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.

Enhanced pathogenicity of Th17 cells due to natalizumab treatment: Implications for MS disease rebound

“…Furthermore, we observed upregulation of FASL on Th17 cells from long-term, but not short-term, NAT patients. Another publication illustrated the relevance of contact-dependent granzyme B-mediated cytotoxicity of Th17 cells upon coculture with human oligodendrocytes, which at least indirectly supports our finding of altered apoptotic pathways induced upon coculture with Th17 cells derived from long-term NAT-treated MS patients ( 44 ). However, at least in the context of NAT treatment, we did observe an altered expression of granzyme B or TRAIL neither on MCAM+CCR6+Th17 cells nor in the supernatants upon coculture with either EC or oligodendrocytes ( SI Appendix , Fig.…”

Regulation of CNS pathology by Serpina3n/SERPINA3: The knowns and the puzzles

“…Pro‐inflammatory T helper 17 cells (Th17) are pathogenic in MS and EAE. Recent studies reported that Th17 cells directly contacted oligodendrocytes in MS and EAE 85 and that Th17 cell‐derived granzyme B was toxic to human oligodendrocytes in vitro 86 . These data suggest that Serpina3n/SERPINA3 expression in oligodendrocytes may protect them from inflammation‐induced injury/death, presumably through dampening lymphocyte‐derived granzyme B—a similar hypothetic mechanism to the one discussed in neuronal cell death/survival (Section 8).…”

“…Recent studies reported that Th17 cells directly contacted oligodendrocytes in MS and EAE 85 and that Th17 cell-derived granzyme B was toxic to human oligodendrocytes in vitro. 86…”

Age-associated CD4 + T cells with B cell–promoting functions are regulated by ZEB2 in autoimmunity

“…S5, C and D, and data file S2). Another CD4 + CTL cluster was observed along with the T H A cluster, likely cytotoxic T H 17 ( 49 – 51 ), because these cells expressed high levels of RORC and CCR6 . ADGRG1 , encoding GPR56 and identified in T H A SEGs using RNA-seq (Fig.…”