Journal: The Journal of Experimental Medicine

Article Title: Chimeric allergen receptor regulatory T cells suppress birch pollen allergic airway inflammation

doi: 10.1084/jem.20252201

Figure Lengend Snippet: CAlleR 8 Tregs do not regulate immunoglobulin production or affect the T cell and dendritic cell compartments in the treatment model. (A) Representative flow cytometry plots showing the gating strategy for neutrophils, monocytes, and eosinophils in the lungs. The expression of SiglecF in lung eosinophils is also shown. (B) Specific anti-Bet v1 IgG1 detection in mouse serum by ELISA ( n = 7–9 from two independent experiments). (C) Total serum IgE quantification by ELISA ( n = 6–10 from two independent experiments). (D) Gating strategy and cumulative data on the percentage of DCs detected in the lungs (gated on CD11c + CD11b + F480 − MHC-II + ) ( n = 7–10 from two independent experiments). (E and F) Cumulative data of the percentage of the different T populations observed in (E) the lungs and in (F) the mLN (CD3 + T cells, CD4 + , CD8 + , and Tregs) and representative plots and cumulative percentage of CD4 + T cells expressing the activation marker CD44 ( n = 7–10 from two independent experiments). (G) Representative flow cytometry plots showing intracellular expression of Th2 cytokines in lung CD4 + T cells. Data are represented as the mean ± SEM, and P values were calculated with one-way ANOVA with Tukey’s multiple comparison test in B, C, and D and with two-way ANOVA with Tukey’s multiple comparison test in E and F. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. DCs, dendritic cells.

Article Snippet: After blocking, cells were washed and extracellular staining was performed for 30 min at 4°C with the following antibodies: anti-mouse CD3 (145-2C11), PE-Cy7 (1:250 dilution) and APC-Cy7 (1:100 dilution), 552774; BD Biosciences (RRID:AB_394460) and 561042 (RRID:AB_2034003), respectively; anti-mouse CD4 (RM4-5), PerCP-Cy5.5 (1:300 dilution) and APC (1:250 dilution), 561090 and 553051; BD Biosciences, respectively; anti-mouse CD8a (53-6.7), FITC (1:100 dilution) and Pacific Blue (1:400 dilution), 553030 and 558106; BD Biosciences, respectively; anti-mouse CD25 (PC61.5), PE (1:200 dilution), 12-0251-83; Invitrogen; anti-mouse CD25 (PC61), PerCP-Cy5.5 (1:250 dilution), 551071; BD Biosciences; anti-mouse CD44 (IM7), FITC (1:100 dilution), 553133; BD Biosciences; anti-mouse CD45 (30-F11), PE-Cy7 (1:250), 552848; BD Biosciences; anti-mouse CD45.2 (104), AF700 (1:100 dilution), 109821; BioLegend; anti-mouse CD69 (H1.2F3), PE (1:400 dilution), 553237; BD Biosciences; anti-mouse CD86 (GL1), PE (1:250 dilution), 553692; BD Biosciences; anti-mouse CD90.1 (Thy1.1; OX-7), PerCP-Cy5.5 (1:250 dilution), 557266; BD Biosciences; anti-mouse CD90.1 (Thy1.1; REA838), FITC (1:500 dilution), 130-112-872; Miltenyi Biotech; anti-mouse CD11c (HL3), APC (1:100 dilution), 550261; BD Biosciences; anti-mouse CD11c (N418), APC-Cy7 (1:600 dilution), 117323; BioLegend; anti-mouse CD11b (M1/70), PECy7 (1:600 dilution), 561098; BD Biosciences; anti-mouse MHC-II (M5/114.15.2), AF700 (1:1,000 dilution), 107621; BioLegend; anti-mouse F480 (BM8), PerCP (1:100 dilution), 123126; BioLegend; anti-mouse Ly6C (HK1.4), Pacific Blue (1:1,000 dilution), 128013; BioLegend; anti-mouse SiglecF (E50-2440), PE (1:100 dilution), 562068; BD Biosciences; anti-mouse Ly6G (1A8), PerCP Cy5.5 (1:100 dilution), 560602; BD Biosciences; biotinylated rBet v1 (1 μg/ml); streptavidin, APC (1:500 dilution) and PE (1:1,000 dilution), 554067 and 554061; BD Biosciences, respectively; Aqua (Live/Dead), AmCyan (1:1,000 dilution), L34957 ; Invitrogen; DAPI (Live/Dead), Invitrogen (1:2,500 dilution), D1306.

