Journal: The Journal of Experimental Medicine

Article Title: CD161 (NKR-P1A) Costimulation of CD1d-dependent Activation of Human T Cells Expressing Invariant Vα24JαQ T Cell Receptor α Chains

doi:

Figure Lengend Snippet: Expression of NK cell–associated proteins by Vα24 invt T cells. Representative FACS ® analysis of DN Vα24 invt T cell clones DN2.D5 ( top ) and DN1.10B3 ( bottom ) ∼3 wk after stimulation with PHA and irradiated feeders. T cells were stained with mAb against the antigens shown and with anti-IgG FITC conjugate before gating on live cells. Left to right , Normal mouse serum ( outline ) and CD28 ( solid line ); CD69; CD94; CD161.

Article Snippet: Kurrle, Boehringwerke, Marburg, Germany); anti-CD3 (SPV-T3b [provided by Dr. H. Spits, Netherlands Cancer Center, Amsterdam, Netherlands] and OKT3 [American Type Culture Collection, Rockville, MD]); anti-CD4 (OKT4; American Type Culture Collection); anti-CD8α (OKT8; American Type Culture Collection); anti-CD8β (2ST8-5H7; provided by Dr. E. Reinherz, Dana-Farber Cancer Institute, Boston, MA); anti-CD28 (9.3 [gift of Dr. J. Hansen, Hutchinson Cancer Center, Seattle, WA] and CD28.2 [ PharMingen , San Diego, CA]); anti-CD69 (FN50; PharMingen ); anti-CD94 mAb (DX-22 [gift of Dr. L. Lanier, DNAX, Palo Alto, CA], HP-3D9 [ PharMingen ], HP-3B1 [Coulter Corp., Miami, FL], and IgA NKH3 [provided by Drs. M. Robertson, Indiana University Medical Center, Indianapolis, IN, and J. Ritz, Dana-Farber Cancer Institute, Boston, MA]); anti-CD161 (DX-1 and DX12 [also provided by Dr. Lanier], HP-3G10 [provided by Dr. M. Lopez-Botet, Hospital de la Princesa, Madrid, Spain], and 191.B8 [gift of Dr. A. Poggi, Instituto Nazionale per la Ricerca sul Cancro, Genoa, Italy]); anti-p40 (NKTA255; provided by Dr. A. Poggi); p38 (C1.7; Coulter Corp.), Fifth Leukocyte Workshop, NK Section, mAb against killer inhibitory receptors p58 (GL183, EB6, CH-L, and HP-3E4) and p70 (DX-9; provided by Dr. Lanier); anti-MHC class I (W6/32; American Type Culture Collection), anti-CD1b (4A7.6.5, IgG 2a ; gift of Dr. D. Olive, Institut Nationale de la Santé et de la Recherche Médicale, Marseilles, France), and isotype control mAbs (P3, IgG 1 ; MPC-11, IgG 2b ; American Type Culture Collection); rat anti–murine NK1.1 (PK136; PharMingen ); and normal mouse and rat sera. p56 Lck was detected with a mixture of antibodies (#42 rabbit serum [provided by Drs. B. Krise and J.

Techniques: Expressing, Clone Assay, Irradiation, Staining

Journal: The Journal of Experimental Medicine

Article Title: CD161 (NKR-P1A) Costimulation of CD1d-dependent Activation of Human T Cells Expressing Invariant Vα24JαQ T Cell Receptor α Chains

doi:

