(A–C) IL-10 expression of Tregs purified from Foxp3RFP/Il10GFP dual reporter mice (see also Figure 1—figure supplement 1) cultured for 3 days with the designated stimulants as analyzed by flow …
(A) Gating strategy for flow cytometry sorting and analyzing IL-10+/- Tregs from Foxp3RFP/Il10GFP dual-reporter mice. Freshly harvested spleens underwent CD4+ magnetic bead selection prior to …
(A) IL-10 expression and production by purified Tregs stimulated for 3 days with the indicated cytokines while titrating IL-2 and analyzed by flow cytometry and ELISA, respectively. 1× IL-2 = 728 …
Luminex analysis of cytokines secreted into the media following the designated stimulation conditions grouped in cytokines that (A) synergistically increase with combinatorial cytokine stimulation, …
(A) IL-10 and FoxP3 expression of purified IL-10+/- Tregs that underwent culture with αCD3ε and combined IL-2/IL-4for 3 days, as analyzed by flow cytometry. N = 3. Related to Figure 1K. (B) Analysis …
(A and B) Treg proliferation as measured by CellTrace signal following 3 days of culture with αCD3ε and all combinations of IL-2 and IL-4, with division and proliferation indices indicated. N = 3 …
(A) Flow cytometry gating schematic for FoxP3+ Treg proliferation analyses. Cells are first gated on FSC-A and SSC-A, then a single cell gate made on FSC-A and FSC-H. Sytox red negative events …
(A–C) Proliferation (flow cytometry) and cytokine output (ELISA) of freshly isolated and Celltrace-stained Tconv co-cultured with αCD3ε and the indicated ratios of WT Tregs separately and previously …
Numeric values of the heatmaps in Figure 3B–C (A), Figure 3E–F (B), Figure 3H–I (C), and Figure 3K–L (D).
Values were obtained by analyzing the suppression assay supernatants by ELISA.
(A and B) Mouse colon photographs and their lengths as measured by the distance between anus (at 0) and cecum.
(A–C) pSTAT3, pSTAT6, and pSTAT5 expression of purified Tregs stimulated with the indicated culture conditions as analyzed by flow cytometry. The histograms are representative and heatmaps of median …
(A) pSTAT5 expression following IL-2 stimulation at the indicated concentrations, with 1× = 728 pM, and as analyzed by flow cytometry. (B–E) Analysis of viability (Sytox Red), IL-10 expression, and …
(A) Expression of 14 parameters as shown in an unpaired principle component analysis of Tregs purified and stimulated with αCD3ε and all cytokine conditions for 3 days unless otherwise noted in the …
Proliferation of purified Tregs stimulated with αCD3ε and all cytokine conditions for 3 days with or without neutralizing IL-10R monoclonal antibody or isotype control blockade, as analyzed by …
(A and B) Analysis of HDM-induced airway inflammation in WT C57Bl/6J mice by cell differentials of the Bronchial Alveolar Lavage (BAL, LY = lymphocytes, Mono = monocytes, Gran = granulocytes) and …
(A) Challenge and treatment schedule for the therapeutic HDM-induced acute asthma trials in Figure 6A and B,D–F. Mice were challenged with HDM (or PBS) for 2 weeks (d7-11, d14-18). IL-2/IL-4 was …
(A) Challenge and treatment schedule for the preventative HDM-induced acute asthma trials in Figure 6G. IL-2/IL-4 was delivered i.n. for five consecutive days (d0–5), followed by 2 weeks of HDM (or …
(A) Challenge and treatment schedule for therapeutic TAC-induced chronic asthma trial in Figure 6H. Mice were sensitized to the triple antigens once weekly for the first 3 weeks. Afterwards, all …
(A and B) EAE clinical score and FFPE-processed spinal cords sectioned and stained with H and E and LFB from mice challenged with myelin oligodendrocyte glycoprotein (MOG) peptide with and without …
(A) EAE was induced in mice with s.c. injections of myelin oligodendrocyte glycoprotein emulsified in Complete Freund’s Adjuvant (CFA) and d0. Pertussis toxin (PTX) was injected i.p. on days 0 and …
(A) Analysis of IL-10 expression in CNS FoxP3- Tconv cells of Foxp3RFP/Il10GFP mice in the EAE trials by flow cytometry. N = 3. (B) Table of p-values for Figure 7I. Red font represents p≤0.05. Black …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Mus musculus) | C56Bl/6J | JAX | JAX 000664, RRID:IMSR_JAX:000664 | |
Strain, strain background (Mus musculus) | Il10-/- | JAX | JAX 002251, RRID:IMSR_JAX:002251 | |
Strain, strain background (Mus musculus) | Il10fl/fl | PMID:15534372, PMID:28783045 | from Dr. Werner Muller via Dr. Asma Nusrat | |
Strain, strain background (Mus musculus) | Foxp3creYFP | JAX | JAX 016959, RRID:IMSR_JAX:016959 | |
