Quantibrite pe quantification beads

TCR expression differences were quantified using the anti-TCRβ clone H57–597 PE antibody (eBioscience). Briefly, a few million splenocytes from infected mice were surface stained on ice for 30 minutes with anti-CD11b PerCP Cy5.5 (M1/70; BD), anti-CD11c PerCP Cy5.5 (HL3; BD), anti-CD19 PerCP Cy5.5 (ID3; BD), 7AAD (BD), anti-CD3ε PE CF594 (145–2C11; BD), anti-CD44 PE Cy7 (IM7; Biolegend), anti-CD62L APC Cy7 (MEL-14; BD), anti-CD27 V450 (LG3.A10; BD), anti-CD4 BV510 (RM4–5; Biolegend), anti-PD-1 BV605 (29F.1A12; Biolegend), and anti-CD8 BV785 (53–6.7; Biolegend) antibodies along with the anti-TCRβ antibody. Cells were washed and kept on ice until flow cytometry was carried out using a LSR II (Beckton Dickson). Using the FlowJo software (Tree Star), TCR, CD4 and forward scatter (FSC-A) mean fluorescence intensities (MFIs) of CD4+CD44^hi (antigen experienced) and CD4+CD44^loCD62+ (naïve) cells was quantified and compared between the two infections per experiment. QuantiBRITE PE quantification beads (BD Biosciences) were used per manufacturer instructions to determine the number of TCRs per cell. For further comparison between infections and across different time points, MFI of antigen experienced cells was normalized to the naïve population within the same sample and quantified as % of naïve ((antigen experienced MFI/naïve MFI) x 100) (39 (link)).

The relative two-dimensional affinity (2D MP) of polyclonal IAb:GP_66–77 specific cells from C57BL/6-FOXP3 EGFP mice (JAX) was measured using the previously described 2D- MP assay (25 (link)–27 (link)). Seven days following LCMV infection and AIM assay culture, splenic CD4+ T cells were negatively enriched by magnetic separation (Miltenyi Biotec;130-104-454) and were stained and sorted to obtain FOXP3- (GFP-) CD25-OX40- AIM-negative CD4+ T cells and FOXP3- (GFP-) CD25+OX40+ AIM-positive CD4+ T cells. Human RBCs were coated with IAb:GP_66–77 monomer or with negative control H2Db:NP_366–374 monomer obtained from the NIH tetramer core. Quantification of pMHC density with anti-IA/IE Ab (M5/114/15/2; eBioscience), TCR surface density with anti-mouse TCRβ PE Ab (H57–597; BD Biosciences) were determined using QuantiBRITE PE quantification beads (BD Bio-sciences). Quantification of binding events and TCR:pMHC affinity calculations were calculated as previously described (25 (link)–27 (link)). Each cell was tested to the monomer of interest and then the control monomer to determine antigen specificity of the cell.