Journal: bioRxiv

Article Title: AML/T cell interactomics uncover correlates of patient outcomes and the key role of ICAM1 in T cell killing of AML

doi: 10.1101/2023.09.21.558911

Figure Lengend Snippet: A. Elimination efficiency of U937 cell line after CRISPR-Cas9 mediated knockout (KO) of selected genes by CD4 IL10 cells; each dot represents a CD4 IL10 donor (n=3). WT = wild type; U937 WT: positive control, K562 WT: negative control. Representative FACS plots from one donor against U937- ITGB2 -KO and U937- ICAM1 -KO are shown on the right; numbers indicate percentage in AML gate. B. Degranulation of CD8 + T cells, expressed as % CD8 + CD107a + cells within live singlet CD3 + T cells, against U937 WT and U937- ICAM1 -KO cells; each circle represents a CD8 + T cell donor (n = 7). Representative FACS plots from one donor are shown on the right; numbers indicate percent degranulating CD8 + T cells. C. Elimination efficiency of U937 WT and U937- ICAM1 -KO cells by CD8 + T cells (n = 10). Wilcoxon test, p < 0.05. Representative FACS plots from one donor are shown on the right; numbers indicate percentage in AML gate. D. Elimination efficiency of U937 cell line 24 hours after treatment with LFA-1 inhibitors at indicated concentrations; each line represents a CD4 IL10 donor (n = 3); circles are color-coded by the inhibitor as indicated. Dunn’s post hoc test results are shown, following Friedman ANOVA ( p < 0.0001). E. CD8 + T cells were co-cultured for 24hr with indicated target cells; activation was measured as percent of CD69 + CD25 +/- cells within the live CD8 + T cell gate. F. Elimination efficiency of indicated target cell lines and primary AML cells by CD8 + T cells after 3 days of co-culture in the absence or presence of LFA-1 inhibitor BMS587101 (10 μM). G. Spearman correlation of percent activation of CD8 + T cells and the elimination efficiency of target cells. H. Timeline of mice injections; n = 10 mice per group. I. Graphs showing total flux obtained from imaging data on days 8 and 12. 1-way ANOVA with Bonferroni multiple comparison test; p = n.s. at day 8, p = 0.0075 at day 12.

Article Snippet: After 1 hour, brefeldin A and monensin (BioLegend, CA, USA; used as per manufacturer’s instructions) were added to each well, cells were cultured for 4 more hours, stained with a viability dye and anti-CD3 and anti-CD8 mAb ( Table S2 ), and fixed with 4% formaldehyde until acquisition by flow cytometry.

Techniques: CRISPR, Knock-Out, Positive Control, Negative Control, Cell Culture, Activation Assay, Co-Culture Assay, Imaging, Comparison

Journal: Journal of Neuroinflammation

Article Title: Astrocytes evoke a robust IRF7-independent type I interferon response upon neurotropic viral infection

doi: 10.1186/s12974-023-02892-w

Figure Lengend Snippet: High diversity of infiltrating immune cells in brains of IRF7-deficient mice upon LGTV infection. WT and Irf7 −/− mice were infected intraperitoneally with 10 4 FFU LGTV ( n = 6) and immune cells were isolated from brains for flow cytometric characterization. A representative gating strategy (A-K). FSC and SSC gating of immune cells ( A ). Life cell selection ( B ) and separation from doublets ( C ). Neutrophil granulocytes Ly6G + CD11b + ( D ), differentiation of remaining mononuclear cells by CD45 and CD11b expression ( E ). Identification of NK1.1 + and NK1.1 − CD3 + cells within CD45 + CD11b − population ( F ). NK1.1 − CD3 + cells were gated for CD4 and CD8 ( G ). Remaining NK1.1 − CD3 − cells from ( F ) were separated in B220 + CD11c − B cells ( H ). B220 + CD11c + cells ( H ) which were further assessed for MHC class II (MHCII) and Ly6C expression show plasmacytoid dendritic cells (pDCs) ( I ). CD45 + CD11b + cells from ( E ) were gated for CD45 and CD206 separating brain-resident microglia from border-associated macrophages (BAMs) and infiltrating myeloid cells ( J ). MHCII and CD11c expression of myeloid cells to distinguish conventional dendritic cells (cDCs) and monocytes/macrophages ( K ). Monocytes and macrophages characterization by CD11b and Ly6C expression: Ly6C hi inflammatory monocytes, Ly6Clow macrophages ( L ). Clustering of Infiltrating immune Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP), constructing a framework of cells while preserving the global structure of sample. Cells within clusters were subsequently identified by overlaying previously gated populations ( M ) and compared between groups of LGTV-infected WT and Irf7 −/− mice on day 7 post-infection ( N ). Stacked bar charts display the overall frequency of immune cell subsets within the brains of mice on day 0 and 7 post-infection ( O )

