Anti cd8 bv605

Mononuclear cells were stained with ethidium monoazide (EMA, Sigma-Aldrich), anti-CD161-BV421, anti-CD16-V500, anti-CD8-BV605, anti-CD19-PECy5, anti-CD107a-PE (BD Biosciences), anti-TCRVα7.2-FITC, anti-CD3-AlexaFluor700 and anti-CD14-PE-Cy7 (Biolegend) on the day they were isolated from blood samples and liver biopsies. Anti-CD69-APC-Cy7 (Biolegend), anti-HLA-DR-BV711 and anti-CD107a-PE (BD Biosciences) were included to assess activation (CD69, HLA-DR) and degranulation (CD107a).
MAIT cells were identified as CD3⁺CD161⁺TCRVα7.2⁺ cells and studied in 35 paired blood samples and 32 paired liver biopsies prior to and at week 4 of antiviral therapy. Monocytes were identified as SSC^highHLA-DR⁺CD14⁺ cells and studied in 34 paired blood samples and 29 paired liver biopsies prior to and at week 4 of therapy. Liver biopsies with less than 41 events in the MAIT cell gate were excluded from analysis for activation and degranulation markers. CD14⁺⁺CD16⁻, CD14⁺⁺CD16⁺ and CD14⁺CD16⁺⁺ monocyte subsets were identified as described ^{31 (link)} and analyzed for activation marker expression if their event counts exceeded the 25^th percentile of the respective subset in all liver biopsies (n=33, 33, 23 and 23 events, respectively).

Immune cell subsets were quantified from normal skin using flow cytometry as previously described.^{6 (link)} Briefly, normal human skin was obtained from patients at UCSF undergoing elective surgery, in which healthy skin was discarded as a routine procedure. Subcutaneous fat and hair were removed, skin was minced finely with dissection scissors and mixed in a 6-well plate with 3mL of digestion buffer consisting of 0.8mg/mL Collagenase Type 4 (4188, Worthington, Lakewood, NJ), 0.02mg/mL DNAse (DN25-1G, Sigma-Aldrich, St. Louis, MO), 10% FBS, 1% Hepes and 1% Penicillin/Streptavidin in RPMI medium. Samples were incubated overnight in 5% CO2 and harvested with wash buffer (2% FBS, 1% Penicillin/Streptavidin in RPMI medium) and filtered through a 100µm filter, centrifuged, counted and stained for flow cytometry. The following antibodies were used for flow cytometry: anti117 hCD3 (-FITC, eBioscience, San Diego, CA), anti-hCD4 (-PerCP-eFluor710, eBioscience), anti-CD8 (-BV605, BD, San Jose, CA), anti-hFoxP3 (-eFluor 450, eBioscience), and LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies, South San Francisco, CA). Data was acquired by an LSRFortessa (BD Bioscienes) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).