anti-cd137 (apc, 4b4-1  (Thermo Fisher)

Journal: bioRxiv

Article Title: Th1 cells are critical tissue organizers of myeloid-rich perivascular activation niches

doi: 10.1101/2024.11.24.625073

Figure Lengend Snippet: (A) Schematic of dermal perivascular niches (PVN) in the inflamed dermis (left) and IV-MPM image (right) of the distribution of CXCL10 + PVNs in the dermis of REX CXCL10-BFP X CCR2-RFP +/- mice following OVC/CFA immunization. (B-F) 2.5 x 10 6 OT-II and 2D2 or OT-II and SMARTA Th1 cells were co-transferred i.v. into OVA/CFA-immunized REX3 reporter mice, and their dynamic movement and position relative to the CXCL10 + PVNs determined on d5 of immunization by IV-MPM of the inflamed ear dermis. (B) Images of distribution of antigen and non-antigen specific Th1 cells with respect to PVN. Scale bar, 80 µm. Representative data from three independent experiments, > 8 imaging volumes; >30 tracks per plot. (C and D), Track mean speed (C) and arrest coefficient (D) of OT-II and 2D2 or SMARTA Th1 cells inside of the PVNs. (E) Migratory paths (x-y projections) of OT-II, 2D2, and SMARTA Th1 cells with respect to CXCL10 + PVN. (F) CXCL10 + cell:T-cell contact time according to antigen specificity, 30 min IV-MPM. (G and H) Representative histograms (G) and frequency (H) of CD137 + , CD153 + , and PD1 + Th1s in Non-Niche and Niche regions of the OVA/CFA-immunized dermis. PVNs were identified in the inflamed dermis on d5 p.i. via IV-MPM, mapped, and biopsied for ex vivo analysis via flow cytometry. Gated on CD4 + /Live/Lymphocytes/CD45.1 + . Statistical analysis performed using Mann-Whitney test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Antibodies used include: anti-CD45 BUV395 (1:500, 30-F11, BD Biosciences), anti-CD4 BUV805 (1:500, GK1.5, BD Biosciences), anti-PD1 BV605 (1:100, J43, BD Biosciences), anti-CD137 PE-Cy7 (1:100, 17B5, Invitrogen), anti-CD153 APC (1:100, RM153, Invitrogen), anti-PD1 PE-Cy7 (1:100, J43, Invitrogen), anti-CD137 APC (1:100 CD137, 17B5, Invitrogen), anti-CD64 BV605 (1:200, X54-5/7.1, Biolegend), anti-CD11c BV711 (1:450, N418, Biolegend), anti-CD19 BV786 (1:100, 1D3, BD Biosciences), anti-F4/80 PE-Cy5 (1:200, BM8, Invitrogen), anti-CD88 anti-APC (1:800, 20/70, Biolegend), anti-MHC-II APC-ef780 (1:2000, M5/114.15.2, eBioscience), anti-CD26 PE-Cy7 (1:50, H194-112, Biolegend), anti-XCR1 BV650 (1:200, ZET, Biolegend), anti-Ly6G FITC (1:500, 1A8, Biolegend), anti-CD3e Biotin (1:400, 145-2C11, eBioscience), anti-CD4 BV605 (1:200, RM4-5, Biolegend), anti-CD4 (1:200, RM4-4, Biolegend), anti-CD45.1 BUV563 (1:250, A20, BDbiosciences), and anti-CXCR6 Biotin (1:100, SA051D1, Biolegend).

Techniques: Imaging, Ex Vivo, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Th1 cells are critical tissue organizers of myeloid-rich perivascular activation niches

