Contact-Dependent Granzyme B-Mediated Cytotoxicity of Th17-Polarized Cells Toward Human Oligodendrocytes

Abstract

Multiple sclerosis (MS) is characterized by the loss of myelin and of myelin-producing oligodendrocytes (OLs) in the central nervous system (CNS). Pro-inflammatory CD4⁺ Th17 cells are considered pathogenic in MS and are harmful to OLs. We investigated the mechanisms driving human CD4⁺ T cell-mediated OL cell death. Using fluorescent and brightfield in vitro live imaging, we found that compared to Th2-polarized cells, Th17-polarized cells show greater interactions with primary human OLs and human oligodendrocytic cell line MO3.13, displaying longer duration of contact, lower mean speed, and higher rate of vesicle-like structure formation at the sites of contact. Using single-cell RNA sequencing, we assessed the transcriptomic profile of primary human OLs and Th17-polarized cells in direct contact or separated by an insert. We showed that upon close interaction, OLs upregulate the expression of mRNA coding for chemokines and antioxidant/anti-apoptotic molecules, while Th17-polarized cells upregulate the expression of mRNA coding for chemokines and pro-inflammatory cytokines such as IL-17A, IFN-γ, and granzyme B. We found that secretion of CCL3, CXCL10, IFN-γ, TNFα, and granzyme B is induced upon direct contact in cocultures of human Th17-polarized cells with human OLs. In addition, we validated by flow cytometry and immunofluorescence that granzyme B levels are upregulated in Th17-polarized compared to Th2-polarized cells and are even higher in Th17-polarized cells upon direct contact with OLs or MO3.13 cells compared to Th17-polarized cells separated from OLs by an insert. Moreover, granzyme B is detected in OLs and MO3.13 cells following direct contact with Th17-polarized cells, suggesting the release of granzyme B from Th17-polarized cells into OLs/MO3.13 cells. To confirm granzyme B-mediated cytotoxicity toward OLs, we showed that recombinant human granzyme B can induce OLs and MO3.13 cell death. Furthermore, pretreatment of Th17-polarized cells with a reversible granzyme B blocker (Ac-IEPD-CHO) or a natural granzyme B blocker (serpina3N) improved survival of MO3.13 cells upon coculture with Th17 cells. In conclusion, we showed that human Th17-polarized cells form biologically significant contacts with human OLs and exert direct toxicity by releasing granzyme B.

Keywords: CD4 T lymphocytes; Th17 cells; granzyme B; human oligodendrocytes; multiple sclerosis; neuroinflammation.

Figure 1

Active interactions of Th17-polarized cells with human primary OLs. (A) Representative images (left) and tracks (right, gray lines) of human Th2- or Th17-polarized cells (green) in coculture during 2 h with human OLs in primary culture (magenta), scale bar = 80 μm. (B) Proportion of T cells according to duration of contact with OLs (upper panel: no contact, < 5 min, 5–10 min or > 10 min) and contact behavior among T cells displaying a >10-min contact (lower panel: scanning, crawling, static). (C) Mean speed of polarized T cells in coculture with primary human OLs according to contact duration (left panel) and contact behavior (right panel) among T cells displaying >10-min contacts. Wilcoxon rank-sum test. (A–C) n = 4 different OL preps and 4 T cell donors, n = 526 cells (Th17-polarized) and 485 cells (Th2-polarized), 30-min observation periods. One dot represents one cell, diamond = mean. (D) Representative images and (E) quantification of adherent human Th2- or Th17-polarized cells (green) after 4 h in coculture with human OLs (magenta) in primary culture (scale bar = 40 μm). Wilcoxon test, n = 4 different OL preps and 4 T cell donors, diamond = mean. (F) Number of non-adherent T cells as measured by flow cytometry using AccuCount beads. Paired t-test, p = 0.22. n = 2 OL preps, n = 3 T cell donors. (G) Representative images and (H) quantification of vesicle-like structures per timepoint (TP) in areas of primary human OLs in contact (magenta) or not (blue) with human Th2- or Th17-polarized cells (vesicle number/TP/µm²). T cells are highlighted in green, areas of interest are delimited by dashed line, and vesicle-like structures are pointed by spots (contact in magenta, no contact in blue), n = 3 OL preps and 3 T cell donors, paired t-test. (I) Quantification of human primary OL death after 16 h of direct coculture with polarized T cells (contact) or separated by a permeable membrane (insert) at a 1:2.5 ratio. Control condition = no T cells. Each dot represents one donor, n = 6 different OL preps and 6 T cell donors, one way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