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Activation Assay, Marker, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Chimeric allergen receptor regulatory T cells suppress birch pollen allergic airway inflammation

doi: 10.1084/jem.20252201

Figure Lengend Snippet: CAlleR 8 Tregs do not prevent immunoglobulin production or the activation of T cells and DCs. (A) Specific anti-Bet v1 IgG1 detection in mouse serum by ELISA ( n = 4–7 from two independent experiments). (B) Total serum IgE quantification by ELISA ( n = 7–13 from two independent experiments). (C) Cumulative data on the percentage of DCs detected in the lungs (gated on CD11c + CD11b + F480 − MHC-II + ) ( n = 4–7 from two independent experiments). (D and E) Cumulative data of the percentage of the different T populations observed in (D) the lungs and in (E) the mLN (CD3 + T cells, CD4 + , CD8 + , and Tregs) ( n = 5–9 from two independent experiments). Data are represented as the mean ± SEM, and P values were calculated with one-way ANOVA with Tukey’s multiple comparison test in A–C and with two-way ANOVA with Tukey’s multiple comparison test in D and E. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. DCs, dendritic cells.

Article Snippet: After blocking, cells were washed and extracellular staining was performed for 30 min at 4°C with the following antibodies: anti-mouse CD3 (145-2C11), PE-Cy7 (1:250 dilution) and APC-Cy7 (1:100 dilution), 552774; BD Biosciences (RRID:AB_394460) and 561042 (RRID:AB_2034003), respectively; anti-mouse CD4 (RM4-5), PerCP-Cy5.5 (1:300 dilution) and APC (1:250 dilution), 561090 and 553051; BD Biosciences, respectively; anti-mouse CD8a (53-6.7), FITC (1:100 dilution) and Pacific Blue (1:400 dilution), 553030 and 558106; BD Biosciences, respectively; anti-mouse CD25 (PC61.5), PE (1:200 dilution), 12-0251-83; Invitrogen; anti-mouse CD25 (PC61), PerCP-Cy5.5 (1:250 dilution), 551071; BD Biosciences; anti-mouse CD44 (IM7), FITC (1:100 dilution), 553133; BD Biosciences; anti-mouse CD45 (30-F11), PE-Cy7 (1:250), 552848; BD Biosciences; anti-mouse CD45.2 (104), AF700 (1:100 dilution), 109821; BioLegend; anti-mouse CD69 (H1.2F3), PE (1:400 dilution), 553237; BD Biosciences; anti-mouse CD86 (GL1), PE (1:250 dilution), 553692; BD Biosciences; anti-mouse CD90.1 (Thy1.1; OX-7), PerCP-Cy5.5 (1:250 dilution), 557266; BD Biosciences; anti-mouse CD90.1 (Thy1.1; REA838), FITC (1:500 dilution), 130-112-872; Miltenyi Biotech; anti-mouse CD11c (HL3), APC (1:100 dilution), 550261; BD Biosciences; anti-mouse CD11c (N418), APC-Cy7 (1:600 dilution), 117323; BioLegend; anti-mouse CD11b (M1/70), PECy7 (1:600 dilution), 561098; BD Biosciences; anti-mouse MHC-II (M5/114.15.2), AF700 (1:1,000 dilution), 107621; BioLegend; anti-mouse F480 (BM8), PerCP (1:100 dilution), 123126; BioLegend; anti-mouse Ly6C (HK1.4), Pacific Blue (1:1,000 dilution), 128013; BioLegend; anti-mouse SiglecF (E50-2440), PE (1:100 dilution), 562068; BD Biosciences; anti-mouse Ly6G (1A8), PerCP Cy5.5 (1:100 dilution), 560602; BD Biosciences; biotinylated rBet v1 (1 μg/ml); streptavidin, APC (1:500 dilution) and PE (1:1,000 dilution), 554067 and 554061; BD Biosciences, respectively; Aqua (Live/Dead), AmCyan (1:1,000 dilution), L34957 ; Invitrogen; DAPI (Live/Dead), Invitrogen (1:2,500 dilution), D1306.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Chimeric allergen receptor regulatory T cells suppress birch pollen allergic airway inflammation

