Tcr vbeta21.3 pe clone rea894

For SmB/B'_58-72 peptide titration, a co-culture of 10⁵ TCR1-transduced Tregs or 10⁵ mock-transduced Tregs and 50,000 DR15 + B-LCLs pulsed with SmB/B'_58-72 in five tenfold serial dilutions from 100 μg/mL to 0.01 μg/mL and no peptide was established. Each condition was plated on a 96-well U bottom plate in supplemented RPMI and incubated in a 37 °C, 5% CO₂ humidified incubator for 36 h after which cells were harvested for flow cytometry. Cells were first stained with Live/Dead Blue (Invitrogen) followed by surface marker staining with anti-human antibodies; CD4 R718 (clone SK3, BD), TCR Vbeta21.3 PE (clone REA894, Miltenyi), CD69 BUV395 (clone FN50, BD) and GARP BV786 (clone 7B11, BD). Cells were acquired on a LSR Fortessa X20 flow cytometer (BD) and analyzed in FlowJo 10.6.2. MFI values were exported and analyzed in GraphPad Prism ver.8.3.1.

TCR1-transduced Tconvs (10⁵) were stained with CTV and co-cultured with 50,000 DR15⁺ B-LCLs pulsed with 100μg/mL SmB/B'_58-72 and serial dilutions of either TCR1-transduced Tregs stained with CTFR or mock-transduced Tregs stained with CTFR. Each condition was plated on a 96-well U bottom plate in 200 μL supplemented RPMI and incubated in a 37 °C, 5% CO₂ humidified incubator for 5 days after which cells were harvested for flow cytometry. Cells were first stained with Live/Dead Near Infrared cell viability stain (Invitrogen) followed by surface marker staining with anti-human antibodies; CD3 PerCP (clone SK7, Biolegend), TCR Vbeta21.3 PE (clone REA894, Miltenyi) and CD19 PE-CF594 (clone HI519, BD). The entire sample was acquired on a Cytek Aurora 5 L flow cytometer and analyzed in FlowJo 10.6.2. The gating strategy is found in Supplementary Fig. 8. The Tregs and Tconvs were distinguished by CTV and CTFR dye labels. The % suppression was calculated by: % proliferation of Tconv’s alone minus % proliferation of Tconv’s with Tregs/% proliferation of Tconv’s alone ×100. Graphs were constructed in GraphPad Prism ver.8.3.1.

In vitro-expanded Tregs were stained with Live/Dead Aqua (Life Technologies) followed by the following anti-human antibodies; CD4 BUV496 (clone SK3, BD), CD127 APC-Vio770 (clone REA614, Miltenyi), CD25 APC (clone BC96, Biolegend) and TCR Vbeta21.3 PE (clone REA894, Miltenyi).
For intracellular staining: Tregs were stimulated with PMA/ionomycin and Brefeldin A (Sigma) for 4 h 37 °C after which cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and stained with anti-human antibodies; Foxp3 (clone 236A/E7, BD), Helios (clone 22F6, Biolegend), IFN-γ (clone B27, BD) and IL-17A (clone N49-653, BD). Cells were acquired on a Cytek Aurora 5 L flow cytometer and analysis performed in FlowJo (version 10.6.2). A gating strategy is found in Supplementary Fig. 7. Methylation of the FOXP3 TDSR was performed by pyrosequencing of intron 1 of TDSR region -2330 to -2263 from ATG of FOXP3 and analysis of 9 CpG sites by EpigenDx.