Strain, strain background (Mus musculus) | Il10fl/fl x Foxp3creYFP (IL-10 cKO) | This paper | JAX 016959, RRID:IMSR_JAX:016959 and from Dr. Werner Muller via Dr. Asma Nusrat | We crossed IL-10fl/fl mice obtained from Dr. Asma Nusrat with commercially available FoxP3-creYFP mice. To obtain, contact the Cobb lab. |
Strain, strain background (Mus musculus) | BALB/c Foxp3GFP | JAX | JAX 006769, RRID:IMSR_JAX:006769 | |
Strain, strain background (Mus musculus) | Foxp3RFP | JAX | JAX 008374, RRID:IMSR_JAX:008374 | |
Strain, strain background (Mus musculus) | Il10GFP | JAX | JAX 008379, RRID:IMSR_JAX:008379 | |
Strain, strain background (Mus musculus) | Foxp3RFP x Il10GFP (Xmas) | This paper | JAX 008374, RRID:IMSR_JAX:008374 and JAX 008379, RRID:IMSR_JAX:008379 | We crossed two commercially available mouse lines. To obtain, contact the Cobb lab. |
Antibody | αCD4-magnetic beads (rat, monoclonal) | Miltenyi Biotec | 130-049-201, RRID:AB_2722753 | (1:50–1:100), (50 µL) |
Antibody | αCD3 (Armenian hamster, monoclonal) | eBioscience | 16-0031-85, RRID: AB_468848 | (1:400), (50 µL) |
Antibody | αCD28 (Syrian hamster, monoclonal) | eBioscience | 16-0281-82, RRID: AB_468921 | (1:400), (50 µL) |
Antibody | CD4-PE (rat, monoclonal) | eBioscience | 12-0042-82, RRID: AB_465510 | (1:250–1:500), (50 µL) |
Antibody | CD4-AF647 (rat, monoclonal) | Biolegend | 100530, RRID: AB_389325 | (1:250–1:500), (50 µL) |
Antibody | CD4-BV421 (rat, monoclonal) | BD | 740007, RRID: AB_2739779 | (1:250–1:500), (50 µL) |
Antibody | F4/80-BV711 (rat, monoclonal) | BD | 565612, RRID: AB_2734769 | (1:250–1:500), (50 µL) |
Antibody | CD25-PECy7 (rat, monoclonal) | BD | 561780, RRID: AB_10893596 | (1:250–1:500), (50 µL) |
Antibody | CD25-PE (rat, monoclonal) | eBioscience | 12-0251-82, RRID: AB_465607 | (1:250–1:500), (50 µL) |
Antibody | CD124-BB700 (rat, monoclonal) | BD | 742172, RRID: AB_2871410 | (1:250–1:500), (50 µL) |
Antibody | CD122-APC (rat, monoclonal) | BD | 564924, RRID: AB_2739009 | (1:250–1:500), (50 µL) |
Antibody | CD132-BV421 (rat, monoclonal) | BD | 740039, RRID: AB_2739809 | (1:250–1:500), (50 µL) |
Antibody | CD213a1-Biotin (rabbit, polyclonal) | MyBioSource | MBS2001753 | (1:250–1:500), (50 µL) |
Antibody | pSTAT3-AF647 (mouse, monoclonal) | BD | 557815, RRID: AB_647144 | (1:100–1:200), (25 µL) |
Antibody | pSTAT5-AF647 (mouse, monoclonal) | BD | 612599, RRID: AB_399882 | (1:100–1:200), (25 µL) |
Antibody | pSTAT6-AF647 (mouse, monoclonal) | BD | 558242, RRID: AB_647145 | (1:100–1:200), (25 µL) |
Antibody | IL-10R biotin (rat, monoclonal) | Biolegend | 112704, RRID: AB_313517 | (1:250–1:500), (50 µL) |
Antibody | SA-APCefluor780 | eBioscience | 47-4317-82, RRID: AB_10366688 | (1:250–1:500), (50 µL) |
Antibody | SA-BV605 | BD | 563260, RRID: AB_2869476 | (1:250–1:500), (50 µL) |
Peptide, recombinant protein | Recombinant IL-2 | R and D Systems | 402 ML-020 | |
Peptide, recombinant protein | Recombinant IL-4 | R and D Systems | 404 ML-010 | |
Peptide, recombinant protein | Recombinant IL-10 | R and D Systems | 417 ML | |
Commercial assay, kit | IFNγ ELISA MAX | Biolegend | 430803 | |
Commercial assay, kit | IL-4 ELISA MAX | Biolegend | 431102 | |
Commercial assay, kit | IL-10 ELISA MAX | Biolegend | 431413 | |
Commercial assay, kit | IL-17A ELISA MAX | Biolegend | 432501 | |
Commercial assay, kit | MOG35-55/CFA Emulsion PTX | Hooke Laboratories | EK-2110 | |
Chemical compound, drug | STAT5i | Stemcell | 73852 | |
Chemical compound, drug | House dust mite | Greer | XPB8103A2.5 | |
Chemical compound, drug | Ovalbumin | Sigma-Aldrich | A5503 | |
Chemical compound, drug | Cockroach antigen | Greer | XPB46D3A4 | |
Chemical compound, drug | Methacholine | Sigma-Aldrich | 62-51-1 | |
Software, algorithm | Prism | Graphpad | Versions 5 and 7, RRID:SCR_002798 | |
Software, algorithm | FlowJo | FlowJo | v10.7, RRID:SCR_008520 | |
Other | FACS-ARIA SORP | BD | ||
Other | Attune NxT | Thermo Fisher | ||
Other | Sytox Red | Invitrogen | S34859 | |
Other | Fixable Viability Stain 510 | BD | 564406 | |
Other | MOG I-A(b) tetramer-BV421 | NIH Tetramer Core | GWYRSPFSRVVH | |
Other | Control I-A(b) tetramer-BV421 | NIH Tetramer Core | PVSKMRMATPLLMQA | |
Other | CellTrace Violet | Invitrogen | C34557 or C34571 | |
Other | Europium-conjugated streptavidin | Perkin-Elmer | 1244–360 | |
Other | DELFIA Enhancement solution | Perkin-Elmer | 4001–0010 | |
Other | 10% formalin | Anachemia | 41916–700 | |
Other | Hematoxylin | Ricca | 3536–32 | |
Other | Eosin | EMD Harleco | 588X-75 | |
Other | Solvent Blue 38 | Sigma-Aldrich | S3382 | |
Other | Percoll | GE | 17-0891-02 |