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 immunology

Article Title: Fyn-ADAP signaling via Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells

doi: 10.1038/ni.2708

Figure Lengend Snippet: ( a ) Flow cytometry analyses of H60 (top) and CD137L (bottom) expression in stably-transfected EL4 cells. Open histogram: background expression of H60 or CD137L in parental EL4 cells. Grey histogram: ligand expression in EL4-H60 lo or EL4-CD137L lo . Black histogram: ligand expression in EL4-H60 hi or EL4-CD137L hi . ( b ) Mean percent cytotoxicity with standard deviation of IL-2-cultured WT NK cells following co-culture with indicated target cells. E/T denotes Effector to Target ratio. ( c ) Intracellular IFN-γ staining in IL-2-cultured CD3 + NK1.1 − T and CD3 − NK1.1 + NK cells from WT mice either left unstimulated or stimulated with plate-bound anti-CD137, anti-CD3 mAb alone or in combination for 12 h. ( d ) Quantitative analyses of IFN-γ, GM-CSF, CCL3, CCL4, and CCL5 from WT NK cells following 18 h co-culture with indicated target cells using bioplex assays. * P <0.05, ** P <0.01, *** P <0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance ( b, d ). Data in a is a representative of three independent experiments. Data in b and d are from five and three WT mice, respectively (mean ± s.d. in b and d ).

Article Snippet: IL-2-cultured, Fc-blocked (anti-CD16/CD32 mAb 2.4G2, BD, Franklin Lakes, NJ) NK cells were activated with plate-bound mitogenic antibodies against CD137 (17B5), NKG2D (A10) or Ly49D (4E5) for 18 h. Culture supernatants were analyzed in a Bioplex assay (Bio-Rad, Richmond, CA).

Techniques: Flow Cytometry, Expressing, Stable Transfection, Transfection, Standard Deviation, Cell Culture, Co-Culture Assay, Staining, Two Tailed Test

Journal: Nature immunology

Article Title: Fyn-ADAP signaling via Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells

doi: 10.1038/ni.2708

Figure Lengend Snippet: ( a ) Immunoblot analyses of Lck and Fyn following immunoprecipitation of NKG2D and CD137 receptors in unstimulated, IL-2-expanded WT NK cells. ( b ) Quantitative analysis of IFN-γ production from NK cells after pre-incubation with Lck inhibitor, C8863. Open squares: C8863-treated NK cells with anti-NKG2D mAb activation; filled circles: C8863-treated NK cells with anti-CD137 mAb activation; open circles: C8863-treated NK cells with IL-12 and IL-18 activation. Cytokine production in the vehicle (DMSO)-treated NK cells in response to anti-NKG2D, anti-CD137 mAbs or IL-12 and IL-18 are indicated as closed or half-filled squares. ( c ) Immunoblot for Lck in NK cells following mock, Lck-specific and scrambled siRNA transfections. Fold change in Lck expression was determined by densitometry following normalization with actin. ( d ) Bar diagram represents the mean percent cytotoxicity of NK cells transfected with mock (open), Lck-specific siRNA (black) or scrambled siRNA (grey) against indicated target cells. E/T denotes Effector to Target ratio. ( e ) Quantitative analyses of cytokine and chemokine production, following activation with anti-NKG2D (left), anti-CD137 (middle) mAbs, and IL-12 and IL-18 (right) in WT NK cells transfected with mock, Lck-specific or scrambled siRNA. * P <0.05, ** P <0.01, *** P <0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance ( b ); two-tailed, paired ( d, e ). Data in a and c are representative of three independent experiments. Data in b , d, and e are mean values from three independent experiments consisting of three mice in each (mean ± s.d. in b , d, e ).

Article Snippet: IL-2-cultured, Fc-blocked (anti-CD16/CD32 mAb 2.4G2, BD, Franklin Lakes, NJ) NK cells were activated with plate-bound mitogenic antibodies against CD137 (17B5), NKG2D (A10) or Ly49D (4E5) for 18 h. Culture supernatants were analyzed in a Bioplex assay (Bio-Rad, Richmond, CA).