doi: 10.1084/jem.20252201

Figure Lengend Snippet: CAlleR 8 murine Treg tissue–specific homing. (A) Experimental design of murine CAlleR 8 GFP + and CAR A2 GFP + Treg production. (B) Representative plots of Treg phenotype and transduction efficiency after 7 days of expansion. (C) Experimental design of the allergic airway inflammation mouse model and adoptive Treg transfer. (D) Representative images of CAlleR 8 and CAR A2 Tregs in the mLNs, lung, and spleen of mice exposed to PBS or BPE. White bars represent the scale of 20 μm, and small images show fluorescence channels of the magnified region outlined in white squares. (E) Cumulative data of detection of GFP + cells/mm2 in each organ ( n = 8 in the lung and spleen and n = 5–8 in mLN from four mice with image duplicates). (F) Cumulative data of the percentage of GFP + cells interacting with CD11c + , B220 + APCs or with none of them (no CD11c + /B220 + contact) for each organ ( n = 8 in the lung and spleen and n = 5–8 in the mLN from four mice with image duplicates). In E, data are represented by the median ± interquartile range and in F by the mean ± SEM. P values were calculated with Kruskal–Wallis and Dunn’s multiple comparison test in E and with two-way ANOVA with Tukey’s test in F. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: After blocking, cells were washed and extracellular staining was performed for 30 min at 4°C with the following antibodies: anti-mouse CD3 (145-2C11), PE-Cy7 (1:250 dilution) and APC-Cy7 (1:100 dilution), 552774; BD Biosciences (RRID:AB_394460) and 561042 (RRID:AB_2034003), respectively; anti-mouse CD4 (RM4-5), PerCP-Cy5.5 (1:300 dilution) and APC (1:250 dilution), 561090 and 553051; BD Biosciences, respectively; anti-mouse CD8a (53-6.7), FITC (1:100 dilution) and Pacific Blue (1:400 dilution), 553030 and 558106; BD Biosciences, respectively; anti-mouse CD25 (PC61.5), PE (1:200 dilution), 12-0251-83; Invitrogen; anti-mouse CD25 (PC61), PerCP-Cy5.5 (1:250 dilution), 551071; BD Biosciences; anti-mouse CD44 (IM7), FITC (1:100 dilution), 553133; BD Biosciences; anti-mouse CD45 (30-F11), PE-Cy7 (1:250), 552848; BD Biosciences; anti-mouse CD45.2 (104), AF700 (1:100 dilution), 109821; BioLegend; anti-mouse CD69 (H1.2F3), PE (1:400 dilution), 553237; BD Biosciences; anti-mouse CD86 (GL1), PE (1:250 dilution), 553692; BD Biosciences; anti-mouse CD90.1 (Thy1.1; OX-7), PerCP-Cy5.5 (1:250 dilution), 557266; BD Biosciences; anti-mouse CD90.1 (Thy1.1; REA838), FITC (1:500 dilution), 130-112-872; Miltenyi Biotech; anti-mouse CD11c (HL3), APC (1:100 dilution), 550261; BD Biosciences; anti-mouse CD11c (N418), APC-Cy7 (1:600 dilution), 117323; BioLegend; anti-mouse CD11b (M1/70), PECy7 (1:600 dilution), 561098; BD Biosciences; anti-mouse MHC-II (M5/114.15.2), AF700 (1:1,000 dilution), 107621; BioLegend; anti-mouse F480 (BM8), PerCP (1:100 dilution), 123126; BioLegend; anti-mouse Ly6C (HK1.4), Pacific Blue (1:1,000 dilution), 128013; BioLegend; anti-mouse SiglecF (E50-2440), PE (1:100 dilution), 562068; BD Biosciences; anti-mouse Ly6G (1A8), PerCP Cy5.5 (1:100 dilution), 560602; BD Biosciences; biotinylated rBet v1 (1 μg/ml); streptavidin, APC (1:500 dilution) and PE (1:1,000 dilution), 554067 and 554061; BD Biosciences, respectively; Aqua (Live/Dead), AmCyan (1:1,000 dilution), L34957 ; Invitrogen; DAPI (Live/Dead), Invitrogen (1:2,500 dilution), D1306.

Techniques: Transduction, Fluorescence, Comparison

Journal: Molecular Therapy Oncology

Article Title: Engineered Salmonella -mediated c-di-AMP delivery activates STING to remodel the tumor microenvironment

doi: 10.1016/j.omton.2026.201185

Figure Lengend Snippet: SL disA increases the antigen-presentation capacity of dendritic cells (A) IFN-β levels in BMDC supernatants from media, SL, SL disA , and SL disA +H151 groups. (B, C) BMDCs double-stained with MHC II and CD11c were analyzed by flow cytometry (B), and CD11c + MHCII + cells are shown in the statistical graph (C). Data were expressed as mean ± SEM, n = 3. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA with Tukey’s multiple comparisons tests.

Article Snippet: Cells were subsequently stained with fluorochrome-conjugated antibodies targeting surface markers, including FITC anti-mouse CD11c (clone N418, Elabscience Biotechnology Co., Ltd.) and PE-Cy7 anti-mouse MHC II (clone M5/114, Elabscience Biotechnology Co., Ltd.), and analyzed using a CytoFLEX cytometer (Beckman Coulter, Brea, CA, USA).

Techniques: Immunopeptidomics, Staining, Flow Cytometry