Anti cd3 apc fire 750

PBMCs were seeded (5 × 10⁵ cells/well) on 96-well plates and treated with SZZ-38 (1 µM), LPS (1 µg/mL), both, or the corresponding vehicle (0.1% DMSO) for 24 h. Anti-CD107a FITC antibody and monensin (Biolegend, San Diego, CA, United States) were added to all wells for the last 4 h of incubation. Cells were washed and stained with Live/Dead Fixable Aqua Dead Cell Stain (Invitrogen, Carlsbad, CA, United States). After further washing, Fc receptors were blocked with Human TruStain FcX (Biolegend) and cells were stained for extracellular markers using anti-CD3 APC/Fire 750, anti-CD56 PE, and anti-CD69 PerCP-Cy5.5 antibodies (Biolegend). Intracellular staining was performed with anti-IFN-γ APC antibody (Biolegend) after fixation and permeabilization using the Cyto-Fast Fix/Perm Buffer Set (Biolegend). Samples were analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA, United States) and FlowJo software (Tree Star, Inc., Ashland, OR, United States). Following exclusion of debris and dead cells, CD3⁻ CD56⁺ NK cells were evaluated for expression of CD107a, CD69, and IFN-γ.

This procedure was performed using modified versions of protocols described previously (12 , 13 (link)). Briefly, human PBMCs were stimulated with 17.7 μg/ml of TT lysate (MassBiologics, University of Massachusetts Medical School, MA) for 5-7 days. Spleen cells from DR3 mice immunized with protein or peptide were harvested 10-14 days post immunization. Human PBMCs were stained with 1 μg of DR3 tetramer for 3-4 hours at 37°C; and mouse spleen cells were stained for 2 hours. After tetramer staining, the cells were washed in PBS containing 2% FCS (FACS buffer) and surface markers were detected by anti-CD4-FITC (Biolegend, San Diego, CA) and anti-CD3-APCeFluor™780 (Invitrogen, Waltham, MA) for human cells and anti-CD4-FITC and anti-CD3-APC/Fire™750 (Biolegend, San Diego, CA) for mouse cells on ice for 30 minutes after Fc blocking. Cells were analyzed by the BD LSR Fortessa™ Cell Analyzer (BD, Franklin Lakes, NJ). When determining the percentage of single epitope reactive T cells from dual tetramer staining as seen in Figure 6, for SmD_66-80, the percentage of single SmD_66-80 tetramer positive T cells was the sum of cells in Q1 and Q2; for the ABC_247-261 Mimic and TT peptides, the percentage of single Clostridium tetani epitope tetramer positive T cells was the sum of cells in Q2 and Q3. Cross-reactive T cells between SmD_66-80 and Clostridium tetani epitopes were detected in Q2.

PBMCs were seeded (5 × 10⁵ cells/well) in 96-well
U-bottom plates and treated with the compounds (1 μM) or vehicle
(0.1% DMSO) for 24 h. Anti-CD107a FITC and monensin (Biolegend, San
Diego, CA) were added to all wells for the last 4 h of incubation.
The cells were washed and stained with Live/Dead Fixable Aqua Dead
Cell Stain (Invitrogen, Carlsbad, CA). After further washing, Fc receptors
were blocked with Human TruStain FcX (Biolegend) and cells were stained
for extracellular markers using anti-CD3 APC/Fire 750, anti-CD56 PE,
and anti-CD69 PerCP-Cy5.5 antibodies (Biolegend). Intracellular staining
was performed with an anti-IFN-γ APC antibody (Biolegend) after
fixation and permeabilization using the Cyto-Fast Fix/Perm Buffer
Set (Biolegend). Samples were analyzed using an Attune NxT flow cytometer
(Thermo Fisher Scientific, Waltham, MA) and the FlowJo software (Tree
Star, Inc., Ashland, OR). Following exclusion of dead cells, CD3^- CD56⁺ NK cells were evaluated for expression
of CD107a, CD69, and IFN-γ. The gating strategy is described
in detail in Figure S6. Statistical significance
was determined with one-way ANOVA with subsequent Bonferroni’s
multiple comparisons test.