Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Université de Montréal, Montreal, QC, Canada.
Department of Neurosciences, Faculty of Medicine, Université de Montréal, Montreal, QC, Canada.
Front Immunol. 2022 Apr 11;13:850616. doi: 10.3389/fimmu.2022.850616. eCollection 2022.
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 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.
多发性硬化症（MS）的特征是中枢神经系统（CNS）中髓鞘和产生髓鞘的少突胶质细胞（OLs）的丧失。促炎 CD4 Th17 细胞被认为在 MS 中具有致病性，对 OLs 有害。我们研究了驱动人类 CD4 T 细胞介导的 OL 细胞死亡的机制。通过荧光和明场活细胞成像，我们发现与 Th2 极化细胞相比，Th17 极化细胞与原代人 OLs 和人少突胶质细胞系 MO3.13 的相互作用更大，表现为接触持续时间更长、平均速度更低，以及在接触部位形成囊泡样结构的速率更高。通过单细胞 RNA 测序，我们评估了直接接触或通过插入物分离的原代人 OLs 和 Th17 极化细胞的转录组谱。我们表明，在紧密相互作用时，OLs 上调了编码趋化因子和抗氧化/抗凋亡分子的 mRNA 的表达，而 Th17 极化细胞上调了编码趋化因子和促炎细胞因子（如 IL-17A、IFN-γ 和 granzyme B）的 mRNA 的表达。我们发现，在人 Th17 极化细胞与人 OLs 的共培养物中直接接触会诱导 CCL3、CXCL10、IFN-γ、TNFα 和 granzyme B 的分泌。此外，通过流式细胞术和免疫荧光验证，与 Th2 极化细胞相比，Th17 极化细胞中 granzyme B 的水平上调，并且当 Th17 极化细胞与 OL 或 MO3.13 细胞直接接触时，与通过插入物与 OL 分离的 Th17 极化细胞相比，甚至更高。此外，在 Th17 极化细胞与 OL 或 MO3.13 细胞直接接触后，在 OL 和 MO3.13 细胞中检测到 granzyme B，表明 granzyme B 从 Th17 极化细胞释放到 OL/MO3.13 细胞中。为了证实 granzyme B 对 OLs 的细胞毒性，我们表明重组人 granzyme B 可诱导 OLs 和 MO3.13 细胞死亡。此外，用可逆的 granzyme B 阻断剂（Ac-IEPD-CHO）或天然 granzyme B 阻断剂（serpina3N）预处理 Th17 极化细胞可改善 MO3.13 细胞在与 Th17 细胞共培养时的存活率。总之，我们表明，人类 Th17 极化细胞与人类 OLs 形成生物学上有意义的接触，并通过释放 granzyme B 发挥直接毒性。