Techniques: Western Blot, Immunoprecipitation, Incubation, Activation Assay, Transfection, Expressing, Two Tailed Test

Journal: Nature immunology

Article Title: Fyn-ADAP signaling via Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells

doi: 10.1038/ni.2708

Figure Lengend Snippet: ( a ) Immunoblot analyses of phosphorylated (pTyr530) and total Fyn in NK cells that were left unstimulated or stimulated with plate-bound anti-NKG2D or anti-CD137 mAbs. Fold change in tyrosine phosphorylation is shown. ( b ) Cytotoxicity of WT (open circles) and Fyn −/− (closed circles) NK cells to H60 + and CD137L + target cells. E/T denotes Effector to Target ratio. ( c ) Quantitative analyses of cytokines and chemokines following activation with plate-bound anti-NKG2D or anti-CD137 mAbs or with a combination of IL-12 and IL-18 for 18 h. ( d ) Quantitative analyses of cytokines and chemokines from WT and Fyn −/− NK cells following co-culture with EL4, EL4-H60 hi or EL4-CD137L hi target cells for 18 h. * P <0.05, ** P <0.01, *** P <0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance ( b–d ). Data in a is a representative of three independent experiments. Data in b , c and d are from seven, five and two mice per genotype from four, four, and two independent experiments, respectively (mean ± s.d. in b , c and d ).

Article Snippet: IL-2-cultured, Fc-blocked (anti-CD16/CD32 mAb 2.4G2, BD, Franklin Lakes, NJ) NK cells were activated with plate-bound mitogenic antibodies against CD137 (17B5), NKG2D (A10) or Ly49D (4E5) for 18 h. Culture supernatants were analyzed in a Bioplex assay (Bio-Rad, Richmond, CA).

Techniques: Western Blot, Phospho-proteomics, Activation Assay, Co-Culture Assay, Two Tailed Test

Journal: Nature immunology

Article Title: Fyn-ADAP signaling via Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells

doi: 10.1038/ni.2708

Figure Lengend Snippet: ( a ) Whole cell lysates from WT NK cells, unstimulated or activated with plate-bound anti-NKG2D and anti-CD137 mAbs were immunoprecipitated for Lck or Fyn and probed with anti-PI(3)K-p85α antibody. Fyn and Lck were also analyzed following their respective immunoprecipitation. Fold induction was determined by densitometry following normalization to the respective protein that was immunoprecipitated. ( b ) Immunoblot analysis of phosphorylated and total PI(3)K-p85α following activation of WT NK cells with plate-bound anti-NKG2D or anti-CD137 mAb. Fold induction was determined by densitometry, following normalization to total PI(3)K-p85α. ( c ) Mean percent cytotoxicity of NK cells from WT (open) or PI(3)K-p110δ (D910A) (filled) against indicated target cells. E/T denotes Effector to Target ratio. ( d ) Quantitative analyses of cytokines and chemokines produced by WT (open) or PI(3)K-p110δ (D910A) (filled) NK cells following activation with plate-bound antibodies to NKG2D and CD137 receptors. Cytokines and chemokines produced by NK cells in response to IL-12 and IL-18 stimulation are shown (right). Immunoblot indicating the ( e ) phosphorylated and total PLC-γ2 or ( f ) phosphorylated PKC-θ following activation of WT NK cells. Fold induction in ( e ) and ( f ) was determined by densitometry, following normalization to total PLC-γ2 and actin, respectively. * P <0.05, ** P <0.01, *** P <0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance ( c, d ). Data in c and d are from four and five mice per genotype from three independent experiments, respectively (mean ± s.d. in c and d ). Data in a , b , e , and f are representative of three independent experiments

Article Snippet: IL-2-cultured, Fc-blocked (anti-CD16/CD32 mAb 2.4G2, BD, Franklin Lakes, NJ) NK cells were activated with plate-bound mitogenic antibodies against CD137 (17B5), NKG2D (A10) or Ly49D (4E5) for 18 h. Culture supernatants were analyzed in a Bioplex assay (Bio-Rad, Richmond, CA).