doi: 10.1101/2024.11.24.625073

Figure Lengend Snippet: (A) Schematic of Kaede Th1 time-stamp model. OT-II + Kaede + Th1 cells were transferred into OVA/CFA-immunized REX3 mice. On d5, p.i., all Th1 cells in the inflamed ear were photoconverted to Kaede-red on exposure to 405 nm light. 24 h later, the ear tissue contained Recent recruits, Kaede-green Th1 cells (< 24h in ear), and Previous tissue recruits, Kaede-red Th1 cells (> 24h in ear). (B) IV-MPM images (left) of the distribution of OT-II + Th1 cells relative to the PVN immediately post-photoconversion (0h) or 24h after photoconversion (24h): Kaede-green, Recent recruits; Kaede-red, Previous recruits. Right, quantitation of the frequency of Kaede-green and Kaede-red within and outside of the PVN, 24h after photoconversion. Representative data from three independent experiments. Statistics by Mann-Whitney, **p<0.01. (C) Kaede OT-II Th1 cells were transferred into WT albino animals immunized with either OVA/CFA or KLH/CFA. The inflamed ear was photoconverted 24h prior to harvest on d6 p.i. Representative histograms of frequency (CD137) or MFI (PD-1) by OT-II Th1 cells prior to tissue entry (lymph node, LN), 24h post tissue entry (Recent, Kaede-green) and after persisting within the inflamed dermis for >24h (Previous, Kaede-red), with (OVA) or without (KLH) cognate antigen. Representative data from four independent experiments. (D-E) Spatiotemporal tracking of PVN-specific Th1 cells. OT-II PA-GFP + Th1 cells were transferred into OVA/CFA-immunized mice. On d5, single CXCL10 + PVNs were identified per mouse using IV-MPM and 830nm laser applied to activate GFP within the PVN localized Th1s. 24 h later, the original photoactivation site was reidentified, example image (D), and distances of PA-GFP + Th1 cells from the PVN calculated (E). Representative data from three independent experiments.

Article Snippet: Antibodies used include: anti-CD45 BUV395 (1:500, 30-F11, BD Biosciences), anti-CD4 BUV805 (1:500, GK1.5, BD Biosciences), anti-PD1 BV605 (1:100, J43, BD Biosciences), anti-CD137 PE-Cy7 (1:100, 17B5, Invitrogen), anti-CD153 APC (1:100, RM153, Invitrogen), anti-PD1 PE-Cy7 (1:100, J43, Invitrogen), anti-CD137 APC (1:100 CD137, 17B5, Invitrogen), anti-CD64 BV605 (1:200, X54-5/7.1, Biolegend), anti-CD11c BV711 (1:450, N418, Biolegend), anti-CD19 BV786 (1:100, 1D3, BD Biosciences), anti-F4/80 PE-Cy5 (1:200, BM8, Invitrogen), anti-CD88 anti-APC (1:800, 20/70, Biolegend), anti-MHC-II APC-ef780 (1:2000, M5/114.15.2, eBioscience), anti-CD26 PE-Cy7 (1:50, H194-112, Biolegend), anti-XCR1 BV650 (1:200, ZET, Biolegend), anti-Ly6G FITC (1:500, 1A8, Biolegend), anti-CD3e Biotin (1:400, 145-2C11, eBioscience), anti-CD4 BV605 (1:200, RM4-5, Biolegend), anti-CD4 (1:200, RM4-4, Biolegend), anti-CD45.1 BUV563 (1:250, A20, BDbiosciences), and anti-CXCR6 Biotin (1:100, SA051D1, Biolegend).

Techniques: Quantitation Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: SARS-CoV-2 spike-specific nasal-resident CD49a + CD8 + memory T cells exert immediate effector functions with enhanced IFN-γ production

doi: 10.1038/s41467-024-52689-5

Figure Lengend Snippet: a Summary of the experimental design ( b ) UMAP projections of 10,003 CD137 + T cells from vaccinees without infection ( n = 4) and with BTI ( n = 5), colored according to each cell type. c Feature plots showing the average normalized expressions of marker genes and ADTs in each cell cluster. d Gating strategy to identify the CD49a + CD8 + and CD49a − CD8 + T-cell subsets based on expression levels of ITGA1 transcripts and CD8 protein. Red dots indicate each subset. e Violin plots showing gene expression levels among the CD49a + CD8 + and CD49a − CD8 + T-cell subsets. Statistical analysis was performed using the Mann–Whitney U test ( e ).

Article Snippet: Subsequently, these nasal cells were stained with PE-conjugated anti-CD3 antibody (BD Biosciences), APC-conjugated CD137 antibody (Miltenyi Biotec), and LIVE/DEAD near-IR fluorescent reactive dye (Invitrogen).

Techniques: Infection, Marker, Expressing, Gene Expression, MANN-WHITNEY