Article Snippet: Thereafter, cells were further stained with the following fluorochrome-conjugated antibodies against cell surface markers in FACS buffer (PBS containing 2% fetal bovine serum and 0.1% sodium azide): eFluor™ 450-CD45 (clone 30-F11), FITC-CD4 (clone GK1.5), PerCP/Cy5.5-Ly6C (clone HK1.4), PE/Cy5-Ly6G (clone 1A8), PE/Cy7-CD11c (clone N418), and APC-CD11b (clone M1/70) (all purchased from eBioscience™); Brilliant Violet™ 510-B220 (CD45R) (clone RA3-6B2), Brilliant Violet™ 605-NK1.1 (clone PK136), Brilliant Violet™ 711-MHC Class II (I-A/I-E) (clone M5/114.15.2), PE-CD3 (clone 17A2), PE/Dazzle594™-CD206 (clone C068C2), AlexaFluor ® 700-CD8a (clone 53–6.7) (all purchased from BioLegend).

Techniques: Infection, Isolation, Selection, Expressing, Preserving

Journal: bioRxiv

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1101/2023.09.18.557956

Figure Lengend Snippet: A murine melanoma xenograft model was established in NSG mice by subcutaneous inoculation of 2 × 10 6 A549 tumor cells on the right flank. When the average tumor size reached 200-300 mm 3 , experimental mice were treated with a single dose of either 1 × 10 7 HER2-MR-CAR T cells, 1 × 10 7 HER2-CAR T cells, or Non-transduced T cells (NT). (A) Seven days after treatment, tumor tissues were dissected, and a single-cell suspension was prepared, with the cell wash buffer then subjected to an ADA activity assay to measure inosine concentrations. *P=0.00847 for HER2-MR-CAR verse HER2-CAR. (B) After seven days of treatment, single cell suspensions were prepared from each tumor tissue, and CD3+ cells were sorted using flow cytometry. The number of sorted cells was quantified and presented (n=3). *P=0.000177 for HER2-MR-CAR verse HER2-CAR. (C-D) Single cell suspensions from tumor tissues were stained with antibodies against CD3, CD8, CCR5, IFN-γ, Granzyme B, and Perforin and analyzed by flow cytometry. The resulting data were presented as a flow cytometry dot plot, and the gated cell populations were quantified and presented in a column figure. The figure indicated P value. (E-F) Sorted CD3+ cells were co-cultured with A549 tumor cells at an E:T ratio of 1:1 overnight. After incubation, the culture medium was collected and subjected to an ELISA to measure the expression of IFN-γ. To determine the tumor-killing capacity, a LDH assay was performed. The figure indicated P value. (G) Sorted CD3+ cells were co-cultured with A549 tumor cells in a transwell culture plate to assess the migration capacity of the CD3+ cells. *P=0.00053 for HER2-MR-CAR verse HER2-CAR. (H) Sorted CD3+ cells were labeled with CFSE and then co-cultured with A549 tumor cells at an E:T ratio of 1:1 for 48 hours. After incubation, the cells were collected, stained with anti-CD3 antibody, and analyzed by flow analysis to determine their proliferation.

Article Snippet: The antibodies used for T-cell phenotyping and cytokine production analyses are: CD3-BV785 (BioLegend, 317329), CD8-Pacific Blue (BioLegend, 344717), CCR2-BUV395 (BD, 747854), CCR5-FITC (BioLegend, 313705), CD26-FITC (BD, 555436), ADA1-PE (Santa Cruz, sc-28346), PD-1-Alexa flour 647 (BD, 566851), IL2-BV711 (BD #563946), IFN gama-PE-CF594 (BD, 562392), GZMB-APC (BioLegend, 372203), Perforin-BV711 (BioLegend, 08129), T-bet-BV785 (BioLegend, 644835), IFN-γ-BV570 (BioLegend, 502534), GZMB-PE (BioLegend, 372208) and IL-2-BV711 (BD, 563946).

