Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐25 initiates Th2 differentiation of human CD4 + T cells and influences expression of its own receptor

doi: 10.1002/iid3.87

Figure Lengend Snippet: IL‐25R is highly expressed by in vitro differentiated CRTh2 + Th2 cells. Flow cytometry of (A) surface CRTh2 (solid line) compared with isotype control (dotted line; day 42) and (B) intra‐cellular IL‐4, IL‐13 and IFN‐γ⋅ (C) qRT‐PCR for IL‐25R mRNA from activated CRTh2 + Th2 cells (day 45, black bar, n = 6 independently differentiated lines) were compared with activated non‐polarized CD4 + T cells (day 3, white bar, 61‐fold, n = 5). (D) Representative example of surface IL‐25R expression (solid line) compared to isotype control (dotted line) by differentiated CRTh2 + Th2 cells (day 43) and (E) kinetics of surface IL‐25R expression following TCR activation ( n = 4, three independently differentiated lines). Statistical significance was determined by Student's t test, * P < 0.05.

Article Snippet: On day 14, αCRTh2 antibody coated microbeads (Cat. #130‐091‐274, Miltenyi) were used to isolate CRTh2‐expressing cells.

Techniques: In Vitro, Flow Cytometry, Control, Quantitative RT-PCR, Expressing, Activation Assay

Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐25 initiates Th2 differentiation of human CD4 + T cells and influences expression of its own receptor

doi: 10.1002/iid3.87

Figure Lengend Snippet: IL‐25 mediates acquisition of the Th2 phenotype. Naïve CD4 + T cells were differentiated in non‐polarizing (NP) control conditions (αCD3/αCD28, IL‐2, αIFNγ and αIL‐12) or NP + IL‐4 (IL‐4), NP + IL‐25 (IL‐25) or NP + IL‐4 and IL‐25 (IL‐4+IL‐25). (A) IL‐25R mRNA expression (fold increase over NP; day 3, 7, 10, 14, n = 10 of three independently differentiated lines). (B) Intracellular IL‐4 and (E) surface CRTh2 were assayed following proliferation (day 7, 14, n = 17, 10 independently differentiated lines). (D) GATA3 was assayed after stimulation (day 3, 10, n = 17, 10 independently differentiated lines). (C) IL‐4 mRNA expression by cells cultured in IL‐4 or IL‐25 (fold increase over NP, day 14; n = 4 independently differentiated lines). (F) Cell counts were determined and comparison was made between the four conditions for each day. Statistical significance determined by repeated measures ANOVA, * P < 0.05.

Article Snippet: On day 14, αCRTh2 antibody coated microbeads (Cat. #130‐091‐274, Miltenyi) were used to isolate CRTh2‐expressing cells.

Techniques: Control, Expressing, Cell Culture, Comparison

Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐25 initiates Th2 differentiation of human CD4 + T cells and influences expression of its own receptor

doi: 10.1002/iid3.87

Figure Lengend Snippet: Regulation of IL‐25R on Th2 cells. Differentiated CRTh2 + Th2 cells were cultured with IL‐2 in the presence or absence of IL‐25 and stained for (A) IL‐25R ( n = 5, three independently differentiated cell lines) or assessed for (B) IL‐25R mRNA levels ( n = 3). (C) Surface expression of IL‐25R on CRTh2 + Th2 cells treated (24 h) with a CRTh2 agonist (DK‐PGD 2 ) or IL‐2 ( n = 4). Statistical significance determined by paired t test within each time point (A, B) or between conditions (C), * P < 0.05.

Article Snippet: On day 14, αCRTh2 antibody coated microbeads (Cat. #130‐091‐274, Miltenyi) were used to isolate CRTh2‐expressing cells.

Techniques: Cell Culture, Staining, Expressing

Journal: Immunity, Inflammation and Disease

Article Title: Interleukin‐25 initiates Th2 differentiation of human CD4 + T cells and influences expression of its own receptor

doi: 10.1002/iid3.87

Figure Lengend Snippet: IL‐25 slows re‐expression of CRTh2 on Th2 cells following TCR activation. (A) Differentiated CRTh2 + Th2 cells cultured with IL‐2, αCD3 and αCD28 exhibited time dependent loss of surface CRTh2 expression ( n = 4, two independently differentiated cell lines). (B–G) CRTh2 + Th2 cells were cultured with IL‐2, αCD3 and αCD28 (24 h) and then placed with IL‐2 in the presence or absence of IL‐25 (another 24 h). % of cells expressing (B) CRTh2, (D) CD62L or (F) CCR4 and mean fluorescent intensity (MFI) for (C) CRTh2, (E) CD62L and (G) CCR4 were quantified ( n = 6, two independently differentiated lines). Statistical significance was determined by paired t test (B–G), * P < 0.05.