Figure Lengend Snippet: Costimulation of Vα24 invt T cells by NK locus–encoded C-type lectins. CD161 + CD94 + Vα24 invt T cell clone DN2.D6 (10 5 /well, 2–4 wk after restimulation, except where shown) was stimulated with limiting quantities of plate-bound CD3 mAb (OKT3 at 1 μg/ ml without PMA, or 0.1 μg/ml with PMA at 1 ng/ml as shown) and/or plate-bound or soluble accessory mAb (10 μg/ml, except as shown). For soluble mAb, cross-linking anti-IgG was also added at molar equivalence. T cell proliferation measured by [ 3 H]thymidine incorporation (cpm) was determined in triplicate at 72 h (SEM shown). Similar results were obtained by IL-4 and IFN-γ cytokine ELISA at 48 h (not shown). ( A and B ) Titration of anti-CD3 mAb in the presence of 10 μg/ml potential accessory molecule antibodies without ( A ) or with ( B ) PMA. ( C ) Titration of soluble and plate-bound CD161 costimulatory mAb 191.B8 with 0.1 μg/ml anti-CD3 mAb and PMA. ( D ) 1.0 μg/ml anti-CD3 mAb and/or 10 μg/ml CD161 and/or CD94 costimulation; no PMA. ( E ) Partially rested T cells at only 9 d after restimulation, with 1.0 μg/ml anti-CD3 mAb, 10 μg/ml CD161 and/or CD94 costimulation, and no PMA, or with PHA only.

Article Snippet: Kurrle, Boehringwerke, Marburg, Germany); anti-CD3 (SPV-T3b [provided by Dr. H. Spits, Netherlands Cancer Center, Amsterdam, Netherlands] and OKT3 [American Type Culture Collection, Rockville, MD]); anti-CD4 (OKT4; American Type Culture Collection); anti-CD8α (OKT8; American Type Culture Collection); anti-CD8β (2ST8-5H7; provided by Dr. E. Reinherz, Dana-Farber Cancer Institute, Boston, MA); anti-CD28 (9.3 [gift of Dr. J. Hansen, Hutchinson Cancer Center, Seattle, WA] and CD28.2 [ PharMingen , San Diego, CA]); anti-CD69 (FN50; PharMingen ); anti-CD94 mAb (DX-22 [gift of Dr. L. Lanier, DNAX, Palo Alto, CA], HP-3D9 [ PharMingen ], HP-3B1 [Coulter Corp., Miami, FL], and IgA NKH3 [provided by Drs. M. Robertson, Indiana University Medical Center, Indianapolis, IN, and J. Ritz, Dana-Farber Cancer Institute, Boston, MA]); anti-CD161 (DX-1 and DX12 [also provided by Dr. Lanier], HP-3G10 [provided by Dr. M. Lopez-Botet, Hospital de la Princesa, Madrid, Spain], and 191.B8 [gift of Dr. A. Poggi, Instituto Nazionale per la Ricerca sul Cancro, Genoa, Italy]); anti-p40 (NKTA255; provided by Dr. A. Poggi); p38 (C1.7; Coulter Corp.), Fifth Leukocyte Workshop, NK Section, mAb against killer inhibitory receptors p58 (GL183, EB6, CH-L, and HP-3E4) and p70 (DX-9; provided by Dr. Lanier); anti-MHC class I (W6/32; American Type Culture Collection), anti-CD1b (4A7.6.5, IgG 2a ; gift of Dr. D. Olive, Institut Nationale de la Santé et de la Recherche Médicale, Marseilles, France), and isotype control mAbs (P3, IgG 1 ; MPC-11, IgG 2b ; American Type Culture Collection); rat anti–murine NK1.1 (PK136; PharMingen ); and normal mouse and rat sera. p56 Lck was detected with a mixture of antibodies (#42 rabbit serum [provided by Drs. B. Krise and J.

Techniques: Enzyme-linked Immunosorbent Assay, Titration

Journal: Journal for Immunotherapy of Cancer

Article Title: Restoration of miR-340 controls pancreatic cancer cell CD47 expression to promote macrophage phagocytosis and enhance antitumor immunity