Single-cell RNA sequencing reveals a transcriptional shift of Th17-polarized cells in contact with OLs. (A) Upper panel: UMAP of 9,236 cells. Doublets, cells with high mitochondrial content and residual myeloid cells were removed. Lower panel: Expression of T cells and mature oligodendrocyte (OL) canonical markers. (B) Left: UMAP of the re-clustered T-cell populations (6,954 cells). Right: Proportions of each unbiased cluster across the two conditions. Middle: Top five markers across the five obtained clusters, as found with MAST. (C) Top four most significant biological processes (GO) associated with the significant markers of each cluster. (D) Left: Distribution of the log2Fold changes between conditions across the five T-cell clusters. Right: Total number of differentially expressed genes (DEGs) within each cluster, split by upregulated (purple) and downregulated (green) genes. (E) Venn diagram showing the overlap of DEGs between biologically relevant clusters (cell cycle-associated cluster removed) and network of biological processes (GO) significantly associated with the common core (71 genes). (F) Boxplot representation of the largest (adjusted p value < 0.0001 and log2FC > 1.5 in total Th17-polarized cells contact vs. no contact) upregulated genes upon direct cell contact with OLs across all T-cell clusters (all adjusted p value for contact versus no contact in every cluster < 0.05).

Figure 3

Secretion of pro-inflammatory cytokines, chemokines, and granzyme B upon direct contact between Th17-polarized cells and human OLs. (A) Concentration of analytes measured by a Luminex assay in supernatants from human polarized Th cells in direct contact or not with oligodendrocytes (OLs). Each dot represents one donor and OL prep, n = 6 different OL preps, and 6 T cell donors. Friedman test with Dunn’s method or one way ANOVA with Tukey’s post hoc test according to distribution for each analyte. (B) Representative confocal images (immunofluorescence, scale bar = 5 µm) of granzyme B expression (GzB, green) by CD4⁺ memory T cells ex vivo or after Th2 polarization versus Th17 polarization (magenta) and (C) quantification of the percentage of CD4⁺GrzB⁺-positive cells per field of view (FOV) and (D) the median fluorescence intensity (MFI) per cell in CD4⁺GzB⁺ cells. Wilcoxon rank test, n = 5 donors, diamond = mean. (E) Representative flow cytometry pseudocolor dot plots of GzB staining in polarized T cells and (F) quantification of percentage of CD4⁺ cells expressing GzB and median fluorescence intensity (MFI) in CD4⁺ T cells after 4 h PMA-ionomycin stimulation, paired t-test, n = 8 donors. (G) Percentage of Th17-polarized cells expressing granzyme B gating on IL-17⁺, IFNγ⁺, or double-positive cells compared to their negative counterpart, paired t-test, n = 6 donors. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

Direct contact with Th17-polarized cells is associated with detection of granzyme B in OLs. (A) Representative FACS pseudocolor dot plots and histogram overlay with isotype and (B, C) quantification of the proportion of CD4⁺ T cells expressing granzyme B in (B) Th2-polarized cells or (C) Th17-polarized cells after 16 h of coculture with human primary OL separated by a permeable membrane (insert) or in direct contact, no activation with PMA-ionomycin. Wilcoxon matched-pair test (B) or paired t-test (C), n = 5 different OL preps and 5 T cell donors. (D) Representative immunofluorescent images of polarized T cell–OL coculture after 4 h and quantification of granzyme B (mean intensity) in areas of Th17-polarized cells in direct contact (red) or not (gray) with OL, n = 3 different OL preps and 4 T cell donors, total of 100 cells (25 cells/donor). (E) Quantification of granzyme B⁺ CD4⁺ cells (percentage), scale bar = 5 μm, n = 3 preps, n = 4 donors, Wilcoxon test, diamond = mean. (F) Representative FACS histogram and (G) quantification of the percentage of O4+ cells expressing granzyme B after 16h coculture with human primary OLs, Friedman test with Dunn’s method, n = 6 OL preps, and 6 T cell donors. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

Th17-polarized secretion of granzyme B induces OL death. Percentage of (A) OLs (n = 4 different OL preps) and (B) MO3.13 cell death as measured by FACS (LIVE/DEAD Amcyan staining) after a 16h incubation with activated granzyme B. Each dot represents one independent experiment, n = 4 independent experiments, Friedmann test with Dunn’s method. (C) Percentage of MO3.13 cell death as measured by FACS (LIVE/DEAD Amcyan staining) after a 16h incubation with Th17-polarized cells pretreated with granzyme blocker Ac-IEPD-CHO (Ac) or DMSO control; each dot represents one donor, n = 5–6 T cell donors, diamond = mean, Wilcoxon test (35 μg/ml), and paired t-test (50 μg/ml). *p < 0.05; **p < 0.01.

Figure 6

CD4 T cells express granzyme B in proximity to OLs in MS tissue. Representative confocal images of CD4⁺ cells (green) positive for granzyme B (yellow, upper panel) shown by arrows in proximity to mature OLs (NogoA, magenta) in MS tissue. Representative of two donors, scale bar = 30 μm.