Homeostatic expansion generates normal Treg-cell numbers in the periphery of miR-181a/b-1^−/− mice.

(A) Frequencies and absolute numbers of splenic Treg cells in Foxp3hCD2 reporter mice crossed to miR-181a/b-1 KO mice. Plots are representative of 6 independent experiments. Graph shows summary of all experiments, in which each data point represents one mouse. (B) Competitive BM chimeras. BM cells isolated from miR-181a/b-1+/− (het) or miR-181a/b-1−/− (KO) mice (both CD45.2) were mixed in 1:1 ratio with competitor BM cells, WT (CD45.1/2), and injected into lethally irradiated WT recipients (CD45.1). Chimeras were analyzed 12 weeks after injection for the generation of TCRβ+CD4+Foxp3+ cells in the spl. Plots are representative of 2 independent experiments with n = 8 for each genotype. Graph shows ratio of TCRβ+CD4+Foxp3+ cells within tested to competitive populations. Each data point represents an individual mouse. (C) Higher frequencies of Helios-positive cells in peripheral lymphoid organs of miR-181a/b-1−/− mice. Representative histograms and plots from 2 independent experiments (n = 6–9 for each genotype) are depicted. Numbers indicated average MFI or frequencies of positive cells, ±SD. Statistical analysis was performed using unpaired Student’s t test. (D,E) High-throughput sequencing of TCRα (CDR3 fragment of Vα8–Cα chain) in splenic Treg (D) and tTreg (E) cells sorted from miR-181a/b-1+/− or miR-181a/b-1−/− mice. Left graph shows number of all unique CDR3 sequences (CDR3). Right graphs show ENS calculated for each sample. Depicted are 4 independent sequencing experiments; samples from the same experiment are paired. Statistical analysis was performed using paired Student’s t test. Numerical values are available in S1 Data. BM, bone marrow; CD, cluster of differentiation; comp., competitor; ENS, effective number of species; Foxp3, forkhead box protein P3; hCD2, human CD2; KO, knockout; MFI, mean fluorescence intensity; miR-181, microRNA-181; mLN, mesenteric lymph node; scLN, subcutaneous lymph node; spl, spleen; TCR, T-cell receptor; th, thymus; Treg cell, regulatory T cell; tTreg cell, thymic Treg cell; WT, wild type.