Anti cd45rb clone 16a

Single-cell suspensions of DLN from La- and Lb-infected mice, as well as from control animals (10⁶ cells/well), were surface-stained at 4°C in the dark for 30 minutes with the following mouse monoclonal antibodies: anti-CD3ε (clone 145 2C11; BD Biosciences), anti-CD4 (clone RM45; BD Biosciences), anti-CD8α (clone 53-6.7; BD Biosciences), anti-CD45RB (clone16A; BD Biosciences), and anti-CD62L (clone MEL14; BD Biosciences). The gating strategy used to determine memory T-cell subsets is described in Supplementary Figure 2.
All samples were acquired on FACSFortessa using FACSDiva software (BD Biosciences), and they were then analyzed with FlowJo software version 9.2 (Tree Star; FlowJo, LLC, Ashland, OR, USA). Fluorescence voltages were determined using matched unstained cells. Compensation was carried out with the aid of CompBeads (BD Biosciences) single stained with the monoclonal antibodies described earlier. Samples were acquired to reach at least 300,000 events.

Splenocytes with or without pooled peripheral LNs (axillary, brachial, inguinal) were isolated, and CD4⁺ T cells were purified with the MojoSort mouse CD4 T cell isolation kit (catalog 480005); they were then stained with anti-CD4 (clone RM4-5, BD Biosciences, 558107) plus anti-CD45RB (clone 16A, BD Biosciences, 562848) anti-murine antibodies, and sorted on the basis of antibody and EGFP fluorescence. Sorting was done on a BD FACS AriaFusion flow cytometer (BD Biosciences). Colitis was induced in 6- to 8-week-old Rag1^–/– mice by i.p. injection of 4 × 10⁵ CD4⁺Ly5.1⁺EGFP⁻CD45RB^hi cells isolated from WT or STAT3 GOF mice. In some experiments, mice were treated 21 days after the induction of colitis with 1 × 10⁶ CD4⁺Ly5.2⁺EGFP⁺ WT or STAT3 GOF Tregs. Mice were weighed at least twice weekly and sacrificed when moribund or at the conclusion of the experiment. Mechanistic details for the lamina propria digest and isolation of lymphocytes are provided in the Supplemental Methods.