From the Department of Neurosciences (A.C.M., Y.C.S., F.L., N.F-k., A.L., S.E.J.Z., M.G., P.D., C.L., A.P., N.A.), Université de Montréal and Centre de Recherche du CHUM (CRCHUM) Montreal; MS-CHUM Clinic (B.L., M.G., P.D., C.L., A.P.); CLSC des Faubourgs (J.V.G.), CIUSSS du Centre-Sud-de-l'Ile-de-Montréal; and Department of Microbiology, Infectious Diseases, and Immunology and Department of Pediatrics (E.H.), Université de Montréal, Centre de Recherche du Centre Hospitalier Universitaire Sainte-Justine (CHU Sainte-Justine), Montreal, Quebec, Canada.
Neurol Neuroimmunol Neuroinflamm. 2021 Dec 6;9(1). doi: 10.1212/NXI.0000000000001119. Print 2022 Jan.
We posit the involvement of the natural killer group 2D (NKG2D) pathway in multiple sclerosis (MS) pathology via the presence of specific NKG2D ligands (NKG2DLs). We aim to evaluate the expression of NKG2DLs in the CNS and CSF of patients with MS and to identify cellular stressors inducing the expression of UL16-binding protein 4 (ULBP4), the only detectable NKG2DL. Finally, we evaluate the impact of ULBP4 on functions such as cytokine production and motility by CD8 T lymphocytes, a subset largely expressing NKG2D, the cognate receptor.
Human postmortem brain samples and CSF from patients with MS and controls were used to evaluate NKG2DL expression. In vitro assays using primary cultures of human astrocytes and neurons were performed to identify stressors inducing ULBP4 expression. Human CD8 T lymphocytes from MS donors and age/sex-matched healthy controls were isolated to evaluate the functional impact of soluble ULBP4.
We detected mRNA coding for the 8 identified human NKG2DLs in brain samples from patients with MS and controls, but only ULBP4 protein expression was detectable by Western blot. ULBP4 levels were greater in patients with MS, particularly in active and chronic active lesions and normal-appearing white matter, compared with normal-appearing gray matter from MS donors and white and gray matter from controls. Soluble ULBP4 was also detected in CSF of patients with MS and controls, but a smaller shed/soluble form of 25 kDa was significantly elevated in CSF from female patients with MS compared with controls and male patients with MS. Our data indicate that soluble ULBP4 affects various functions of CD8 T lymphocytes. First, it enhanced the production of the proinflammatory cytokines GM-CSF and interferon-γ (IFNγ). Second, it increased CD8 T lymphocyte motility and favored a kinapse-like behavior when cultured in the presence of human astrocytes. CD8 T lymphocytes from patients with MS were especially altered by the presence of soluble ULBP4 compared with healthy controls.
Our study provides new evidence for the involvement of NKG2D and its ligand ULBP4 in MS pathology. Our results point to ULBP4 as a viable target to specifically block 1 component of the NKG2D pathway without altering immune surveillance involving other NKG2DL.
我们假设自然杀伤细胞组 2D（NKG2D）途径通过存在特定的 NKG2D 配体（NKG2DL）参与多发性硬化症（MS）的发病机制。我们旨在评估 NKG2DL 在 MS 患者中枢神经系统（CNS）和脑脊液（CSF）中的表达，并鉴定诱导 UL16 结合蛋白 4（ULBP4）表达的细胞应激源，ULBP4 是唯一可检测到的 NKG2DL。最后，我们评估 ULBP4 对 CD8 T 淋巴细胞（大量表达 NKG2D 这一受体的亚群）的细胞因子产生和运动等功能的影响。
使用 MS 患者和对照者的尸检脑组织样本和 CSF 来评估 NKG2DL 的表达。使用原代培养的人星形胶质细胞和神经元进行体外实验，以鉴定诱导 ULBP4 表达的应激源。从 MS 供体和年龄/性别匹配的健康对照中分离人 CD8 T 淋巴细胞，以评估可溶性 ULBP4 的功能影响。
我们在 MS 患者和对照者的脑组织样本中检测到编码 8 种已鉴定的人类 NKG2DL 的 mRNA，但仅通过 Western blot 检测到 ULBP4 蛋白表达。与 MS 供体的正常灰质和对照者的白质和灰质相比，MS 患者的 ULBP4 水平更高，特别是在活动期和慢性活动期病变以及正常外观的白质中。我们还在 MS 患者和对照者的 CSF 中检测到可溶性 ULBP4，但与对照者和 MS 男性患者相比，女性 MS 患者 CSF 中显著升高的 25 kDa 较小的分泌/可溶性形式。我们的数据表明，可溶性 ULBP4 影响 CD8 T 淋巴细胞的各种功能。首先，它增强了前炎性细胞因子 GM-CSF 和干扰素-γ（IFNγ）的产生。其次，当在人星形胶质细胞存在的情况下培养时，它增加了 CD8 T 淋巴细胞的运动并有利于类似联会的行为。与健康对照者相比，MS 患者的 CD8 T 淋巴细胞受可溶性 ULBP4 的影响尤其明显。
我们的研究为 NKG2D 及其配体 ULBP4 参与 MS 发病机制提供了新的证据。我们的结果表明 ULBP4 是一个可行的靶点，可以特异性阻断 NKG2D 途径的 1 个组成部分，而不会改变涉及其他 NKG2DL 的免疫监视。
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