ArrayExpress Uploader05529Sorted on CD4+ Foxp3/GFP-CD4+Foxp3- T cells were culture with LP-DCs at a 10:1 ratio with anti-CD3 (1ug/ml) + TGFbeta (0.6ng/ml) + IL-2 (5ng/ml on D2 and D4) for 5 daysC57BL/6-Foxp3/GFPCD4+Foxp3- T cells were obtained from spleen and dendritic cells were obtained from lamina propriaSorted on CD4+ Foxp3/GFP-CD4+Foxp3- T cells were culture with LP-DCs at a 10:1 ratio with anti-CD3 (1ug/ml) + TGFbeta (0.6ng/ml) + IL-2 (5ng/ml on D2 and D4) for 5 daysC57BL/6-Foxp3/GFPCD4+Foxp3- T cells were obtained from spleen and dendritic cells were obtained from lamina propriaSorted on CD4+ Foxp3/GFP+CD4+Foxp3- T cells were culture with LP-DCs at a 10:1 ratio with anti-CD3 (1ug/ml) + TGFbeta (0.6ng/ml) + IL-2 (5ng/ml on D2 and D4) for 5 daysC57BL/6-Foxp3/GFPCD4+Foxp3- T cells were obtained from spleen and dendritic cells were obtained from lamina propriaSorted on CD4+ Foxp3/GFP+CD4+Foxp3- T cells were culture with LP-DCs at a 10:1 ratio with anti-CD3 (1ug/ml) + TGFbeta (0.6ng/ml) + IL-2 (5ng/ml on D2 and D4) for 5 daysC57BL/6-Foxp3/GFPCD4+Foxp3- T cells were obtained from spleen and dendritic cells were obtained from lamina propriaSorted on CD4+ Foxp3/GFP-CD4+Foxp3- T cells were culture with Sp-DCs at a 10:1 ratio with anti-CD3 (1ug/ml) + TGFbeta (0.6ng/ml) + RA (100nM) + IL-2 (5ng/ml on D2 and D4) for 5 daysC57BL/6-Foxp3/GFPCD4+Foxp3- T cells and dendritic cells were obtained from spleenSorted on CD4+ Foxp3/GFP+CD4+Foxp3- T cells were culture with Sp-DCs at a 10:1 ratio with anti-CD3 (1ug/ml) + TGFbeta (0.6ng/ml) + RA (100nM) + IL-2 (5ng/ml on D2 and D4) for 5 daysC57BL/6-Foxp3/GFPCD4+Foxp3- T cells and dendritic cells were obtained from spleenSorted on CD4+ Foxp3/GFP+CD4+Foxp3- T cells were culture with Sp-DCs at a 10:1 ratio with anti-CD3 (1ug/ml) + TGFbeta (0.6ng/ml) + RA (100nM) + IL-2 (5ng/ml on D2 and D4) for 5 daysC57BL/6-Foxp3/GFPCD4+Foxp3- T cells and dendritic cells were obtained from spleenSorted on CD4+ Foxp3/GFP-CD4+Foxp3- T cells were culture with Sp-DCs at a 10:1 ratio with anti-CD3 (1ug/ml) + TGFbeta (0.6ng/ml) + IL-2 (5ng/ml on D2 and D4) for 5 daysC57BL/6-Foxp3/GFPCD4+Foxp3- T cells and dendritic cells were obtained from spleenSorted on CD4+ Foxp3/GFP-CD4+Foxp3- T cells were culture with Sp-DCs at a 10:1 ratio with anti-CD3 (1ug/ml) + TGFbeta (0.6ng/ml) + IL-2 (5ng/ml on D2 and D4) for 5 daysC57BL/6-Foxp3/GFPCD4+Foxp3- T cells and dendritic cells were obtained from spleenSorted on CD4+ Foxp3/GFP+CD4+Foxp3- T cells were culture with Sp-DCs at a 10:1 ratio with anti-CD3 (1ug/ml) + TGFbeta (0.6ng/ml) + IL-2 (5ng/ml on D2 and D4) for 5 daysC57BL/6-Foxp3/GFPCD4+Foxp3- T cells and dendritic cells were obtained from spleenSorted on CD4+ Foxp3/GFP+CD4+Foxp3- T cells were culture with Sp-DCs at a 10:1 ratio with anti-CD3 (1ug/ml) + TGFbeta (0.6ng/ml) + IL-2 (5ng/ml on D2 and D4) for 5 daysC57BL/6-Foxp3/GFPCD4+Foxp3- T cells and dendritic cells were obtained from spleenMemory T cells were sorted based on the expression of the congenic marker (CD45.1 or CD45.2)Congenically marked (CD45.1 or CD45.2) memory T cells were cultured with CD44negCD62LhiCD25-CD4+ (naive) T cells at a 2:1 ratio with anti-CD3/CD28 beads + IL-2 (20 U/ml) + TGFbeta (10 ng/ml) for 48hrsC57BL/6CD44hiCD62L-CD25-CD4+ (Memory) T cells were sorted form spleen and lymph nodes (mesenteric and cervical)Memory T cells were sorted based on the expression of the congenic marker (CD45.1 or CD45.2)Congenically marked (CD45.1 or CD45.2) memory T cells were cultured with CD44negCD62LhiCD25-CD4+ (naive) T cells at a 2:1 ratio with anti-CD3/CD28 beads + IL-2 (20 U/ml) + TGFbeta (10 ng/ml) for 48hrsC57BL/6CD44hiCD62L-CD25-CD4+ (Memory) T cells were sorted form spleen and lymph nodes (mesenteric and cervical)Memory