and inducible regulatory T-cell subsets during interferon-β therapy of multiple sclerosis patients

Marco Chiarini et al. J Transl Med. .

Abstract

Background: The mechanisms underlying the therapeutic activity of interferon-β in multiple sclerosis are still not completely understood. In the present study, we evaluated the short and long-term effects of interferon-β treatment on different subsets of regulatory T cells in relapsing-remitting multiple sclerosis patients biologically responsive to treatment because of mixovirus resistance protein A inducibility.

Methods: In this prospective longitudinal study, subsets of natural regulatory T cells (naïve, central memory and effector memory) and inducible regulatory T cells (Tr1), as well as in vitro-induced regulatory T cells (Tr1-like cells), were simultaneously quantified by flow cytometry in samples prepared from 148 therapy-naïve multiple sclerosis patients obtained before and after 6, 12, 18, and 24 months of interferon-β-1a treatment. mRNA for interleukin-10 and Tr1-related genes (CD18, CD49b, and CD46, together with Cyt-1 and Cyt-2 CD46-associated isoforms) were quantified in Tr1-like cells.

Results: Despite profound inter-individual variations in the modulation of all regulatory T-cell subsets, the percentage of natural regulatory T cells increased after 6, 12, and 24 months of interferon-β treatment. This increase was characterized by the expansion of central and effector memory regulatory T-cell subsets. The percentage of Tr1 significantly enhanced at 12 months of therapy and continued to be high at the subsequent evaluation points. Patients experiencing relapses displayed a higher percentage of naïve regulatory T cells and a lower percentage of central memory regulatory T cells and of Tr1 before starting interferon-β therapy. In addition, an increase over time of central memory and of Tr1 was observed only in patients with stable disease. However, in vitro-induced Tr1-like cells, prepared from patients treated for 24 months, produced less amount of interleukin-10 mRNA compared with pre-treatment Tr1-like cells.

Conclusion: Interferon-β induces the expansion of T regulatory subsets endowed with a high suppressive activity, especially in clinically stable patients. The overall concurrent modulation of natural and inducible regulatory T-cell subsets might explain the therapeutic effects of interferon-β in multiple sclerosis patients.

Keywords: Interferon-β; Multiple sclerosis; Regulatory T cells; Treg subsets.

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Conflict of interest statement

D Bertoli, M.Z. Conti, S. Galgani, V. Giustini, C. Scarpazza, F. Serana, C. Solaro, and A. Sottini have no competing interests. M. Chiarini has received a fellowship from Merck Serono. R. Capra has received lecture fees and/or travel grants from Biogen, Celgene, Genzyme-Sanofi, Novartis, and Teva. D. Ferraro has received travel grants and/or speaker honoraria from Biogen, Genzyme-Sanofi, Merck Serono, Novartis, and Teva and has participated to scientific Advisory Board for Biogen, Novartis, and Roche. L. Imberti has received speaker honoraria from Biogen, Genzyme-Sanofi, Merck Serono, and Novartis and has participated to scientific Advisory Board for Biogen. She has received research funding from FISM (Fondazione Italiana Sclerosi Multipla) and research support from Genzyme-Sanofi and Merck Serono. V. Torri Clerici has participated to scientific Advisory Board for Biogen, Merck Serono, Novartis, and Roche and has received funding for traveling and honoraria for speaking or writing from Almirall, Genzyme-Sanofi, Novartis, Roche, and Teva. She received support for research project by Almirall. A. Visconti is employed of Merck Serono S.p.A., an affiliate of Merck KGaA, Darmstadt, Germany.

Figures

Fig. 1
Fig. 1
Flow cytometry quantification of nTreg and Tr1 in IFN-β-treated patients. Modulation of nTreg (a), naïve Treg (b), TregCM (c), TregEM (d), CD120b+ nTreg (e), and Tr1 (f) after IFN-β treatment was investigated at T0: before therapy initiation and T6, T12, T18, and T24: after 6, 12, 18, and 24 months of IFN-β, respectively. Estimated means are shown, connected by black continuous line, along with error bars representing the 95% confidence interval. Other lines indicate statistical significance (always p < 0.05): long dash lines designate differences between consecutive time points and the pre-therapy point; dotted lines designate differences between previous time points among patients
Fig. 2
Fig. 2
Tr1-like cell induction after in vitro anti-CD3/CD46 MoAb stimulation. Mean percentage of in vitro induced Tr1-like cells (a) and MFI mean of CD18 (b) and CD49b (c) markers analyzed by flow cytometry in CD4+ cells in IFN-β-treated patients obtained before therapy initiation (T0) and then after 12 (T12) and 24 (T24) months of therapy, and in healthy controls (Ctrl). Cells were stimulated with anti-CD3 and anti-CD46 MoAbs. mRNA levels of CD46 (d) in CD4+ cells pre- and post- in vitro stimulation. Results are calculated as normalization ratio (NR), relative to a common standard sample. Estimated means are shown, connected by continuous lines, along with error bars representing the 95% confidence interval. Other lines indicate statistical significance
Fig. 3
Fig. 3
Cyt-1, Cyt-2, and IL-10 mRNA quantification after in vitro anti-CD3/CD46 MoAb stimulation. mRNA levels of Cyt-1 (a), Cyt-2 (b), Cyt-2 and Cyt-1 ratio (c), and IL-10 mRNA levels (d) in CD4+ cells pre- and post- in vitro stimulation. CD4+ cells obtained from IFN-β-treated patients before therapy initiation (T0) and then after 12 (T12) and 24 (T24) months of therapy and healthy controls (Ctrl) and stimulated with anti-CD3 and anti-CD46 MoAbs. Results are calculated as normalization ratio (NR), relative to a common standard sample. Estimated means are shown, connected by continuous lines, along with error bars representing the 95% confidence interval. Other lines indicate statistical significance
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