Journal: Nature Communications

Article Title: PD-L1 + and XCR1 + dendritic cells are region-specific regulators of gut homeostasis

doi: 10.1038/s41467-021-25115-3

Figure Lengend Snippet: a Mass cytometry t-SNE analysis in the LP of each intestinal region. b Mass cytometry t-SNE plots of different regions of gut LP. CD11b and CD11c clusters of CD45 + gated cells are shown. c t-SNE plots comparing duodenal and colonic LP cells. All t-SNE plots correspond to concatenated data of singlets, live, CD45 + , and Ter119 − gated cells. N = 10 mice/region of the gut over three independent experiments. d Immunofluorescence of duodenum, ileum, and colon. DAPI (blue) e-cadherin (green) and CD11c (red) (×40), the total size of fluorescently labeled CD11c from each sample was obtained using an ImageJ custom macro code. e Immunohistochemistry of CD11c + cells in duodenum and colon (×10). Percentage of area showing marked CD11c + DCs in the LP considering the entire tissue area. f Quantification of DCs using flow cytometry (live, CD45 + F4/80/CD64 − CD11c + ) per mg of tissue and counting beads. N = 5 over two independent experiments. Bars represent mean ± SEM. One-way ANOVA followed by Tukey post hoc test for multiple comparisons was used for IF comparison. Unpaired two-tailed Student’s t test was used for IHC and flow cytometry. * p = 0.02, ** p = 0.008, *** p = 0.0007, *** p = 0.005 (CD11c),**** p < 0.0001. Antigen presentation cells (APC).

Article Snippet: Cells isolated from human biopsies were incubated with Human Fc Block (1:100; BD Bioscience) and stained with anti-human HLA-DR APC-Cy7-conjugated mAb (L243; 1:300; Biolegend), PE-conjugated mAb to CD11c (B-ly6; 1:200; BD Bioscience), PerCP-C5.5-conjugated mAbs to CD3 (UCHT1), CD19 (HIB19) and CD14 (M5E2) (all at 1:300; BD Bioscience); PE-Cy7-conjugated mAb to CD1c (L161; 1:300; Biolegend), FITC-conjugated mAb to CD274/PD-L1 (MIH2; 1:100; Biolegend), APC-conjugated mAb to CD141 (1A4; 1:300; BD Bioscience), BV421-conjugated mAb to XCR1 (S15046E; 1:200; Biolegend).

Techniques: Mass Cytometry, Immunofluorescence, Labeling, Immunohistochemistry, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: PD-L1 + and XCR1 + dendritic cells are region-specific regulators of gut homeostasis

doi: 10.1038/s41467-021-25115-3

Figure Lengend Snippet: a Diagram showing upregulation and downregulation of genes found in RNA-seq analysis from DCs sorted from duodenal, jejunal, ileal, cecal, and colonic LP of 8 to 10-week-old naive C57BL/6 J mice. b PCoA, c heatmap, and d volcano plots of genes found in RNA-seq analysis. N = 5. e RT-qPCR from sorted DCs of different regions of the gut showing Cd274, Cd209, Xcr1, and S100a4 expression. ( N = 6). Bars represent mean ± SEM. One-way ANOVA followed by Tukey post hoc test for multiple comparisons. * p = 0.02, ** p = 0.003, Duo vs Jej *** p = 0.006 Duo vs Ile *** p = 0.004; **** p < 0.0001. f Mass and flow cytometry data showing the percentage of CD101, PD-L1, CD209b, Sirpα, XCR1, TLR3, ICOSL, CX3CR1 expression on DCs and CD103 + CD11b − and CD103 + CD11b + cells gated on singlets, live, CD45 + CD19 − B220 − Nk1 . 1 − Ly − 6G − CD3 − F4/80 − CD64 − CD11c High . g Dot plots showing the percentage of PD-L1 + , XCR1 + , and Sirpα + DCs LP from duodenum and colon by mass cytometry. N = 5; a pool of three mice/region of the gut over at least three independent experiments. Source data are provided as a Source Data file. Duodenum (DUO), jejunum (JEJ), ileum (ILE), cecum (Cec) colon (COL).

