Cd49d clone 9f10

The following conjugated Abs were used for cell surface staining: CD3-Pacific Blue (clone UCHT1), CD4-Alexa Fluor 700 (clone RPA-T4), CD8-APC-H7 (clone SK1), CD160-Alexa Fluor 647 (clone BY55), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3)-PE-CF594 (clone 7D3), 2B4-FITC (clone 2–69) (BD Biosciences, San Jose, CA), and programmed cell death protein-1 (PD-1)-PE (clone J105) (Thermo Fisher Scientific). Conjugated Abs for intracellular staining included the following: cytotoxic T lymphocyte antigen-4 (CTLA-4)-PE-Cy5 (clone BNI3), granzyme B-PE-CF594 (clone GB11), interferon-gamma (IFN-γ)-PE-Cy7 (clone B27), interleukin-2 (IL-2)-APC (clone MQ1-17H12), tumor necrosis factor-alpha (TNF-α)-Alexa Fluor 488 (clone MAb11) (BD Biosciences, San Diego, CA), and perforin-PE (clone B-D48) (Abcam, Cambridge, UK). All conjugated Abs were titrated and multicolor panels for flow cytometry assays were approached as previously reported [38 (link)]. The following purified (No azide/Low endotoxin) Abs were used in cultured cells: CD28 (clone CD28.2) and CD49d (clone 9F10) (BD Biosciences).

For staining of the cell surface, the following conjugated antibodies were used: CD3-Pacific Blue (clone UCHT1), CD4-Alexa Fluor 700 (clone RPA-T4), CD8-APC-H7 (clone SK1), CD160-Alexa Fluor 647 (clone BY55), TIM-3-PE-CF594 (clone 7D3), 2B4-FITC (clone 2–69) (BD Biosciences, San Jose, CA), and PD-1-PE (clone J105) (Thermo Fisher Scientific). Intracellular staining was performed with the following antibodies: CTLA-4-PE-Cy5 (clone BNI3), granzyme B-PE-CF594 (clone GB11), IFN-γ-PE-Cy7 (clone B27), IL-2-APC (clone MQ1-17H12), TNF-α-Alexa Fluor 488 (clone MAb11) (BD Biosciences, San Diego, CA), and perforin-PE (clone B-D48) (Abcam, Cambridge, UK). All conjugated antibodies were titrated as previously reported [29 (link)]. The multicolor panels used for the flow cytometry assays were utilized according to the fluorescence minus one control (FMO) method as recommended [29 (link)]. In addition, the following purified NA/LE antibodies were used for cell culture: CD28 (clone CD28.2) and CD49d (clone 9F10) (BD Biosciences).

Myelin-specific CD49d⁺CD154⁺ cell proliferation was analyzed with the use of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE, BD Biosciences) [23 (link)]. CFSE-labelled PBMCs were incubated in 6-well culture plates at the density of 2 × 10⁶ cells per well with or without pep(MOG/MBP/PLP) or pre-incubated with pep(MOG/MBP/PLP) (21 h, each 25 μg/mL) maturating OPCs (1:50). For neutralizing experiments, 5 μg/mL anti-hCD40 (clone 40804) or isotype control (clone 20102R, all R&D Systems) were added 10 min before pep(MOG/PLP/MBP) stimulations or 10 min to pre-incubated with pep(MOG/PLP/MBP) hOPCs and washed in RPMI. After 24, 48, and 72 h of incubation, cells were labelled with CD49d (clone 9F10, BD Biosciences), CD154 (clone 89-76, BD Biosciences), or mouse IgG1 isotype control (clone 11711, R&D system) antibodies and analyzed by BD LSR Flow Cytometer.