Isolation and Analysis of Lymphocyte Subsets

Single cell suspensions of spleen, mesenteric lymph nodes (mLNs), or various subcutaneous lymph nodes (scLN) (Schallenberg et al., 2010 (link)) were prepared using 70 μm cell strainers (BD Biosciences). mAbs to CD4 (RM4–5, GK1.5), CD25 (PC61, 7D4), CD62L (MEL-14) and Thy1.1 (OX-7), as well as Fc receptor–blocking mAbs to CD16/32 (93) and Pacific Blue- and APC-conjugated streptavidin were obtained from eBioscience or BD Biosciences. The mAb to TCR-HA_107–119 (6.5) was purified and conjugated to Alexa Fluor 647 (Invitrogen) in our laboratory according to standard protocols. Before FACS, for some experiments, CD4⁺ CD25⁺ cells were enriched using biotinylated mAbs against CD4 or CD25, respectively, streptavidin-conjugated microbeads and the AutoMACS (Miltenyi Biotec). Samples were analyzed on a LSR II or sorted using a FACSAria II or III (BD). Data were analyzed using FlowJo (Tree Star, Inc.).