Article Snippet: Cells isolated from human biopsies were incubated with Human Fc Block (1:100; BD Bioscience) and stained with anti-human HLA-DR APC-Cy7-conjugated mAb (L243; 1:300; Biolegend), PE-conjugated mAb to CD11c (B-ly6; 1:200; BD Bioscience), PerCP-C5.5-conjugated mAbs to CD3 (UCHT1), CD19 (HIB19) and CD14 (M5E2) (all at 1:300; BD Bioscience); PE-Cy7-conjugated mAb to CD1c (L161; 1:300; Biolegend), FITC-conjugated mAb to CD274/PD-L1 (MIH2; 1:100; Biolegend), APC-conjugated mAb to CD141 (1A4; 1:300; BD Bioscience), BV421-conjugated mAb to XCR1 (S15046E; 1:200; Biolegend).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Mass Cytometry

Journal: Nature Communications

Article Title: PD-L1 + and XCR1 + dendritic cells are region-specific regulators of gut homeostasis

doi: 10.1038/s41467-021-25115-3

Figure Lengend Snippet: a Flow cytometry analysis of PD-L1 + , XCR1 + , Sirpα + , and CD103 + in LP DC from XCR1 venus mice in five segments of the gut. b Absolute number of PD-L1 + and c XCR1 + DCs from C57BL/6 mice gated on singlets, live, CD45 + , dump − (CD19, B220, CD90.2, F4/80, CD64) CD11c + per gram of tissue upper Small Intestine (uSI) (duodenum and proximal jejunum) and colon (Col). N = 5 mice/group. Graphs represent mean ± SEM. Unpaired two-tailed Student’s t test. ** p = 0.003 (PD-L1), ** p = 0.005 (XCR1). d – f Heatmap ( d ), volcano plot ( e ), and Gene Set Enrichment Analysis (GSEA) pathways ( f ) of modulated genes found in RNA-seq analysis from DCs sorted from uSI LP of Pd-l1 −/− and littermate controls, and colonic LP of XCR1 DTA and littermate controls. N = 3 of a pool of N = at least three mice over at least three independent experiments. Source data are provided as a Source Data file. Wild-type (WT); gene set enrichment analysis (GSEA).

Article Snippet: Cells isolated from human biopsies were incubated with Human Fc Block (1:100; BD Bioscience) and stained with anti-human HLA-DR APC-Cy7-conjugated mAb (L243; 1:300; Biolegend), PE-conjugated mAb to CD11c (B-ly6; 1:200; BD Bioscience), PerCP-C5.5-conjugated mAbs to CD3 (UCHT1), CD19 (HIB19) and CD14 (M5E2) (all at 1:300; BD Bioscience); PE-Cy7-conjugated mAb to CD1c (L161; 1:300; Biolegend), FITC-conjugated mAb to CD274/PD-L1 (MIH2; 1:100; Biolegend), APC-conjugated mAb to CD141 (1A4; 1:300; BD Bioscience), BV421-conjugated mAb to XCR1 (S15046E; 1:200; Biolegend).

Techniques: Flow Cytometry, Two Tailed Test, RNA Sequencing Assay

Journal: Nature Communications

Article Title: PD-L1 + and XCR1 + dendritic cells are region-specific regulators of gut homeostasis

doi: 10.1038/s41467-021-25115-3

Figure Lengend Snippet: a Histological damage index (HDI) and Hematoxylin and Eosin (H&E) staining from distal jejunum from littermate controls and CD11c Cre xPd-l1 flox/flox mice after 72 h of 5-FU administration (10X); N = at least five * p = 0.032. b Flow cytometry representative dot plots and quantification of LP cells (monocytes, neutrophils, IL-17-producing CD4 T cells, Treg cells, and RORγt expressing CD4 T cells) isolated from upper small intestine (duodenum+jejunum) of CD11c Cre xPd-l1 flox/flox mice and littermate controls. Unpaired two-tailed Student’s t test * p = 0.01, ** p = 0.002, ** p = 0.007 (IL-17), *** p = 0.0006. c Weight loss * p = 0.041, Disease Activity Index (DAI) * p = 0.048, ** p = 0.0015, ** p = 0.0029 (day8) and H&E from the colon in DSS-treated littermate controls and XCR1 DTA mice (×10), N = 5. d Flow cytometry (FC) representative dot plots and quantification of LP cells as shown in b isolated from colonic LP cells from XCR1 DTA mice and littermate controls. Boxes and dots represent mean ± SEM. * p = 0.044, * p = 0.014 (IL-17), ** p = 0.001, *** p = 0.0001, **** p < 0.0001. Non-parametric Student’s t test with unpaired two-tailed Mann–Whitney post test was used for histological analysis, unpaired two-tailed Student’s t test was used for FC and two-way-ANOVA followed by Sidak post hoc test for multiple comparisons was used for DAI and weight loss analysis. Experiments were performed independently in duplicate. Source data are provided as a Source Data file. Wild-type (WT).

Article Snippet: Cells isolated from human biopsies were incubated with Human Fc Block (1:100; BD Bioscience) and stained with anti-human HLA-DR APC-Cy7-conjugated mAb (L243; 1:300; Biolegend), PE-conjugated mAb to CD11c (B-ly6; 1:200; BD Bioscience), PerCP-C5.5-conjugated mAbs to CD3 (UCHT1), CD19 (HIB19) and CD14 (M5E2) (all at 1:300; BD Bioscience); PE-Cy7-conjugated mAb to CD1c (L161; 1:300; Biolegend), FITC-conjugated mAb to CD274/PD-L1 (MIH2; 1:100; Biolegend), APC-conjugated mAb to CD141 (1A4; 1:300; BD Bioscience), BV421-conjugated mAb to XCR1 (S15046E; 1:200; Biolegend).