Techniques: Immunoprecipitation, Western Blot, Activation Assay, Produced, Two Tailed Test

Journal: Nature immunology

Article Title: Fyn-ADAP signaling via Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells

doi: 10.1038/ni.2708

Figure Lengend Snippet: ( a ) Immunoblot showing the MAP3K7 expression in IL-2-cultured NK cells obtained from poly (I:C)-treated floxed Map3k7 fx/fx and Mx1-Cre + Map3k7 fx/fx mice. Fold change in MAP3K7 expression was determined by densitometry following normalization with actin. ( b ) Mean percent cytotoxicity with standard deviation of NK cells obtained from poly I:C-treated Map3k7 fx/fx mice (open circle) or from poly (I:C) untreated (open square) and treated (filled square) Mx1-Cre + Map3k7 fx/fx mice against the indicated targets. E/T denotes Effector to Target ratio. ( c ) Quantitative analyses of cytokines and chemokines produced by NK cells derived from poly (I:C)-treated Map3k7 fx/fx (open) and Mx1-Cre + Map3k7 fx/fx (filled) mice following 18 h of activation with plate-bound anti-CD137 mAb (left) or with IL-12 and IL-18 (right). * P <0.05, ** P <0.01, *** P <0.001; NS, not significant versus WT (Student’s t-test; two-tailed, two sample unequal variance versus NK cells from Mx1-Cre + Map3k7 fx/fx mice that were untreated with poly (I:C) ( b ); two-tailed, two sample equal variance versus NK cells from Map3k7 fx/fx mice that were treated with poly (I:C) ( c) . Data in a is a representative of three independent experiments. Data in b, c are from three mice per genotype from three independent experiments (mean ± s.d.).

Article Snippet: IL-2-cultured, Fc-blocked (anti-CD16/CD32 mAb 2.4G2, BD, Franklin Lakes, NJ) NK cells were activated with plate-bound mitogenic antibodies against CD137 (17B5), NKG2D (A10) or Ly49D (4E5) for 18 h. Culture supernatants were analyzed in a Bioplex assay (Bio-Rad, Richmond, CA).

Techniques: Western Blot, Expressing, Cell Culture, Standard Deviation, Produced, Derivative Assay, Activation Assay, Two Tailed Test

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 immunology

Article Title: Fyn-ADAP signaling via Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells

doi: 10.1038/ni.2708

Figure Lengend Snippet: ( a ) Flow cytometry analyses of H60 (top) and CD137L (bottom) expression in stably-transfected EL4 cells. Open histogram: background expression of H60 or CD137L in parental EL4 cells. Grey histogram: ligand expression in EL4-H60 lo or EL4-CD137L lo . Black histogram: ligand expression in EL4-H60 hi or EL4-CD137L hi . ( b ) Mean percent cytotoxicity with standard deviation of IL-2-cultured WT NK cells following co-culture with indicated target cells. E/T denotes Effector to Target ratio. ( c ) Intracellular IFN-γ staining in IL-2-cultured CD3 + NK1.1 − T and CD3 − NK1.1 + NK cells from WT mice either left unstimulated or stimulated with plate-bound anti-CD137, anti-CD3 mAb alone or in combination for 12 h. ( d ) Quantitative analyses of IFN-γ, GM-CSF, CCL3, CCL4, and CCL5 from WT NK cells following 18 h co-culture with indicated target cells using bioplex assays. * P <0.05, ** P <0.01, *** P <0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance ( b, d ). Data in a is a representative of three independent experiments. Data in b and d are from five and three WT mice, respectively (mean ± s.d. in b and d ).

Article Snippet: Antibodies for NK1.1 (PK136), CD3ε (17A2, 145-2C11), NKG2D (A10), CD137 (17b5), CD137L (TKS-1), anti-human IFN-g (MG1.2), and anti-human CD107a (1D4B) were obtained from e-Bioscience (San Diego, CA).