Techniques: Suspension, Activity Assay, Flow Cytometry, Staining, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Lactate Dehydrogenase Assay, Migration, Labeling

Journal: Nano-Micro Letters

Article Title: Artificial Macrophage with Hierarchical Nanostructure for Biomimetic Reconstruction of Antitumor Immunity

doi: 10.1007/s40820-023-01193-4

Figure Lengend Snippet: Antitumor immunity in vivo and in vitro. a Illustration of the transwell model to study the in vitro antigen capture of BaSO 4 @ZIF-8/TRF NMΦs. 4T1 tumor cells and J774A.1 macrophages (MΦ) were seeded in the upper and lower chambers of the transwells, respectively. b Confocal fluorescence images showing the locations of tumor antigens and nanoparticles in macrophages (scale bars = 15 μm). c Populations of M1 and M2 macrophages were analyzed by flow cytometry after different treatments: (1) control, (2) LPS, (3) BaSO 4 , (4) ZIF-8/TRF, (5) BaSO 4 @ZIF-8, (6) BaSO 4 @ZIF-8/TRF. The secretions of d TNF-α, e IL-6, and f IL-10 from J774.1A after different treatments. g The percentages of DC maturation, h populations of T helper cells (CD3 + /CD4 + , T h cells), and i cytotoxic T cells (CD3 + /CD8. + , CTLs) in vivo after different treatments: (1) control, (2) BaSO 4 , (3) ZIF-8/TRF, (4) BaSO 4 @ZIF-8, (5) BaSO 4 @ZIF-8/TRF. ** p < 0.01 (n = 5)

Article Snippet: FITC-conjugated CD86, APC-conjugated CD206, PE-conjugated CD80, APC-conjugated CD11c, PE-conjugated CD4, APC-conjugated CD8a, FITC-conjugated CD3, PE-conjugated F4/80, FITC-conjugated CD44, PerCP/Cy5.5-conjugated CD62L, and PE-conjugated CD8a antibodies were obtained from Biolegend (San Diego, USA).

Techniques: In Vivo, In Vitro, Fluorescence, Flow Cytometry

Journal: Nano-Micro Letters

Article Title: Artificial Macrophage with Hierarchical Nanostructure for Biomimetic Reconstruction of Antitumor Immunity

doi: 10.1007/s40820-023-01193-4

Figure Lengend Snippet: Anti-lung metastasis and immune memory effect of NMΦ in vivo. a Schedule for the in vivo studies of immune memory effect. b In vivo bioluminescence images of lung metastasis after different treatments. c Digital photos of metastatic nodes in the lungs with different treatments. d Lungs were examined through H&E staining on day 42 (scale bar = 100 μm). e Statistical results of metastatic nodules in the lungs. f Flow cytometry analysis of effector memory T cells (T EM ) in splenic lymphocytes (gating on CD8. + ), and g corresponding statistical results after different treatments. h Blood biochemistry analysis of mice in different groups: A white blood cell; B lymphocyte; C mononuclear cell; D neutrophile granulocyte; E hemoglobin; F red blood cell; G hematocrit; H mean corpuscular volume; I mean corpuscular hemoglobin; J mean corpuscular hemoglobin concentration; K red blood cell distribution width-standard deviation; L red blood cell volume distribution width the coefficient; M platelet count; N platelet crit; O mean platelet volume; P platelet distribution width. Groups: (1) control, (2) BaSO 4 , (3) αPD-1,(4) ZIF-8/TRF, (5) BaSO 4 @ZIF-8, (6) BaSO 4 @ZIF-8/TRF, (7) ZIF-8/TRF + αPD-1, (8) BaSO 4 @ZIF-8/TRF + αPD-1. ** p < 0.01 (n = 5)

Article Snippet: FITC-conjugated CD86, APC-conjugated CD206, PE-conjugated CD80, APC-conjugated CD11c, PE-conjugated CD4, APC-conjugated CD8a, FITC-conjugated CD3, PE-conjugated F4/80, FITC-conjugated CD44, PerCP/Cy5.5-conjugated CD62L, and PE-conjugated CD8a antibodies were obtained from Biolegend (San Diego, USA).