Techniques: Staining, Flow Cytometry, Expressing, Isolation, Two Tailed Test, Activity Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: PD-L1 + and XCR1 + dendritic cells are region-specific regulators of gut homeostasis

doi: 10.1038/s41467-021-25115-3

Figure Lengend Snippet: a Flow cytometry dot plots from duodenal and colonic human biopsies showing DC phenotype, and immunofluorescence showing CD1c and CD141 colocalization in colonic biopsies (20X). b Graphs represent the percentage of PD-L1 + and XCR1 + DCs gated on singlets, live, HLA-DR High , dump − (CD19, CD3, CD14) and CD11c + cells; N = 8, paired two-tailed Student’s t test, * p = 0.031. Source data are provided as a Source Data file. Biorender was used for illustration.

Article Snippet: Cells isolated from human biopsies were incubated with Human Fc Block (1:100; BD Bioscience) and stained with anti-human HLA-DR APC-Cy7-conjugated mAb (L243; 1:300; Biolegend), PE-conjugated mAb to CD11c (B-ly6; 1:200; BD Bioscience), PerCP-C5.5-conjugated mAbs to CD3 (UCHT1), CD19 (HIB19) and CD14 (M5E2) (all at 1:300; BD Bioscience); PE-Cy7-conjugated mAb to CD1c (L161; 1:300; Biolegend), FITC-conjugated mAb to CD274/PD-L1 (MIH2; 1:100; Biolegend), APC-conjugated mAb to CD141 (1A4; 1:300; BD Bioscience), BV421-conjugated mAb to XCR1 (S15046E; 1:200; Biolegend).

Techniques: Flow Cytometry, Immunofluorescence, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: In vivo signaling through the neurokinin 1 receptor favors transgene expression by Langerhans cells and promotes the generation of Th1- and Tc1-biased immune responses.

doi: 10.4049/jimmunol.178.11.7006

Figure Lengend Snippet: FIGURE 2. Skin DCs constitu- tively express the NK1R. A–C and D–F are cross-sections of skin (ear) from two independent experiments showing the expression of the NK1R (green) by keratinocytes, epidermal LCs (arrowheads), and DDCs (ar- rows) (the latter two identified by their expression of CD11c in red). C and F, The yellow fluorescence is due to the overlap of red (CD11c) and green (NK1R). Cell nuclei were counterstained with 4,6-diamidino- 2-phenylindole (blue). Immunofluo- rescence, magnification: 200. G, The green histogram demonstrates the expression of NK1R by freshly isolated LCs gated on CD11c expres- sion. The gray histogram corresponds to negative control cells. The num- bers in the histogram represents the percentage of NK1R-positive LCs and the mean fluorescent intensity (between parentheses). Data are representative of two independent experiments.

Article Snippet: Flow cytometric analysis of single-cell suspensions LC-enriched epidermal cell suspensions were blocked with 10% normal donkey serum and incubated (30 min, 4°C) with PE anti-CD11c mAb in combination with goat anti-mouse NK1R polyclonal Ab, recognizing the N terminus epitope of the human and mouse NK1R (Santa Cruz Biotechnology), followed by FITC-conjugated donkey anti-goat IgG, F(ab )2 (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Isolation, Negative Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: In vivo signaling through the neurokinin 1 receptor favors transgene expression by Langerhans cells and promotes the generation of Th1- and Tc1-biased immune responses.

