GoKawiilMice
Six-to-eight-week-old female and male C57BL/6 J mice (IMSR_JAX:000664), as well as other strains, were purchased from The Jackson Laboratory. OT-II (IMSR_JAX:004194) and Thy1.1 (IMSR_JAX:000406) mice were crossed to generate OT-II Thy1.1 mice. Foxp3-GFP mice (IMSR_JAX:006772) were crossed with OT-II Thy1.1 mice. 2D2 mice (IMSR_JAX:006912) were crossed with Thy1.1 mice. All animals were housed in AAALAC-accredited facilities. Sample sizes were not predetermined but are reported with each result, and randomization was performed across littermates.
Flow cytometry
The following antibodies were purchased from BioLegend: mouse CD45.2 (109839), CD3 (100206), CD4 (100453, 100430, 100428, 100451), CD8 (100706), NK1.1 (156506), TCRvα2 (127806, 127822), TCRvα3.2 (135404), Thy1.1 (202528, 202522), CTLA4 (106310), CD62L (104453), CD25 (102012, 102022, 102047, 102038), CD44 (103026, 103032), SIGLECF (155534), CD73 (127215), ICOS (313550), CD69 (104530), CD11b (101259), CXCR3 (126514), CD39 (143806), NRP1 (145218), CD11c (117318), CD103 (110910), GITR (126316), CXCR6 (151117), CCR6 (129819), IL-17A (506928), GM-CSF (505406), IFNγ (505832, 505826), IL-10 (505034, 505026, 505034), BLIMP1 (150008), Helios (137214), Ki-67 (151212, 652406), TNFα (506346); human CD3 (317324), CD4 (980806) and FOXP3 (320126). The following antibodies were purchased from BD Biosciences: mouse BCL6 (562401), RORγt (564722, 562682, 562683), SMAD2 (pS465/pS467)/SMAD3 (pS423/pS425) (562696) and STAT5 (pY694) (612599). The following antibodies and reagents were purchased from Invitrogen: mouse PD-1 (48-9985-82), FOXP3 (12-5773-82, 17-5773-82, 404-5773-82), T-bet (25-5825-82), c-MAF (53-9855-82), GATA3 (46-9966-42) and Fixable Viability Dye (65-0865-18). PE- and Brilliant Violet 421-labelled I-Ab OVA 328–337 tetramers (HAAHAEINEA) were provided by the NIH Tetramer Core Facility.
For surface marker staining, live cells were incubated with antibodies and viability dye in PBS at 4 °C for 1 h. Dead cells were excluded on the basis of viability dye staining. For I-Ab OVA 328–337 tetramer staining, live cells were first incubated with tetramers in PBS at 37 °C for 1 h, followed by surface antibody and viability dye staining. For transcription factor and cytokine staining, cells were fixed and permeabilized using the FOXP3/Transcription Factor Staining Buffer Set (00-5521-00, Invitrogen), followed by intracellular staining at room temperature for 2 h. Prior to cytokine staining, cells were stimulated with Cell Stimulation Cocktail (00-4970-03, Invitrogen) and Protein Transport Inhibitor Cocktail (00-4980-03, Invitrogen) at 37 °C for 5 h. For pSMAD2/3 and pSTAT5 staining, freshly isolated T cells were stimulated ex vivo with the indicated proteins for 25 min, then were immediately fixed with Cytofix Fixation Buffer (554655, BD) and permeabilized using Phosflow Perm Buffer III (558050, BD). Cells were resuspended in PBS and analysed on CytoFLEX flow cytometer (Beckman Coulter). Data analysis was performed using FlowJo software v.10.10.0.
Protein production
Recombinant proteins were cloned into the pD649 mammalian expression vector (ATUM), which includes a haemagglutinin (HA) secretion signal peptide, an N-terminal MSA fusion, and a C-terminal 6×His tag. Expression constructs were transfected into Expi293F cells using the Expi293 Expression System (Gibco). After 3–4 days of culture, proteins were purified from supernatants by Ni-NTA Agarose (Qiagen), followed by size-exclusion chromatography using a Superdex 200 column in ÄKTA chromatography system (Cytiva). Endotoxin was removed using the Proteus NoEndo HC Spin Column Kit (VivaProducts), and levels were confirmed to be acceptable using the Pierce Chromogenic Endotoxin Quant Kit (Thermo Fisher Scientific). Final protein preparations were formulated and concentrated in sterile PBS, flash-frozen in liquid nitrogen, and stored at −80 °C until use.
CD4+ T cell isolation and in vitro differentiation
Mouse lymph nodes and spleens were collected and mechanically dissociated to obtain single-cell suspensions. Red blood cells were lysed using ACK lysis buffer (A10492-01, Gibco), followed by magnetic isolation of CD4+ T cells using the EasySep Mouse CD4+ T Cell Isolation Kit (19852, STEMCELL). Naive CD4+ T cells (CD4+CD44−CD25−Foxp3-GFP−) and activated CD4+ T conv cells (CD4+CD44+CD25−Foxp3-GFP−) were subsequently sorted using a Sony SH800S Cell Sorter. The purity of the sorted populations was consistently greater than 99%. Human CD4+ T cells were isolated from frozen PBMCs using the EasySep Human CD4+ T Cell Isolation Kit (17952, STEMCELL). Complete medium was prepared using RPMI 1640 with GlutaMAX supplement (Gibco, 61870036) and supplemented with 10% fetal bovine serum (Gibco, A5256701), 10 mM HEPES (Gibco, 15630080), 1% sodium pyruvate (Gibco, 11360070), 1% penicillin–streptomycin (Gibco, 15140122) and 0.1% 2-mercaptoethanol (Gibco, 21985023).
For the in vitro mouse CD4+ T cell differentiation assay, naive CD4+ T cells were plated at 0.75 × 106 cells per ml in flat-bottom 96-well plates pre-coated overnight with 5 μg ml−1 InVivoMAb anti-mouse CD3 (Bio X Cell, BE0002) and 5 μg ml−1 InVivoMAb anti-mouse CD28 (Bio X Cell, BE0015-1). For OT-II cell differentiation, mouse splenocytes were plated at 1.5 × 106 cells per ml in flat-bottom 96-well plates and stimulated with 0.05 μg ml−1 OVA 323–339 peptide (GenScript, RP10610). For human CD4+ T cell differentiation, CD4+ T cells were plated at 0.75 × 106 cells per ml in flat-bottom 96-well plates pre-coated overnight with InVivoMAb anti-human CD3 (Bio X Cell, BE0001-2) and 5 μg ml−1 InVivoMAb anti-human CD28 (Bio X Cell, BE0248). Recombinant proteins were added at the indicated concentrations at the start of the culture, and cells were incubated at 37 °C for ~4 days.
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