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, CD4 + CD25 + T lymphocytes (175 000 cells in a volume of 50 ml DMEM supplemented with 1% FBS) were plated in the upper chamber and incubated overnight at 37 C. After incubation, T cells in the upper and lower chamber were recovered separately and incubated with 0.5 ml of 10 mm carboxylated beads (Polyscience, #18133) and DAPI (3 mM). T cell counting was performed by FACS using precision beads for normalization and expressed as percentage of migration being the ratio of the T cell number in the lower chamber by the total number of cells. The overall percentage of T cell survival shown is the ratio of T cells alive (DAPI -) by the total number of cells, considering both low and upper chambers, Transwell Migration Assay For migration assays, 20 000 cells of CAF-S4, CAF-S1 or CAF-S1 transiently transfected with siCTR, siCCL11, siCXCL12, siCXCL13 or siCXCL14 were plated in 200 ml of DMEM supplemented with 1% FBS, in the lower chamber of the transwell (5 mm pore size, Corning HTS Transwell 96 wells, #CLS3388)

, All interactions, including short and persistent contacts, were quantified considering at least 1 contact observed during a time window of 10 frames (bin=10 and minC=1). 4 Videos corresponding to 28h (210 frames, 8 min per frame) were analyzed for each condition. Co-Culture of T Cells with CAF-S1 or CAF-S4 and FOXP3 Induction To study the impact of CAF-S1 or CAF-S4 on CD4 + CD25 + T cells, we performed co-cultures. 50 000 primary CAF-S1 or CAF-S4 were plated in 24-well plates in DMEM supplemented with 10% FBS and used non-transfected or siRNA-transfected (siCTR, siCD276/ B7H3, siNT5E/CD73, siDPP4 and siTNFSF4/OX40L). The medium was replaced by fresh DMEM supplemented with 1% FBS just before 500 000 CD4 + CD25 + T lymphocytes were added to CAF-S4, CAF-S1, or to CAF-S1 + siRNA 30h post-transfection. Cocultures of CAF-S1 or CAF-S4 and CD4 + CD25 + T cells were incubated for 16 h at 37 C, 20%O 2 . Negative control was incubation of CD4 + CD25 + T cells in absence of CAF. After incubation, T cells were collected and analyzed by FACS, Cells with CAF-S1 Time-Lapse Video Microscopy For co-culture experiments for time-lapse video microscopy, CAF-S1 (50-60 000 cells) were transiently transfected with siRNA (listed above) and plated in 12-well plates in DMEM supplemented with 10% FBS. Purified CD4 + CD25 + T lymphocytes were freshly added to CAF-S1 cells, 30h post-transfection, to reach a ratio of 1:5 (CAF:T cell). Just before adding the T cells, the media was replaced to DMEM supplemented with 2.5% FBS

, #130-091-024) for 15 min at RT. The detection of FOXP3 was performed using the FOXP3 staining buffer set kit (eBioscience, #00-5523-00) for fixation and permeabilization according to manufacturer's instructions followed by incubation with anti-Foxp3-Alexa Fluor 488 (eBioscience, #53-4776) for 30 min at RT. For CD25 and FOXP3 staining, the corresponding isotype controls were #130-092-215 and #53-4321 for CD25 and FOXP3, respectively. Analyses were performed in the BD LSR II flow cytometer (BD Biosciences) and data was then analyzed using FlowJo version 9, Treg Cell Suppressive Assay Treg Cell Suppressive Assay, 2016.

, The negative fraction (CD4 + CD25 -cells containing effector T cells, Teff) was also recovered and kept overnight at 4 C in TexMACS medium, CD4+CD25+ were isolated from healthy donor PBMC, as described above, pp.130-097

, BD Pharmigen #555489) proteins together with DAPI. After a washing step, DAPI -CD4 + CD25 high CD127 -CD45RA low cells (enriched in regulatory T cells, Tregs) were sorted on FACSARIA (BD Biosciences) and pre-incubated overnight at 37 C, 5% CO 2 , 20% O 2 into 24-well plates, with previously plated 50 000 primary CAF-S1 or CAF-S4 fibroblasts in 1 ml of DMEM supplemented with 1% FBS, or with 1 ml of DMEM supplemented with 1% FBS only (control condition), + CD25 + cells were then stained with a pool of fluorescent-conjugated primary antibodies recognizing CD4 (1:20, Miltenyi Biotec, vol.127, p.20