Tcr vβ repertoire kit

Individual Vβ clones were detected in PBMC using TCR-Vβ repertoire kit from Beckman Coulter as per manufacturer’s instructions. Cells were stained simultaneously with for CD3 and IFN-γ, and a set of three antibodies directed against TCR-Vβ repertoires and analyzed by flow cytometry. Cells were gated on CD3⁺cells, individual TCR families were then analyzed for IFN-γ positive CD3⁺ T cells.
Staining for HER2 BATs in the peripheral blood. The percentage of anti-CD3 x anti-HER2 positive cells was quantitated by using goat PE conjugated anti-mouse IgG2a antibodies to detect OKT3 in the BiAb as reported previously [21 (link)].

Normal donors were recruited under a Stanford University IRB-approved protocol. Informed consent was obtained. Standard blood draws in green-top tube were obtained for each time point. 1-5mL of whole blood was enriched for CD4⁺ cells using RosetteSep Human CD4⁺ T Cell Enrichment Cocktail (StemCell Technology) as described (Buenrostro et al., 2013 (link)). PBMCs were prepared by Ficoll-Hypaque density-gradient centrifugation. PBMCs were stained with fluorochrome-labeled anti–human monoclonal antibodies (Biolegend Inc) to CD45 (clone HI30), CD4 (clone RPA-T4), and CD3 (clone HIT3a). T-cell receptor (TCR) Vβ clonality was determined with the TCR Vβ Repertoire Kit (Beckman-Coulter). Antibody-stained patient lymphocytes were sorted into CD3⁺CD4⁺Vβ⁺ and CD3⁺CD4⁺Vβ^- fractions with the use of an Influx flow cytometer (Becton Dickinson) (Dummer et al., 1996 (link)). At least 50,000 CD4⁺ T cells were enriched by negative selection without ex vivo expansion. For CD4⁺ T helper cell subtypes, cells were sorted as previously described(Morita et al., 2011 (link)). Briefly, naive cells were sorted as CD4⁺CD25^-CD45RA⁺, Th1 cells as CD4⁺CD25^-CD45RA^-CXCR3⁺CCR6^-, Th2 cells as CD4⁺CD25^-CD45RA^-CXCR3^-CCR6^-, Th17 cells as CD4⁺CD25^-CD45RA^-CXCR3^-CCR6⁺, and Treg cells as CD4⁺CD25⁺CD127lo. >95% post-sort purities were confirmed prior to ATAC-seq.