Stress Signal ULBP4, an NKG2D Ligand, Is Upregulated in Multiple Sclerosis and Shapes CD8⁺ T-Cell Behaviors

(A) Representative images for the costaining of ULBP4 (cyan) and GFAP (magenta) in paraffin-embedded sections from untreated MS donors (NAGM and WM lesion) and controls (no brain related disease); n = 4 for each group. Nuclei were stained with 4',6-diamidino-2-phenylindole (blue). Corresponding isotype controls are shown. Yellow arrows indicate ULBP4⁺GFAP⁺ cells, and red arrows indicate ULBP4-negative GFAP⁺ cells. Scale bars = 25 μM. (B) Representative enlarged image showing colocalization of GFAP and ULBP4 in the brain section. Scale bars = 10 µM. GFAP = glial fibrillary acidic protein; MS = multiple sclerosis; NAGM = normal-appearing gray matter; WM = white mater; ULBP4 = UL16-binding protein 4.

(A–C) Flow cytometry analysis of ULBP4 expression by human astrocytes. (A) Gating strategy from 1 representative donor. Cell debris, doublets, and dead cells were excluded, and GFAP+ cells were selected for analysis. (B) Representative dot plots showing isotype control (B.a) or ULBP4 detection on living GFAP+ gated cells. Astrocytes were either kept under normal culture conditions (NIL) (B.b) or exposed to sodium arsenite (Na Arsenite) for 2 (B.c) and 6 hours (B.d). Percentage of ULBP4+ cells is indicated. (C) Quantification of ULBP4 expression on GFAP+ cells after exposure to sodium arsenite (C.a), tunicamycin (C.b), or proinflammatory cytokines (C.c). Each dot represents 1 donor. Data are shown as mean or mean +SEM, n = 7–9. Friedman test and Dunn multiple comparison test comparing NIL vs Na Arsenite or DMSO vs tunicamycin; 1-way analysis of variance comparing NIL vs cytokine *p < 0.05, **p < 0.01 ***p < 0.001. DMSO = dimethylsulfoxide; GFAP = Glial fibrillary acidic protein; MS = multiple sclerosis; IFNγ = interferon-γ; TNF = tumor necrosis factor; ULBP4 = UL16-binding protein 4.

(A and B) Western blot analysis of ULBP4 and albumin levels in CSF from patients with MS and controls (Ctl). (A) Representative Western blot including 5 untreated MS donors (1–5) and 2 controls (A and B) illustrating expression of ULBP4 and albumin. (B) Quantification of 45–50 kDa (B.a) and 25 kDa (B.b) bands of ULBP4 relative to albumin according to sex (♀: female; ♂: male) and disease (MS vs control). Each dot represents 1 donor. Mean ± SEM, n = 5 female untreated MS, n = 5 male untreated MS, n = 4 female controls, and n = 4 male controls. (C and D) Production of GM-CSF and IFNγ by CD8⁺ T lymphocytes stimulated with or without rhULBP4. (C) Diagram illustrating culture conditions: isolated CD8⁺ T lymphocytes were stimulated overnight with plate-bound anti-CD3 in the presence or absence of sULBP4 before collecting supernatants for ELISA measurements. (D) Quantification of secreted GM-CSF (D.a) and IFNγ (D.b) levels (pg/mL) in the presence (blue bars) or absence (white bars) of rhULBP4. Each dot represents 1 donor. Mean, n = 10 per group. Paired t test for GM-CSF production and Wilcoxon test for IFNγ production comparing NIL vs sULBP4; *p < 0.05 and **p < 0.01. GM-CSF = granulocyte-macrophage colony-stimulating factor; MS = multiple sclerosis; rhULBP4 = recombinant human ULBP4; sULBP4 = soluble UL16-binding protein 4; ULBP4 = UL16-binding protein 4.

(A–D) Time-lapse imaging of activated CD8⁺ T lymphocytes cocultured with human astrocytes in the presence or absence of sULBP4. (A) Diagram illustrating culture conditions. CD8⁺ T lymphocytes were activated overnight on anti-CD3 coated plates in the presence of anti-CD28 and interleukin-15, CFSE-labeled, and then added to astrocytes in the presence or absence of sULBP4 and imaged for 2 hours. (B) Three-dimensional time-lapse view at T = 0, T = 1, and T = 2 hours of activated CD8⁺ T lymphocytes (green) cocultured with astrocytes (magenta). (C and D) Analysis of the motility and behavior of CD8⁺ T lymphocytes from 3 untreated patients with MS and 3 sex- and age-matched healthy controls on coculture with astrocytes in the presence or absence of sUBLP4. Presented data are pooled from 3 independent experiments. (C) Coefficient of arrest (%) of individual CD8⁺ T lymphocytes; each dot represents 1 cell. Kruskal-Wallis test comparing sULBP4 vs NIL for each donors' group ****p < 0.0001. (D) Proportion of CD8⁺ T lymphocytes exhibiting the scanning, dancing, poking, and round behaviors when cocultured with astrocytes in the presence or absence of sULBP4. One way analysis of variance comparing NIL vs sULBP4 for each donors' group, *p < 0.05, **p < 0.01. CFSE = carboxyfluorescein succinimidyl ester; MS = multiple sclerosis; sULBP4 = soluble UL16-binding protein 4; ULBP4 = UL16-binding protein 4.