Techniques: Flow Cytometry, Expressing, Stable Transfection, Transfection, Standard Deviation, Cell Culture, Co-Culture Assay, Staining, Two Tailed Test

Journal: Nature immunology

Article Title: Fyn-ADAP signaling via Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells

doi: 10.1038/ni.2708

Figure Lengend Snippet: ( a ) Immunoblot analyses of Lck and Fyn following immunoprecipitation of NKG2D and CD137 receptors in unstimulated, IL-2-expanded WT NK cells. ( b ) Quantitative analysis of IFN-γ production from NK cells after pre-incubation with Lck inhibitor, C8863. Open squares: C8863-treated NK cells with anti-NKG2D mAb activation; filled circles: C8863-treated NK cells with anti-CD137 mAb activation; open circles: C8863-treated NK cells with IL-12 and IL-18 activation. Cytokine production in the vehicle (DMSO)-treated NK cells in response to anti-NKG2D, anti-CD137 mAbs or IL-12 and IL-18 are indicated as closed or half-filled squares. ( c ) Immunoblot for Lck in NK cells following mock, Lck-specific and scrambled siRNA transfections. Fold change in Lck expression was determined by densitometry following normalization with actin. ( d ) Bar diagram represents the mean percent cytotoxicity of NK cells transfected with mock (open), Lck-specific siRNA (black) or scrambled siRNA (grey) against indicated target cells. E/T denotes Effector to Target ratio. ( e ) Quantitative analyses of cytokine and chemokine production, following activation with anti-NKG2D (left), anti-CD137 (middle) mAbs, and IL-12 and IL-18 (right) in WT NK cells transfected with mock, Lck-specific or scrambled siRNA. * P <0.05, ** P <0.01, *** P <0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance ( b ); two-tailed, paired ( d, e ). Data in a and c are representative of three independent experiments. Data in b , d, and e are mean values from three independent experiments consisting of three mice in each (mean ± s.d. in b , d, e ).

Article Snippet: Antibodies for NK1.1 (PK136), CD3ε (17A2, 145-2C11), NKG2D (A10), CD137 (17b5), CD137L (TKS-1), anti-human IFN-g (MG1.2), and anti-human CD107a (1D4B) were obtained from e-Bioscience (San Diego, CA).

Techniques: Western Blot, Immunoprecipitation, Incubation, Activation Assay, Transfection, Expressing, Two Tailed Test

Journal: Nature immunology

Article Title: Fyn-ADAP signaling via Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells

doi: 10.1038/ni.2708

Figure Lengend Snippet: ( a ) Immunoblot analyses of phosphorylated (pTyr530) and total Fyn in NK cells that were left unstimulated or stimulated with plate-bound anti-NKG2D or anti-CD137 mAbs. Fold change in tyrosine phosphorylation is shown. ( b ) Cytotoxicity of WT (open circles) and Fyn −/− (closed circles) NK cells to H60 + and CD137L + target cells. E/T denotes Effector to Target ratio. ( c ) Quantitative analyses of cytokines and chemokines following activation with plate-bound anti-NKG2D or anti-CD137 mAbs or with a combination of IL-12 and IL-18 for 18 h. ( d ) Quantitative analyses of cytokines and chemokines from WT and Fyn −/− NK cells following co-culture with EL4, EL4-H60 hi or EL4-CD137L hi target cells for 18 h. * P <0.05, ** P <0.01, *** P <0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance ( b–d ). Data in a is a representative of three independent experiments. Data in b , c and d are from seven, five and two mice per genotype from four, four, and two independent experiments, respectively (mean ± s.d. in b , c and d ).

Article Snippet: Antibodies for NK1.1 (PK136), CD3ε (17A2, 145-2C11), NKG2D (A10), CD137 (17b5), CD137L (TKS-1), anti-human IFN-g (MG1.2), and anti-human CD107a (1D4B) were obtained from e-Bioscience (San Diego, CA).

