TCR internalization assay

Lymph node cells from DA and DA^Mut rats were stained with mouse anti-rat CD3 (Clone 1F4) conjugated with Fitc (Biolegend) at 4 °C. Labeled cells were added to plates coated with anti-rat αβTCR (Clone R73) and anti-rat CD28(Clone JJI319) in D-MEM supplemented with HEPES (GIBCO), streptomycin/d-penicillin (104 IU/ml penicillin, 10 mg/ml streptomycin; Invitrogen Life Technologies), b-mercaptoethanol (GIBCO), 10% fetal calf serum (GIBCO), and incubated for 15, 30, and 60 min at 37 °C or left on ice for zero time point. Cells were washed and spilt into two fractions, were one fraction was stripped of surface anti-CD3-Alexa488 antibodies with PBS (GIBCO) pH 2 for 1 min and the other fraction left untreated. For the TCR internalization assay with the CRISPR Jurkat T cells the cells were stained with anti-human CD3 (Clone UTCH1) conjugated with Alexa488 (BD Biosciences) at 4 °C. Labeled cells were added to plates coated with anti-human CD3 (Clone Hit3a, BD Biosciences) and anti-human CD28(Clone CD28.2, BD Biosciences) in RPMI1640 (GIBCO), supplemented streptomycin/D-penicillin (104 IU/ml penicillin, 10 mg/ml streptomycin; Invitrogen Life Technologies) and 10% fetal calf serum (GIBCO) and incubated for 15, 30, and 60 min at 37 °C or left on ice for zero time point. Cells were washed and spilt into two fractions, were one fraction was quenched of surface anti-CD3-Alexa488 signal with unlabeled anti-Alexa488 (MolecularProbes, Invitrogen) and the other fraction left untreated. For the Brefeldin A inhibitor experiment lymph node cells from SH3gl1^−/− and SH3gl1^+/+ mice were stained with Armenian hamster anti-mouse TCRb (Clone H57-597, Biolegend) conjugated with Alexa488 at 4 °C. Labeled cells were added to plates coated with anti-mouse CD3e (Clone 145-2C11, BD Biosciences) and anti-mouse CD28(Clone 37.51, BD Biosciences) in D-MEM supplemented with HEPES (GIBCO), streptomycin/D-penicillin (104 IU/ml penicillin, 10 mg/ml streptomycin; Invitrogen Life Technologies), b-mercaptoethanol (GIBCO), 10% fetal calf serum (GIBCO) and together with 1, 25ug/ml Brefeldin A (Sigma) and incubated for 30 min at 37 °C or left on ice for zero time point. Cells were washed and spilt into two fractions, quenched or no quench, and subsequently stained with rat anti-mouse CD45R-PE-Cy7 (Clone RA3-6B2, BD Biosciences), rat anti-mouse CD4-BV605 (Clone RM4.5, BD Biosciences), rat anti-mouse CD3-PacificBlue (Clone 17A2, Biolegend) and anti-Alexa488 (MolecularProbes, Invitrogen) to half of the samples (quenched). Cells were acquired on a LSRII (BD Biosciences, Franklin Lakes, NJ, USA) using the BD FACSDiva™ software (BD Biosciences) with gates set to exclude doublets and include all viable cells determined as LIVE/DEAD™ -Near-IR (Invitrogen) negative cells and later analyzed by FlowJo (Tree Star, Inc.) software. Percentage of internalized CD3-Alexa488 was calculated as (Qx-Q0)/(Tt-Q0)x100, where Qx is the mean fluorescence of cells quenched with anti-Alexa488 at each time point, Q0 is the mean fluorescence of cells quenched at time zero, and Tt is the mean fluorescence of cells that were not quenched.