Alterations of Thymus-Derived Tregs in Multiple Sclerosis

Abstract

Background and objectives: Multiple sclerosis (MS) is considered a prototypic autoimmune disease of the CNS. It is the leading cause of chronic neurologic disability in young adults. Proinflammatory B cells and autoreactive T cells both play important roles in its pathogenesis. We aimed to study alterations of regulatory T cells (Tregs), which likely also contribute to the disease, but their involvement is less clear.

Methods: By combining multiple experimental approaches, we examined the Treg compartments in 41 patients with relapsing-remitting MS and 17 healthy donors.

Results: Patients with MS showed a reduced frequency of CD4⁺ T cells and Foxp3+ Tregs and age-dependent alterations of Treg subsets. Treg suppressive function was compromised in patients, who were treated with natalizumab, while it was unaffected in untreated and anti-CD20-treated patients. The changes in natalizumab-treated patients included increased proinflammatory cytokines and an altered transcriptome in thymus-derived (t)-Tregs, but not in peripheral (p)-Tregs.

Discussion: Treg dysfunction in patients with MS might be related to an altered transcriptome of t-Tregs and a proinflammatory environment. Our findings contribute to a better understanding of Tregs and their subtypes in MS.

Figure 1. Treg Phenotype in Patients With Relapsing-Remitting Multiple Sclerosis and Healthy Donors

(A) CD25⁺CD127lo cells were gated on CD3⁺CD4⁺ cells. The expression of Foxp3, CD39, CD49d, and CD103 was evaluated on this subset as percentage of positive cells or mean fluorescence intensity (MFI) (only Foxp3 expression is shown). (B–K) Patients with MS (MS) were compared with HDs (HD) and included untreated (green), natalizumab-treated (red), and anti-CD20–treated (blue) patients. (B–C) Frequency of CD3⁺CD4⁺ cells and CD25⁺CD127lo cells. (D) Frequency of Foxp3+ cells in CD25⁺CD127lo cells. (E) The expression of CD127 was analyzed on CD25+Foxp3+ cells as MFI. (F) Percentage of CD25⁺CD127lo cells expressing CD39. (G–H) MFI of CD103 and CD49d on CD25⁺CD127lo cells. (I–K) Multiple comparison between untreated (UNT), natalizumab-treated (NAT), anti-CD20-treated patients (a-CD20), and HDs (HD). Frequency of Foxp3+ cells and MFI of CD127 and CD49d are shown, respectively. (B–K) Dots represent frequency of each donor, boxes extend from the 25th to 75th percentiles, and whiskers from min to max. The line in the middle of the box is the median. Unpaired t test or Mann-Whitney test was used to compare 2 sets of data. One-way ANOVA or Kruskal-Wallis test followed by the Tukey and Dunn statistical hypothesis testing, respectively, were adopted to compare more than 2 sets of data. *p < 0.05; **p < 0.01.

Figure 2. Transcriptome Analysis of Thymic Tregs and Peripheral Tregs in 9 Patients With Multiple Sclerosis and 3 Healthy Donors

(A) Exemplary dot plots and histogram of Foxp3, CD45RA, and GPA33 expression in Tregs isolated from a HD showing the gating strategy designed to define 2 subsets: p-Tregs (CD45RA-GPA33-) and t-Tregs (CD45RA+GPA33+). A minor cell subset was CD45RA-GPA33+. The first 2 subsets (red squares) were analyzed by RNA sequencing. The third subset was speculated to contain activated (Act.) t-Tregs but was not further investigated by transcriptome analysis. (B) Heatmap showing the expression counts of the top 50 differentially expressed genes across t-Tregs and p-Tregs. Both positive and negative log fold changes are displayed. (C) Genes belonging to different Gene Ontology (GO) Biological Process (BP) terms were upregulated in p-Tregs and t-Tregs, respectively. (D) Volcano plot showing genes involved in Treg homeostasis, homing, activation, and function differentially expressed between t-Tregs and p-Tregs (log2 fold change threshold >1.5; false discovery rate <0.05).

