Anti cd44 bv785 im7

Single‐cell suspensions from spleens and lymph nodes of OVA‐immunized mice were incubated for 15 min at 4°C with anti‐CD16/CD32 Fc‐receptor block (2.4G2, eBiosciences), followed by incubation for 45 min at 4°C with anti‐CXCR5‐PE (L138D7, Biolegend), anti‐CD62L‐PE‐Cy7 (MEL‐14, Biolegend), anti‐PD‐1‐BV605 (J43, BD Biosciences), anti‐CD4‐FITC (GK1.5, Biolegend), anti‐CD44‐BV785 (IM7, Biolegend), and anti‐ICOS‐BV421 (7E.17G9, BD Biosciences) antibody, all diluted in PBS containing 2% FCS. Dead cells were excluded using 7‐Aminoactinomycin D (7AAD, life technologies). Single T cells from defined subpopulations were isolated into 384‐well plates containing 2 µL/well of ice‐cold Sort/RHP mix (4 mM DTT, 0.69% NP‐40, 20 ng/µL random‐hexamer‐primers (RHP, Roche), 1.87 U/µL RNAsin (Promega) in 0.25× PBS) with an AriaIII cell sorter and index‐sort option (BD Biosciences).

IL-7Rα internalisation assay was performed similar as described before^{30 (link)}. In brief, CD4+ T cells were purified from the spleens of wild-type or T-Atg7^−/− mice. After incubation with biotin-conjugated anti-IL-7Rα antibody (BioLegend, A7R34) 1:50 diluted in DPBS on ice for 30 min, cells were extensively washed and fractionated into the following groups (5 × 10⁶ cells/group): 90 min on ice, 90, 60 and 30 min at 37 °C, 5% CO₂. After incubation, cells were stained with AF647-Streptavidin (BioLegend, 1:1000), anti-CD4 BV605 GK1.5 (BioLegend, 1:400), anti-CD44 BV785 IM7 (BioLegend, 1:400), LIVE/DEAD Fixable Aqua (Invitrogen) stain for 20 min on ice and fixed with 2% PFA. The % internalised IL-7Rα is calculated as the Eq. (1) (MFI: mean fluorescence intensity): $\%\mathrm{internalised\; IL}\text{-}7\mathrm{R}\mathrm{\alpha }=100-\left[\left(\frac{\mathrm{MFI\; sample}}{\mathrm{MFI}90\mathrm{minice}}\right)\times 100\right]$