Reduced Expression of PD-1 in Circulating CD4+ and CD8+ Tregs Is an Early Feature of RRMS

Abstract

Altered regulatory T cell (Treg) function could contribute to MS. The expression of activating and inhibitory receptors influences the activity of Tregs. Our aim was to investigate T cell phenotypes in relapsing-remitting MS (RRMS) patients at an early phase of the disease. We examined the influence of demographic parameters on the distribution of CD4+ and CD8+ T cell subclasses by generalized linear modeling. We also studied the expression of the following markers-CTLA-4, GITR, PD-1, FoxP3, Helios, CD28, CD62L, CD103-on T cell subsets from peripheral blood with a 14-color flow cytometry panel. We used an antibody array to define the profiles of 34 Th1/Th2/Th17 cytokines in the serum. Expression of PD-1 and GITR on CD4+ and CD8+ Tregs was decreased in RRMS patients. The proinflammatory factors IFN-γ, IL-17, IL-17F, TGFβ-1, TGFβ-3, IL-1SRII, IL-12 p40, sgp130, IL-6sR were significantly increased in RRMS patients. Therefore, a deficiency of PD-1 and GITR immune checkpoints on CD4+ and CD8+ Tregs is a feature of RRMS and might underlie impaired T cell control.

Keywords: T cells; cytokines; relapsing–remitting multiple sclerosis; suppressive markers.

Figure 1

Gating strategy for CD4+ and CD8+ Treg populations. Gating strategy for flow cytometric analysis of lymphocyte population to identify CD4+CD25+FoxP3+, CD8+CD25+FoxP3+ and CD8+CD122+Helios+ Tregs. A time gate was initially applied to exclude any electronic noise and artifacts (not shown here). Next, based on size and granularity, lymphocytes were gated in a forward scatter area (FSC-A) versus side scatter area (SSC-A) plot (A). Then, doublet cells were excluded using FSC-A/FSC-height (FSC-H), FSC-A/FSC-Width (FSC-W) and SSC-A/SSC-height (SSC-H) parameters (B). Next, viable lymphocytes were gated and cells that expressed monocyte, NK cell and B cell lineage markers CD14, CD16 and CD19, respectively were excluded (“dump channel”, dump negative gate) (C), followed by expression of CD3 (D). CD8+CD122+Helios+ Tregs were identified as CD8+CD122+ cells (E) with FoxP3^+/− and Helios co-expression (F). CD8+CD25+FoxP3+ Tregs were identified by CD8 and CD25 (G) and Foxp3 (H) expression. CD4+CD25+FoxP3+ Tregs were identified as CD4⁺CD25⁺FoxP3⁺ cells (I,J). Representative plots are presented.

Figure 2

Frequency of CD4+ and CD8+ Treg populations. Comparison of the frequencies of CD4+CD25+FoxP3+ (among CD4+CD25+), CD8+CD25+FoxP3+ (among CD8+CD25+) and CD8+CD122+Helios+ (among CD8+CD122+) Treg populations in healthy controls (HC) and RRMS patients. * p ≤ 0.05.

Figure 3

Expression of suppressive markers on CD4+ and CD8+ Treg populations. Comparison of the percentage and expression (MFI) of CTLA-4+ (A,B), GITR+ (C,D) and PD-1+ (E,F) cells within CD4+CD25+FoxP3+, CD8+CD25+FoxP3+ and CD8+CD122+Helios+ Treg populations in healthy controls (HC) and RRMS patients. * p ≤ 0.05.

Figure 4

Identification of serum Th1/Th2/Th17 cytokines by antibody arrays. The serum cytokines differentially expressed between patients at pre-treatment (RRMS) and healthy controls (HC). Colored boxes indicate the locations of ten significantly different proteins on the arrays, and different colored boxes represent respective cytokines. Only those cytokines whose expression level (Relative Signal Intensity) differed in RRMS patients compared to the control are shown. Horizontal lines indicate significantly different groups ^a Mann–Whitney U-test or ^b t-Student test. RRMS n = 5, HC n = 5.