Techniques: Western Blot, Activation Assay, Co-Culture Assay, Two Tailed Test

Journal: Nature immunology

Article Title: Fyn-ADAP signaling via Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells

doi: 10.1038/ni.2708

Figure Lengend Snippet: ( a ) Whole cell lysates from WT NK cells, unstimulated or activated with plate-bound anti-NKG2D and anti-CD137 mAbs were immunoprecipitated for Lck or Fyn and probed with anti-PI(3)K-p85α antibody. Fyn and Lck were also analyzed following their respective immunoprecipitation. Fold induction was determined by densitometry following normalization to the respective protein that was immunoprecipitated. ( b ) Immunoblot analysis of phosphorylated and total PI(3)K-p85α following activation of WT NK cells with plate-bound anti-NKG2D or anti-CD137 mAb. Fold induction was determined by densitometry, following normalization to total PI(3)K-p85α. ( c ) Mean percent cytotoxicity of NK cells from WT (open) or PI(3)K-p110δ (D910A) (filled) against indicated target cells. E/T denotes Effector to Target ratio. ( d ) Quantitative analyses of cytokines and chemokines produced by WT (open) or PI(3)K-p110δ (D910A) (filled) NK cells following activation with plate-bound antibodies to NKG2D and CD137 receptors. Cytokines and chemokines produced by NK cells in response to IL-12 and IL-18 stimulation are shown (right). Immunoblot indicating the ( e ) phosphorylated and total PLC-γ2 or ( f ) phosphorylated PKC-θ following activation of WT NK cells. Fold induction in ( e ) and ( f ) was determined by densitometry, following normalization to total PLC-γ2 and actin, respectively. * P <0.05, ** P <0.01, *** P <0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance ( c, d ). Data in c and d are from four and five mice per genotype from three independent experiments, respectively (mean ± s.d. in c and d ). Data in a , b , e , and f are representative of three independent experiments

Article Snippet: Antibodies for NK1.1 (PK136), CD3ε (17A2, 145-2C11), NKG2D (A10), CD137 (17b5), CD137L (TKS-1), anti-human IFN-g (MG1.2), and anti-human CD107a (1D4B) were obtained from e-Bioscience (San Diego, CA).

Techniques: Immunoprecipitation, Western Blot, Activation Assay, Produced, Two Tailed Test

Journal: Nature immunology

Article Title: Fyn-ADAP signaling via Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells

doi: 10.1038/ni.2708

Figure Lengend Snippet: ( a ) Immunoblot showing the MAP3K7 expression in IL-2-cultured NK cells obtained from poly (I:C)-treated floxed Map3k7 fx/fx and Mx1-Cre + Map3k7 fx/fx mice. Fold change in MAP3K7 expression was determined by densitometry following normalization with actin. ( b ) Mean percent cytotoxicity with standard deviation of NK cells obtained from poly I:C-treated Map3k7 fx/fx mice (open circle) or from poly (I:C) untreated (open square) and treated (filled square) Mx1-Cre + Map3k7 fx/fx mice against the indicated targets. E/T denotes Effector to Target ratio. ( c ) Quantitative analyses of cytokines and chemokines produced by NK cells derived from poly (I:C)-treated Map3k7 fx/fx (open) and Mx1-Cre + Map3k7 fx/fx (filled) mice following 18 h of activation with plate-bound anti-CD137 mAb (left) or with IL-12 and IL-18 (right). * P <0.05, ** P <0.01, *** P <0.001; NS, not significant versus WT (Student’s t-test; two-tailed, two sample unequal variance versus NK cells from Mx1-Cre + Map3k7 fx/fx mice that were untreated with poly (I:C) ( b ); two-tailed, two sample equal variance versus NK cells from Map3k7 fx/fx mice that were treated with poly (I:C) ( c) . Data in a is a representative of three independent experiments. Data in b, c are from three mice per genotype from three independent experiments (mean ± s.d.).

Article Snippet: Antibodies for NK1.1 (PK136), CD3ε (17A2, 145-2C11), NKG2D (A10), CD137 (17b5), CD137L (TKS-1), anti-human IFN-g (MG1.2), and anti-human CD107a (1D4B) were obtained from e-Bioscience (San Diego, CA).

