Anti perforin bv 421

CD8⁺ T cells isolated as above were washed with FACS buffer (PBS containing 2% FBS and 2 mM EDTA) and labeled for 30 min in dark at 4 °C with the following monoclonal antibodies (TONBO biosciences) anti-CD3-FITC, anti-CD8a-PE, anti-TLR4-PE Cy7 (eBioscience, CA, USA), anti-CD28-PerCpCy5.5, anti-CD45RO-FITC(TONBO biosciences). After surface staining with anti-TLR4-PECy7 antibody, CD8⁺ T cells were then fixed for 30 min with 2% PFA, permeabilized with Perm buffer (TONBO biosciences) and labeled with anti-Granzyme B-PE (eBioscience), anti-Perforin-BV 421, anti-TNFα-PE, anti-IFNγ-APC/Alexa Fluor 700 antibodies (BD Biosciences). Expression levels were measured using BD LSRFortessa (BD Biosciences), and data were analyzed using FlowJo software (version 10; Tree Star). CD3⁺CD8⁺ T cells, directly gated in whole blood, (Supplementary Fig. 1b) were also analyzed for TLR4 surface protein expression.