Mice were housed in a specific pathogen-free facility in accordance with institutional guidelines. BALB/c mice were purchased from Charles River Laboratories and the Jackson Laboratory. IL-2−/− (3
), CD25−/− (4
)and Bim−/− (8
) mice were back-crossed >12 generations to BALB/c; Foxp3/GFP mice (9
) were back-crossed ≥7 generations.
The following clones were used: 145-2C11 (for CD3), GK1.5, RM4-5 (CD4), PC-61 (CD25), UC10-4F10-11 (CTLA-4), FJK-16s (Foxp3), JES6-1A12 (IL-2), 24DMS1 (CD39), eBioTY/11.8 (CD73), JES5-16E3 (IL-10), and B56 (Ki-67).
Anemia and anti-erythrocyte antibodies
Hematocrits, the percentages of blood volume filled with erythrocytes, were measured using a Hemavet 950 instrument (Drew Scientific). Endogenous anti-erythrocyte antibodies were detected using flow cytometry by staining erythrocytes with FITC-conjugated anti-IgM Ab or anti-IgG F(ab′)2 (Jackson ImmunoResearch Laboratories) (10
Cells were cultured in RPMI 1640 media (Sigma-Aldrich) with 1mM each L-glutamine, non-essential amino acids, sodium pyruvate, HEPES, penicillin, streptomycin (Life Technologies), 50mM 2-ME, and 10% FCS (Omega Scientific). To measure cytokines, cells were activated for 4h with 75 ng/mL Phorbol Myristate Acetate (PMA) plus 750 ng/mL Ionomycin (Sigma-Aldrich) and, for the last 2h, 10mg/mL Brefeldin A (Epicentre Biotechnologies).
After washing, blocking and staining for surface antigens, cells were fixed with 2% paraformaldehyde. For intracellular staining, fixed cells were permeabilized with0.5% saponin (Sigma-Aldrich) or processed using Cytofix/Cytoperm and Perm/Wash buffers(BD Biosciences). Foxp3 was detected using the Foxp3 Staining Set (eBioscience). Flowcytometry was performed on a FACSCalibur or LSR II instrument using CellQuest or FACSDiva software (BD Bioscience).
Co-culture suppression assay
Lymph node CD4+ GFP+ (Foxp3+) Tregs and CD4+ CD25- GFP- (Foxp3-) responder T cells were purified with matching GFP levels using a MoFlo high-speed cell sorter (DakoCytomation). APCs were prepared from erythrocyte depleted, Mitomycin C (Sigma-Aldrich) treated WT spleens. Cultures were set up in U bottom96 well plates with 4 × 104 APCs, 2 × 104 responder T cells, 3 μg/mL soluble anti-CD3 Ab, and a titrated number of Tregs per well, incubated 48h, pulsed with 1.25 mCi [3H] Thymidine, and harvested at 60h using a Harvester 96 Mach III instrument (Tomtec). Proliferation was measured by scintillation counting using a Trilux 1450 Microbeta instrument (Wallac-Perkin Elmer).
Treatment with IL-2 immune complexes
IL-2-containing immune complexes (6
) were made by incubating 1.5 μg mouse rIL-2 with 50 μg functional grade purified anti-IL-2 Ab, clone JES6-1A12, (eBioscience) perdose for 15–30 min at 37° in PBS. Mice were treated by i.p. injection beginning at 9–18 dof age 1/d for 3 d, then 3/wk. Treatment was decreased to 2/wk in 4–5 wk old mice whose hematocrits stabilized, or was stopped if hematocrits fell below 15 and mice became moribund.
Data were analyzed using Prism 5.02 for Windows software (GraphPad Software) applying: t test with Welch’s correction (two-tailed , S1B, S2B, one-tailed , ), Mann-Whitney test (, , , S1C), Log-rank test (), 1-way ANOVA (), and Fisher’s exact test (). Significance is indicated as p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p >0.05not significant (ns).
Bim deficiency restores peripheral Foxp3+ cells in IL-2-deficient mice
Treatment with IL-2/anti-IL-2-antibody complexes rescues IL-2−/− Bim−/− mice and expands Tregs
Treatment with IL-2/anti-IL-2-antibody complexes increases the expression of putative suppressive molecules in Tregs
Rescued Foxp3+ T cells fail to protect IL-2−/− Bim−/− and CD25−/− Bim−/− mice from autoimmune disease and are less suppressive in culture