Techniques: Western Blot, Expressing, Cell Culture, Standard Deviation, Produced, Derivative Assay, Activation Assay, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: CD137 expression correlated with intrarenal lymphangiogenesis and fibrosis in IgAN. (A) IHC analysis of CD137 expression (40×); Masson's trichome staining showing collagen deposition(40×); (B) Representative images and double-positive analyses for immunofluorescence-labeled D2-40 (green) and CD137 (red) in IgA patients (n=85) and controls (n=5). Scale bar, 20µm. (C) Relationships between CD137 + lymphatic vessels in the interstitium and Serum urea nitrogen, Serum Creatinine, Serum uric acid, proteinuria, Plasma albumin, HB and eGFR. HB, hemoglobin; eGFR, estimated glomerular filtration rate; (D) The difference of density of lymphatic vessels and CD137 + lymphatic vessels between patients with different T and S grades. T, tubular atrophy/interstitial fibrosis; S, segmental glomerulosclerosis.

Article Snippet: The renal fibrosis model was induced by unilateral ureteral obstruction (UUO) as previously reported. anti-CD137 blocking antibody (4-1BB, Clone:17B5, Bio X Cell, Lebanon NH) were injected (i.p.) at 10 mg/kg/dose twice weekly.

Techniques: Expressing, Staining, Immunofluorescence, Labeling, Clinical Proteomics, Filtration

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: Demographic and clinical parameters between patients with D2-40 and CD137 + D2-40 expression.

Article Snippet: The renal fibrosis model was induced by unilateral ureteral obstruction (UUO) as previously reported. anti-CD137 blocking antibody (4-1BB, Clone:17B5, Bio X Cell, Lebanon NH) were injected (i.p.) at 10 mg/kg/dose twice weekly.

Techniques: Expressing, Clinical Proteomics

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: Histopathologic features from renal biopsies between patients with low or high levels of CD137 + D2-40 expression.

Article Snippet: The renal fibrosis model was induced by unilateral ureteral obstruction (UUO) as previously reported. anti-CD137 blocking antibody (4-1BB, Clone:17B5, Bio X Cell, Lebanon NH) were injected (i.p.) at 10 mg/kg/dose twice weekly.

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: CD137 is increased in the obstructed kidney. (A) IHC staining showing LYVE-1 and CD137 expression in the sham control and UUO mice (400×). The expression levels of Prox-1 and LYVE-1 were detected by Western blot (B), and the expression levels of CD137 were detected by real-time PCR (C). (D) Immunofluorescence staining showing CD137 (red) expression in UUO kidneys in the interstitium on days 7 and 14 and colocalization with LYVE-1 (green) original magnification, ×400. n = 6 per group. The error bars represent the SEM. ***P < 0.001.

Article Snippet: The renal fibrosis model was induced by unilateral ureteral obstruction (UUO) as previously reported. anti-CD137 blocking antibody (4-1BB, Clone:17B5, Bio X Cell, Lebanon NH) were injected (i.p.) at 10 mg/kg/dose twice weekly.

Techniques: Immunohistochemistry, Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: Demographic and clinical parameters between patients with low or high levels of CD137 + D2-40 expression.

Article Snippet: The renal fibrosis model was induced by unilateral ureteral obstruction (UUO) as previously reported. anti-CD137 blocking antibody (4-1BB, Clone:17B5, Bio X Cell, Lebanon NH) were injected (i.p.) at 10 mg/kg/dose twice weekly.