Figure 3. Balance Between t-Tregs, p-Tregs, and CD45RA-GPA33+ t-Tregs in Patients With Relapsing-Remitting Multiple Sclerosis

(A–H) Patients with MS (MS) were compared with HDs (HD). Patients included untreated (green), natalizumab-treated (red), and anti-CD20–treated (blue) patients. (A) Age-dependent course of t-Tregs, p-Tregs, and CD45RA-GPA33+ t-Tregs in patients with MS (dark gray line) and HDs (black line) (XY correlation). (B–C) Frequency of t-Tregs in younger and older patients, respectively. (D–E) Frequency of CD45RA-GPA33+ t-Tregs in younger and older patients, respectively. (F–G) Frequency of CD45RA-GPA33+ t-Tregs in younger and older patients, respectively. (B–G) Dots represent the frequency of each donor, boxes extend from the 25th to 75th percentiles, and whiskers from min to max. The line in the middle of the box is the median. Unpaired t test or Mann-Whitney U test. *p < 0.05; **p < 0.01. (H) Multiple comparison of the frequency of CD45RA-GPA33+ t-Tregs between untreated (UNT), natalizumab-treated (NAT), anti-CD20–treated patients (a-CD20), and HDs (HD). (I) Multiple comparison of the median MFI of Foxp3 in t-Tregs, p-Tregs, and CD45RA-GPA33+ t-Tregs (Act. t-Tregs) between patients and HDs. UNT: untreated. NAT: natalizumab-treated. a-CD20: anti-CD20–treated. (H–I) Scatterplot with bar graph. One-way ANOVA or Kruskal-Wallis test. *p < 0.05; **p < 0.01.

Figure 4. Treg Function in Multiple Sclerosis

(A) Schematic representation of suppression assay: polyclonally stimulated Teffs were cocultured with different ratio of Tregs, and the proliferation was measured at day 5 (created with Biorender.com). The mean Treg inhibitory capacity calculated at 5 Treg:Teff ratios is shown. (B) Concentrations of cytokines were detected in the supernatants harvested from the suppression assay at ratio Treg:Teff 1:1 by a bead-based immunoassay. (C) XY correlation between Treg suppressive capacity and frequency of t-Tregs within CD25⁺CD127lo subset. (D) XY correlation between Treg suppressive function and IFN-γ concentration in the supernatants. (E) XY correlation between Treg function and frequency of FOXP3+ cells within CD25⁺CD127lo fraction. (F) XY correlation between frequency of t-Tregs and CD39⁺ cells within CD25⁺CD127lo subset. (A–B) Scatterplot with bar graph. Two-way ANOVA test. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. (A–F) UNT: untreated (green). NAT: natalizumab-treated (red). a-CD20: anti-CD20–treated (blue). HD: healthy donor (yellow).

Figure 5. Transcriptomic Analysis of t-Tregs Derived From Natalizumab-Treated Patients and Healthy Donors

(A) Comparison of average gene expression showing significantly differentially expressed genes between t-Tregs derived from natalizumab-treated patients and HDs. Present (black) indicates genes with read counts ≥10 in more than one sample, absent (gray) indicates all genes that are not flagged as present, and significant (blue) indicates genes with log2 ratio >0.5 and a p value <0.01. Log2 ratio indicates the fold-change in gene expression. (B) Heatmap showing the expression counts of the top 50 differentially expressed genes across t-Tregs derived from natalizumab-treated patients and HDs. Both positive and negative log fold changes are displayed. (C) Genes belonging to different Gene Ontology (GO) Biological Process (BP) terms were upregulated in t-Tregs derived from natalizumab-treated patients and HDs, respectively. (D) Volcano plot showing genes involved in T-cell activation, proliferation, metabolism, and RNA processing differentially expressed between t-Tregs derived from natalizumab-treated patients and HDs (log2 fold change threshold >1.5; false discovery rate <0.05).