doi: 10.1136/jitc-2019-000253

Figure Lengend Snippet: Overexpression of miR-340 promotes phagocytosis of tumor cells by macrophages. Panc02 cells overexpressing miR-NC or miR-340 were incubated with mouse peritoneal cavity-derived macrophages or BMDMs for 4 hours, stained with F4/80-APC antibody and analyzed by flow cytometry. Phagocytosis was described as the percentage of F4/80 + GFP + phagocytosed cancer cells by F4/80 + macrophages. (A, C) Representative plots show the percentage of F4/80 + GFP + macrophages phagocytosing cancer cells among F4/80 + peritoneal cavity-derived macrophages and BMDMs. (B, D) Statistical analysis of phagocytosis of pancreatic cancer cells by both peritoneal cavity-derived macrophages and BMDMs. (E) Representative plots show the percentage of F4/80 + GFP + macrophages phagocytosing cancer cells among F4/80 + BMDMs treated with isotype control or CD47 blocking antibody. (F) Statistical analysis of phagocytosis of Panc02 cells by BMDMs with or without CD47 blocking by flow cytometry. Panc02 cells overexpressing miR-NC or miR-340 were incubated with BMDMs for 4 hours, stained with F4/80-APC antibody and analyzed by immunofluorescence staining. (G) Statistical analysis of phagocytosis of Panc02 cells by BMDMs treated with isotype control or CD47 blocking antibody for immunofluorescence staining. (H) Immunofluorescence staining shows representative images of mouse BMDMs engulfing Panc02 cells with miR-NC and miR-340 overexpression with or without CD47 blocking. The white arrows point to macrophages that phagocytose cancer cells. Macrophages were stained red (F4/80 + ), cancer cells were green (GFP + ) and nuclei were blue (DAPI + ). Magnification: 100×. The error bars were shown as mean±SEM and the data were analyzed by two-tailed, unpaired t-test. *P<0.05; **p<0.01 and ***p<0.001. The experiments were performed three times with similar results. BMDM, bone marrow-derived macrophages.

Article Snippet: Tumors were allowed to grow for 7–10 days and treated with isotype control IgG (200 μg/day intravenously, Clone No. MPC-11, BioXcell) or anti-mouse CD47 mAb (200 μg/day intravenously, Clone No. MIAP301, BioXcell) in vivo .

Techniques: Over Expression, Incubation, Derivative Assay, Staining, Flow Cytometry, Control, Blocking Assay, Immunofluorescence, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Restoration of miR-340 controls pancreatic cancer cell CD47 expression to promote macrophage phagocytosis and enhance antitumor immunity

doi: 10.1136/jitc-2019-000253

Figure Lengend Snippet: High expression of miR-340 notably enhances antitumor immunity. An orthotopic model of PDAC was built with Panc02 cells stably overexpressing miR-NC or miR-340, and the tumor and peripheral immune microenvironment were analyzed by flow cytometry 3 weeks after orthotopic implantation (n=7 per group). (A) The frequency and MFI of M1-like (CD11b + F4/80 + MHC-II + ) macrophages in the tumors from mice bearing Panc02 cells with miR-NC and miR-340 overexpression. (B) The ratio of M1-like and M2-like macrophages in the tumor microenvironment of tumor-bearing mice. (C) The proportion and MFI of M1-like (CD11b + F4/80 + MHC-II + ) macrophages in the spleens from mice bearing Panc02 cells with miR-NC and miR-340 overexpression. (D) The ratio of M1-like and M2-like macrophages in the spleens of tumor-bearing mice. (E) The frequencies of CD4 + T cells in the tumor microenvironment, (F) the frequencies of CD8 + T cells in the tumor microenvironment, (G) the frequencies of CD4 + T cells in the spleen, (H) the frequencies of CD8 + T cells in the spleen, (I) the frequencies of CD4 + T cells in the blood and (J) the frequencies of CD8 + T cells in the blood in mice bearing Panc02 cells with miR-NC and miR-340 overexpression. (K) The orthotopic model of PDAC was established, and tumor-bearing mice were treated with isotype control mouse IgG or an anti-mouse CD47 antibody. Representative plots and statistical analysis showed the percentage of F4/80 + GFP + macrophages phagocytosing cancer cells among macrophages with or without anti- CD47 antibody blockade in vivo . The error bars were shown as mean±SEM and the data were analyzed by two-tailed, unpaired t-test. *P<0.05; **p<0.01 and ***p<0.001. PDAC, pancreaticductal adenocarcinoma.

Article Snippet: Tumors were allowed to grow for 7–10 days and treated with isotype control IgG (200 μg/day intravenously, Clone No. MPC-11, BioXcell) or anti-mouse CD47 mAb (200 μg/day intravenously, Clone No. MIAP301, BioXcell) in vivo .