Techniques: Expressing, Clinical Proteomics

Journal: Nature Communications

Article Title: Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting

doi: 10.1038/s41467-023-40115-1

Figure Lengend Snippet: a Representative H&E and CD3 IHC in solid tumor masses collected from the upper omental region of i.p. ID8-mSTn tumor-bearing mice treated with indicated T cells. Scale bar = 100 µm. b Representative flow cytometric analysis of tumor cells (CD3-CD45- double negative) in peritoneal ascites. c Quantification of tumor cells (CD3− CD45− double negative) and d immune subsets (CD45+, CD3+, CD11b+, and NK+) as cells/mL in peritoneal ascites. e Representative flow cytometric analysis of percent CAR T cells (CD3+ CD19t+) and f quantification counts of CD4+ and CD8+ CAR T cells/mL in peritoneal ascites. g Quantification of mean fluorescent intensity (MFI) of CD137+ in CAR T cells in peritoneal ascites. h Quantification of percent CD62L+ CD44+ (Tcm) in both CAR+ and CAR− T cells in peritoneal ascites. i Quantification of myeloid cell counts (Ly6G+, Ly6C+, Ly6G−/C− double negative tumor-associated macrophages (TAM) and CD11c+ CD103+ dendritic cells (DC) as cells/mL in peritoneal ascites gated from total CD11b+ cells. Representative flow cytometric analysis of percent ( j ) and quantification of MFI ( k ) on CD103+ MHC Class II+ double positive DC in peritoneal ascites. All analyses represent data collected from ascites of ID8-mSTn tumor-bearing mice at 7 days post treatment, n = 3/group. All data are presented as mean values ±SEM. P values indicate differences between TAG72-CAR and TAG72-CAR/mbIL12 using a two-tailed Student’s t test.

Article Snippet: Antibodies against CD3 (BD Biosciences, Cat: 563109, Clone: SK7), CD4 (BD Biosciences, Cat: 340443, Clone: SK3), CD8 (BD Biosciences, Cat: 347313, Clone: SK1), CD19 (BD Biosciences, Cat: 557835, Clone: SJ25C1), mouse CD45 (BioLegend, Cat: 103145, Clone: 30-F11), CD45 (BD Biosciences, Cat: 555484, Clone: 2D1), CD69 (BD Biosciences, Cat: 341652, Clone: L78), CD137 (BD Biosciences, Cat: 555956, Clone: 4B4-1), mouse CD137 (Thermofisher, Cat: 25-1371-82, Clone: 17B5), mouse NK1.1 (BioLegend, Cat: 108733, Clone: PK163), mouse PD-1 (Thermofisher, Cat: 69-9985-80, Clone: J43), mouse LAG3 (BioLegend, Cat: 125227, Clone: C9B7W), mouse TIM-3 (BioLegend, Cat: 119704, Clone: RMT3-23), mouse CD11b (BioLegend, Cat: 101237, Clone: M1/70), CD44 (BioLegend, Cat: 103010, Clone: IM7), CD62L (BioLegend, Cat: 104412, Clone: MEL-14), CD80 (BD Biosciences, Cat: 740130, Clone: 16-10A1), mouse I-A/I-E (MHC Class II) (Thermofisher, Cat: 64-5321-80, Clone: M5/114.15.2), mouse CD274 (PD-L1) (BioLegend, Cat: 124312, Clone: 10 F.962), Ly6C (BioLegend, Cat: 128029, Clone: HK1.4), mouse CD11c (BioLegend, Cat: 117316, Clone: N418), mouse Ly6G (Biolegend, Cat: 127623, Clone: 1A8), mouse CD103 (BioLegend, Cat: 121426, Clone: 2E7), mouse F4/80 (BioLegend, Cat: 123127, Clone: BM8), mouse IL-12/IL-23 p40 (Thermofisher, Cat: 12-7123-41, Clone:17.8), Ep-CAM/CD326 (BioLegend, Clone: 9C4) (Cat: 324208), human IL-12 p40/p70 (BD Biosciences, Cat: 554575, Clone: C11:5), biotinylated Protein L (GenScript USA, Cat: M00097) (25), TAG72 (Novus Biologicals), Clone, Cat: NBP2-33128, muCC49), Donkey Anti-Rabbit Ig (Invitrogen, Cat: A-31573), Goat Anti-Mouse Ig (BD Biosciences, Cat: 550589), and streptavidin (BD Biosciences, Cat: 349023, 1:20 dilution) were used.

Techniques: Two Tailed Test