T cells were sorted based on the expression of the congenic marker (CD45.1 or CD45.2)Congenically marked (CD45.1 or CD45.2) memory T cells were cultured with CD44negCD62LhiCD25-CD4+ (naive) T cells at a 2:1 ratio with anti-CD3/CD28 beads + IL-2 (20 U/ml) + TGFbeta (10 ng/ml) for 48hrsC57BL/6CD44hiCD62L-CD25-CD4+ (Memory) T cells were sorted form spleen and lymph nodes (mesenteric and cervical)Memory T cells were sorted based on the expression of the congenic marker (CD45.1 or CD45.2)Congenically marked (CD45.1 or CD45.2) memory T cells were cultured with CD44negCD62LhiCD25-CD4+ (naive) T cells at a 2:1 ratio with anti-CD3/CD28 beads + IL-2 (20 U/ml) + TGFbeta (10 ng/ml) + Retinoic Acid (100nM) for 48hrsC57BL/6CD44hiCD62L-CD25-CD4+ (Memory) T cells were sorted form spleen and lymph nodes (mesenteric and cervical)Memory T cells were sorted based on the expression of the congenic marker (CD45.1 or CD45.2)Congenically marked (CD45.1 or CD45.2) memory T cells were cultured with CD44negCD62LhiCD25-CD4+ (naive) T cells at a 2:1 ratio with anti-CD3/CD28 beads + IL-2 (20 U/ml) + TGFbeta (10 ng/ml) + Retinoic Acid (100nM) for 48hrsC57BL/6CD44hiCD62L-CD25-CD4+ (Memory) T cells were sorted form spleen and lymph nodes (mesenteric and cervical)Memory T cells were sorted based on the expression of the congenic marker (CD45.1 or CD45.2)Congenically marked (CD45.1 or CD45.2) memory T cells were cultured with CD44negCD62LhiCD25-CD4+ (naive) T cells at a 2:1 ratio with anti-CD3/CD28 beads + IL-2 (20 U/ml) + TGFbeta (10 ng/ml) + Retinoic Acid (100nM) for 48hrsC57BL/6CD44hiCD62L-CD25-CD4+ (Memory) T cells were sorted form spleen and lymph nodes (mesenteric and cervical)arrayexpress_sid6CD4(+)Foxp3(+) regulatory T (Treg) cells originate primarily from thymic differentiation, but conversion of mature T lymphocytes to Foxp3...19006694E-GEOD-13306/profile/8773/arrayexpressuploader417cellcytokineDec.12, 2014Falseretinoic-acid-enhances-foxp3-induction-indirectlyE-GEOD-13306_A-AFFY-45Retinoic Acid Enhances Foxp3 Induction Indirectly by Relieving Inhibition from CD4(+)CD44(hi) Cells.Nov.10, 2014CD4(+)Foxp3(+) regulatory T (Treg) cells originate primarily from thymic differentiation, but conversion of mature T lymphocytes to Foxp3 positivity can be elicited by several means, including in vitro activation in the presence of TGF-beta. Retinoic acid (RA) increases TGF-beta-induced expression of Foxp3, through unknown molecular mechanisms. We showed here that, rather than enhancing TGF-beta signaling directly in naive CD4(+) T cells, RA negatively regulated an accompanying population of CD4(+) T cells with a CD44(hi) memory and effector phenotype. These memory cells actively inhibited the TGF-beta-induced conversion of naive CD4(+) T cells through the synthesis of a set of cytokines (IL-4, IL-21, IFN-gamma) whose expression was coordinately curtailed by RA. This indirect effect was evident in vivo and required the expression of the RA receptor alpha. Thus, cytokine-producing CD44(hi) cells actively restrain TGF-beta-mediated Foxp3 expression in naive T cells, and this balance can be shifted or fine-tuned by RA. All gene expression profiles were obtained from highly purified T cell populations sorted by flow cytometry. To reduce variability, cells from multiple mice were pooled for sorting, and replicates were generated for essentially all groups. RNA from 0.5-3 x 105 cells was amplified, labeled, and hybridized to Affymetrix M430v2 microarrays. Raw data were preprocessed with the RMA algorithm in GenePattern, and averaged expression values were used for analysis.http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-13306mousehttp://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-13306/samples/