Article Snippet: On day 14, αCRTh2 antibody coated microbeads (Cat. #130‐091‐274, Miltenyi) were used to isolate CRTh2‐expressing cells.

Techniques: Expressing, Activation Assay, Cell Culture

Journal: Cancer cell

Article Title: The allergy mediator histamine confers resistance to immunotherapy in cancer patients via activation of the macrophage histamine receptor H1.

doi: 10.1016/j.ccell.2021.11.002

Figure Lengend Snippet: Figure 4. Histamine-HRH1 activation promotes VISTA membrane localization (A) Percentages of IFN-g+ CD8+ T cells co-cultured with EO771 TCM-treated WT or HRH1/ BMDMs in direct contact or separately in transwells (n = 6, t test). (B) Heatmap depicting relative expression of co-stimulatory/inhibitory molecules on naive or TCM-treated WT or HRH1/ BMDMs measured by flow cytometry. The MFI of each molecule was normalized to the MFI of the naive WT group. (C) Percentages of IFN-g+ CD8+ T cells co-cultured with EO771 TCM-treated WT or HRH1/ BMDMs pre-treated with IgG, anti-TIM-3 (10 mg/mL), and/or anti- VISTA (10 mg/mL) antibodies (n = 3–4, one-way ANOVA). (D) Flow cytometry analysis of VISTA+ TAMs (CD45+ CD11b+ F4/80+) from EO771 and B16-GM tumors growing in WT versus HRH1/ mice, and vehicle-treated versus FEXO-treated WT mice (n = 5–6, t test). (E) Western blot analysis of total VISTA and membrane VISTA expression on naive or TCM-treated WT or HRH1/ BMDMs. b-actin and CD11b were used as loading controls. (F) Percentages of VISTA+ BMDMs after treatment with 10 mM BAPTA-AM (an intracellular calcium chelator) or 1 mg/mL ionomycin-Ca2+ (n = 3, t test). All in vitro experiments were performed at least twice. Mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Recombinant Murine IFN-g PeproTech Cat # 315-05 Recombinant Murine IL-4 PeproTech Cat # 214-14 Fixable Viability Dye eFluor 450 eBioscience Cat # 65-0863-14 CD11b MicroBeads Miltenyi Biotec Cat # 130-049-601 Cell-ID Cisplatin Fluidigm Cat # 201064 Critical commercial assays Histamine ELISA kits Enzo Life Sciences Cat # ENZ-KIT140-0001 iScript cDNA Synthesis Kit Bio-rad Cat # 1708891 Intracellular Fixation & Permeabilization Buffer Set eBioscience Cat # 88-8824-00 Maxima SYBR Green qPCR Master Mix Thermo Fisher Scientific Cat# K0253 QIAGEN Plasmid Maxi Kit QIAGEN Cat# 12163 ImmunoSpot Kits Cellular Technology Limited Mouse IFN-g Single-Color ELISPOT Deposited data RNA-seq with mouse BMDM (Raw and analyzed data) This paper GEO: GSE161484 RNA-seq with human melanomas Hugo et al., 2016 GEO: GSE78220 Single-cell RNA-seq of human melanoma Jerby-Arnon et al., 2018 GEO: GSE115978 Single-cell RNA-seq with CD45+ immune cells isolated from EO771 tumors This paper SRA Run Selector Accession: PRJNA756466 Experimental models: Cell lines 293T ATCC Cat# ACS-4500; RRID:CVCL_4V93 4T1 ATCC Cat# CRL-2539; RRID:CVCL_0125 B16/BL6 ATCC Cat# CRL-6475; RRID:CVCL_0159 BT20 ATCC Cat# HTB-19; RRID:CVCL_0178 BT549 ATCC Cat# HTB-122; RRID:CVCL_1092 CT26 ATCC Cat# CRL-2638; RRID:CVCL_7256 EMT6 ATCC Cat# CRL-2755; RRID:CVCL_1923 EO771 ATCC Cat# CRL-3461; RRID:CVCL_GR23 HCC1806 ATCC Cat# CRL-2335; RRID:CVCL_1258 HS578T ATCC Cat# HTB-126; RRID:CVCL_0332 L929 ATCC Cat# CCL-1; RRID:CVCL_0462 LLC1 ATCC Cat# CRL-1642; RRID:CVCL_4358 MDA-MB-231 ATCC Cat# HTB-26; RRID:CVCL_0062 MDA-MB-435 ATCC Cat# HTB-129; RRID:CVCL_0622 MDA-MB-436 ATCC Cat# HTB-130; RRID:CVCL_0623 THP-1 ATCC Cat# TIB-202; RRID:CVCL_0006

Techniques: Activation Assay, Membrane, Cell Culture, Expressing, Cytometry, Flow Cytometry, Western Blot, In Vitro