Techniques: In Vivo, Staining, Flow Cytometry, Concentration Assay, Standard Deviation

Journal: Nano-Micro Letters

Article Title: Artificial Macrophage with Hierarchical Nanostructure for Biomimetic Reconstruction of Antitumor Immunity

doi: 10.1007/s40820-023-01193-4

Figure Lengend Snippet: Antitumor immunity in vivo and in vitro. a Illustration of the transwell model to study the in vitro antigen capture of BaSO 4 @ZIF-8/TRF NMΦs. 4T1 tumor cells and J774A.1 macrophages (MΦ) were seeded in the upper and lower chambers of the transwells, respectively. b Confocal fluorescence images showing the locations of tumor antigens and nanoparticles in macrophages (scale bars = 15 μm). c Populations of M1 and M2 macrophages were analyzed by flow cytometry after different treatments: (1) control, (2) LPS, (3) BaSO 4 , (4) ZIF-8/TRF, (5) BaSO 4 @ZIF-8, (6) BaSO 4 @ZIF-8/TRF. The secretions of d TNF-α, e IL-6, and f IL-10 from J774.1A after different treatments. g The percentages of DC maturation, h populations of T helper cells (CD3 + /CD4 + , T h cells), and i cytotoxic T cells (CD3 + /CD8. + , CTLs) in vivo after different treatments: (1) control, (2) BaSO 4 , (3) ZIF-8/TRF, (4) BaSO 4 @ZIF-8, (5) BaSO 4 @ZIF-8/TRF. ** p < 0.01 (n = 5)

Article Snippet: FITC-conjugated CD86, APC-conjugated CD206, PE-conjugated CD80, APC-conjugated CD11c, PE-conjugated CD4, APC-conjugated CD8a, FITC-conjugated CD3, PE-conjugated F4/80, FITC-conjugated CD44, PerCP/Cy5.5-conjugated CD62L, and PE-conjugated CD8a antibodies were obtained from Biolegend (San Diego, USA).

Techniques: In Vivo, In Vitro, Fluorescence, Flow Cytometry

Journal: Nano-Micro Letters

Article Title: Artificial Macrophage with Hierarchical Nanostructure for Biomimetic Reconstruction of Antitumor Immunity

doi: 10.1007/s40820-023-01193-4

Figure Lengend Snippet: Anti-lung metastasis and immune memory effect of NMΦ in vivo. a Schedule for the in vivo studies of immune memory effect. b In vivo bioluminescence images of lung metastasis after different treatments. c Digital photos of metastatic nodes in the lungs with different treatments. d Lungs were examined through H&E staining on day 42 (scale bar = 100 μm). e Statistical results of metastatic nodules in the lungs. f Flow cytometry analysis of effector memory T cells (T EM ) in splenic lymphocytes (gating on CD8. + ), and g corresponding statistical results after different treatments. h Blood biochemistry analysis of mice in different groups: A white blood cell; B lymphocyte; C mononuclear cell; D neutrophile granulocyte; E hemoglobin; F red blood cell; G hematocrit; H mean corpuscular volume; I mean corpuscular hemoglobin; J mean corpuscular hemoglobin concentration; K red blood cell distribution width-standard deviation; L red blood cell volume distribution width the coefficient; M platelet count; N platelet crit; O mean platelet volume; P platelet distribution width. Groups: (1) control, (2) BaSO 4 , (3) αPD-1,(4) ZIF-8/TRF, (5) BaSO 4 @ZIF-8, (6) BaSO 4 @ZIF-8/TRF, (7) ZIF-8/TRF + αPD-1, (8) BaSO 4 @ZIF-8/TRF + αPD-1. ** p < 0.01 (n = 5)

Article Snippet: FITC-conjugated CD86, APC-conjugated CD206, PE-conjugated CD80, APC-conjugated CD11c, PE-conjugated CD4, APC-conjugated CD8a, FITC-conjugated CD3, PE-conjugated F4/80, FITC-conjugated CD44, PerCP/Cy5.5-conjugated CD62L, and PE-conjugated CD8a antibodies were obtained from Biolegend (San Diego, USA).

Techniques: In Vivo, Staining, Flow Cytometry, Concentration Assay, Standard Deviation