Techniques: Expressing, Stable Transfection, Flow Cytometry, Over Expression, Control, In Vivo, Two Tailed Test

Journal: PLoS Pathogens

Article Title: FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure

doi: 10.1371/journal.ppat.1004894

Figure Lengend Snippet: ( A ) Sorted transitional cells (CD19 + CD10 + ), CD20 + atMBCs (IgG + CD21 - CD27 - CD19 + ), classical MBCs (IgG + CD21 + CD27 + CD19 + ), and CD27 - plasmablasts (CD20 - IgG + CD21 - CD27 - CD19 + ) were cultured on anti-IgG ELISpot plates for 18 h without additional stimulation. ( B ) Gating strategy and frequencies of CD38 hi cells in the above plasmablast gating strategy.

Article Snippet: Isotype controls included mouse IgG1 (clone MOPC-21) (Tonbo Biosciences), and IgG2a (clone MOPC-173) and IgG2b (clone MPC-11) (BioLegend).

Techniques: Cell Culture, Enzyme-linked Immunospot

Journal: PLoS Pathogens

Article Title: FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure

doi: 10.1371/journal.ppat.1004894

Figure Lengend Snippet: ( A ) Surface expression, expressed as median fluorescence intensity (MFI), of CD85d, CD120b, CD360, CD11c, and IgG (BCR) on IgG + atMBCs and IgG + classical MBCs. Lines between symbols denote MBC subsets from the same subject. Wedges represent means. ( B ) Labeling of SVT2 mouse fibroblast cell lines that express full-length human FCRL4 or FCRL5 protein by monoclonal antibodies 2A6, 1A3, and 7D11. ( C ) Labeling of human atMBCs with monoclonal antibodies 2A6, 1A3, and 7D11. ( D ) Isotype-subtracted MFI of FCRL family member expression (“Net MFI”) on atypical and classical MBCs from highly P . falciparum -exposed individuals. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05; **, p < 0.01

Article Snippet: Isotype controls included mouse IgG1 (clone MOPC-21) (Tonbo Biosciences), and IgG2a (clone MOPC-173) and IgG2b (clone MPC-11) (BioLegend).

Techniques: Expressing, Fluorescence, Labeling, Bioprocessing

Journal: PLoS Pathogens

Article Title: FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure

doi: 10.1371/journal.ppat.1004894

Figure Lengend Snippet: ( A ) Representative plot showing heterogeneous expression of FCRL5 on IgG + atypical MBCs. Individual FCRL5 + atypical and classical MBC frequencies were determined using gates set with a “fluorescence minus one” control with IgG2b isotype control antibody. ( B ) Proportion of atypical and classical IgG + MBCs expressing FCRL5. Reported frequencies have been subtracted for isotype-labeled background. ( C ) Median fluorescence intensity (MFI) of surface markers on FCRL5 + vs. FCRL5 - atMBCs and FCRL5 + vs. FCRL5 - classical MBCs. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Isotype controls included mouse IgG1 (clone MOPC-21) (Tonbo Biosciences), and IgG2a (clone MOPC-173) and IgG2b (clone MPC-11) (BioLegend).

Techniques: Expressing, Fluorescence, Control, Labeling

Journal: PLoS Pathogens

Article Title: FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure

doi: 10.1371/journal.ppat.1004894

Figure Lengend Snippet: Sorted FCRL5 + and FCRL5 - , atypical (CD20 + CD21 - CD27 - IgG + ) and classical (CD20 + CD21 + CD27 + IgG + ) MBCs were stimulated for 4 days with CpG, F(ab’) 2 anti-IgG, and autologous T cells. IgG-secreting cells were detected by IgG ELISpot and are reported as the number of IgG secreting cells per 1000 cells sorted on day 0. ASC, antibody-secreting cells. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05.

Article Snippet: Isotype controls included mouse IgG1 (clone MOPC-21) (Tonbo Biosciences), and IgG2a (clone MOPC-173) and IgG2b (clone MPC-11) (BioLegend).

Techniques: Enzyme-linked Immunospot