rhil 27  (R&D Systems)

Journal: Nature Immunology

Article Title: Fungal sensing by dectin-1 directs the non-pathogenic polarization of T H 17 cells through balanced type I IFN responses in human DCs

doi: 10.1038/s41590-022-01348-2

Figure Lengend Snippet: a , b , Real-time PCR analyses of IFNG, TBX21, IL1R1, CXCL3, CCL3, CSF2, IL22, CCL5, IL17A, IL17F, RORC, IL10, MAF and PTGDS normalized mRNA levels in IL-17 + T cells, isolated by flow cytometry-based sorting after restimulation of T cells, outgrown in vitro by co-culture of memory CD4 + T cells with immature DCs (iDC) or DCs primed for 48 h with curdlan ( a,b ) or C. albicans ( a ), in the presence of blocking IFN-α/βR antibodies (Ab, a ), rhIFN-β ( a ) (both during DC stimulation), neutralizing IL-27 antibodies ( a ), rhIL-27 ( a ), blocking TGF-βR antibodies ( a ), rhTGF-β1 ( a,b ) (all during DC-T co-culture), blocking αvβ8 antibodies (during DC stimulation) ( a ), or after overexpression of BST2 (eGFP + cells) by transfecting DCs with mammalian expression plasmid pCG–BST2-IRES–eGFP and subsequent sorting based on eGFP expression (eGFP − , eGFP + ) 24 h later ( b ), at day 11–14 ( a , untreated n = 12; d , IFN-α/βR Ab n = 11, rhIFN-β n = 4; e , n = 4; f , TGF-βR Ab n = 6, rhTGF-β1 n = 4, αvβ8 Ab n = 3; b , n = 3). Results from RT–PCR were quantified using standard curves for all genes and normalized to the expression of reference household gene ACTB . These data are an addendum to the data presented in Fig. ( a ), Fig. ( a ), Fig. ( a ), Fig. ( a ) and Fig. ( b ). Data represent mean ± s.d. of independent donors. ** P < 0.01, * P < 0.05 (Student’s t -test, paired, two-tailed), calculated between untreated and treated samples that were likewise stimulated ( a,b ) and eGFP − and eGFP + DCs primed with curdlan as indicated by brackets ( b ).

Article Snippet: During co-culture, cells were treated with either rhIL-27 (300 ng ml; −1 R&D Systems), rhTGF-β1 (20 ng ml −1 ; R&D Systems), neutralizing antibodies (5 μg ml −1 ) to IL-1β (AF-201-NA, R&D Systems), IL-23 (AF1716, R&D Systems), IL-12 (AF-219-NA, R&D Systems), IL-27 (AF2526, R&D Systems) or TGF-βRII (AF-241-NA, R&D Systems) or normal goat IgG (AB-108-C; R&D Systems) as a control.

Techniques: Real-time Polymerase Chain Reaction, Isolation, Flow Cytometry, In Vitro, Co-Culture Assay, Blocking Assay, Over Expression, Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Nature Immunology

Article Title: Fungal sensing by dectin-1 directs the non-pathogenic polarization of T H 17 cells through balanced type I IFN responses in human DCs

doi: 10.1038/s41590-022-01348-2

Figure Lengend Snippet: a , b , ELISA for quantification of IL-1β, IL-6, IL-23, IL-12 and IL-27 in the supernatant of unstimulated DCs or after stimulation of DCs with curdlan or C. albicans , in the presence of blocking IFN-α/βR antibodies ( a , b ) or rhIFN-β ( b ) at 28 h ( a , n = 5; b , IFN-α/βR antibody n = 5, rhIFN-β n = 3). Data in a , b represent mean ± s.d. of independent donors. ** P < 0.01, * P < 0.05 (paired, two-tailed Student’s t -test), calculated between untreated and treated samples that were likewise stimulated. c – e , Flow cytometry analyses of T H polarization by staining for intracellular IL-17 and IFN-γ expression (FI) in restimulated T cells, outgrown in vitro by co-culture of memory CD4 + T cells with iDC or DCs primed for 48 h with curdlan or C. albicans , in the presence of neutralizing IL-27 antibodies or rhIL-27 (both during DC-T co-culture), at day 11–14 (IL-27 antibody n = 7, rhIL-27 n = 4). In c , represe n tative dot plots for independent donors are shown, with percentage positive cells indicated in each quadrant. In d , the percentage IL-17 + cells per total amount of T cells are shown and in e , the percentage IFN-γ − and percentage IFN-γ + cells per IL-17 + T cells are shown. Data in d , e represent mean ± s.d. of independent donors.

Article Snippet: During co-culture, cells were treated with either rhIL-27 (300 ng ml; −1 R&D Systems), rhTGF-β1 (20 ng ml −1 ; R&D Systems), neutralizing antibodies (5 μg ml −1 ) to IL-1β (AF-201-NA, R&D Systems), IL-23 (AF1716, R&D Systems), IL-12 (AF-219-NA, R&D Systems), IL-27 (AF2526, R&D Systems) or TGF-βRII (AF-241-NA, R&D Systems) or normal goat IgG (AB-108-C; R&D Systems) as a control.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Two Tailed Test, Flow Cytometry, Staining, Expressing, In Vitro, Co-Culture Assay