doi: 10.4049/jimmunol.178.11.7006

Figure Lengend Snippet: FIGURE 5. The NK1R agonist enhances the abilities of the GG to promote sDLN inflammation and homing of activated LCs. A–D, Structure of sDLNs (inguinal) excised from nontreated control mice (A) and from animals treated (24 h prior) with: the NK1R agonist (i.d.) (B), GG (pCMV-Luc) (C), or GG plus NK1R agonist (D). sDLNs from treated mice show sinus hyperplasia characterized by the presence of cells with abundant pale cytoplasm (some with morphological features of DCs indicated by arrows and detailed in the insets) located mainly within the subcortical and paracortical areas (dotted lines). Additionally, 1-m gold beads were detected in the cytoplasm of DCs 24 h after GG treatments alone or in combination with the NK1R agonist (illustrated in insets of C and D and indicated by asterisks). H&E stain: magnification, 400; insets, 1000. E–H, Identification of the population of LCs coexpressing langerin (CD207 (green)) and CD11c (red) (arrows) in the paracortical areas of sDLNs (inguinal) excised from nonimmunized control mice (E) and from animals treated (24 h prior) with NK1R agonist (i.d.) (F), GG (pCMV-Luc) (G), or GG plus NK1R agonist (H). Insets show CD11clangerin LCs at higher magnification. Immunofluorescence: magnification, 200; insets, 1000. I, Quantification by flow cytometry of CD11cCD11bMHC-II (IAb)highlan- gerin epidermal LCs and CD11cCD11bMHC-II (IAb)highlangerin DDCs in the sDLNs (inguinal) of nonimmunized control mice and animals treated (24 h prior) with NK1R agonist (i.d.), GG (pCMV-Luc), or GG plus NK1R agonist. Data are representative of two independent experiments. J, Comparative analysis of tg Luc expression in sDLNs 24 h after GG delivery of pNFB-Luc or pCMV-Luc in the presence (or not; control) of the NK1R agonist. Means 1 SD of the fold increase of RLU compared with background levels are illustrated. Three independent experiments were performed.

Article Snippet: Flow cytometric analysis of single-cell suspensions LC-enriched epidermal cell suspensions were blocked with 10% normal donkey serum and incubated (30 min, 4°C) with PE anti-CD11c mAb in combination with goat anti-mouse NK1R polyclonal Ab, recognizing the N terminus epitope of the human and mouse NK1R (Santa Cruz Biotechnology), followed by FITC-conjugated donkey anti-goat IgG, F(ab )2 (Jackson ImmunoResearch Laboratories).

Techniques: Control, Staining, Cytometry, Expressing

Journal: The Journal of Experimental Medicine

Article Title: AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma

doi: 10.1084/jem.20200962

Figure Lengend Snippet: AIM2 exerts an immunosuppressive effect in the melanoma microenvironment. (A–F) WT and Aim2 −/− mice were inoculated s.c. with 1.0 × 10 6 B16F10 cells on day 0. On day 13, tissues were harvested. (A) Tumor growth over time (top; n = 10). Sample photo of B16F10 tumor on day 13 (bottom). Scale bar, 10 mm. (B–D) Flow cytometry analysis of TILs ( n = 10). (B) The numbers of CD8 + and CD4 + T cells among 10 4 live singlet cells. Percentage of FoxP3 + cells in CD4 + T cells and CD8/T reg cell ratio. (C) Representative contour plot for FoxP3 among CD4 + T cells. (D) Percentages of IFN-γ + and TNF-α + in CD8 + T cells. (E and F) IFN-β ( n = 7; E), IL-1β, and IL-18 ( n = 6; F) protein levels within the tumor. (G–L) WT and Aim2 −/− mice were inoculated s.c. with 1.0 × 10 6 YUMM1.7 cells on day 0. On day 17, tissues were harvested. (G–J) Similar analysis as in A–D was performed on YUMM1.7 tumor–bearing WT and Aim2 −/− mice ( n = 11). (G) Tumor growth over time (top; n = 11). Sample photo of YUMM1.7 tumor on day 17 (bottom). Scale bar, 10 mm. (K and L) IFN-β ( n = 8; K), IL-β, and IL-18 ( n = 7; L) protein levels within the tumor. (M) The numbers of CD11c + and AIM2 + CD11c + cells and the percentage of AIM2 + cells in CD11c + cells in HPF of primary lesions of human thin ( n = 15) and thick ( n = 16) melanomas. (N) Immunofluorescence microscopy of primary lesions of human thin and thick primary melanomas, visualized for CD11c, AIM2, and DAPI. Scale bar, 100 µm. Data are shown as mean ± SEM and are pooled from four (M), three (A, B, D, G, H, and J), or two (E, F, K, and L) experiments or are representative of at least three independent experiments (C, I, and N). *, P < 0.05; **, P < 0.01; ****, P < 0.0001; two-way ANOVA with Sidak’s multiple-comparisons test (A and G) or Mann–Whitney test (B, D–F, H, and J–M).

Article Snippet: Nonspecific binding was blocked in 1% goat serum for 30 min at room temperature, and sections were incubated with the following primary Abs diluted in PBS with Tween 20: rabbit anti-AIM2 polyclonal Ab (eBioscience; 1:800) and mouse anti-CD11c mAb (2F1C10; Proteintech; 1:200), mouse anti-CD141 mAb (141C01; Invitrogen; 1:50), or mouse anti-CD1c mAb (OTI2F4; Abcam; 1:150) overnight at 4°C.

Techniques: Flow Cytometry, Immunofluorescence, Microscopy, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma

doi: 10.1084/jem.20200962

Figure Lengend Snippet: Enhanced anti-melanoma immunity of vaccination with AIM 2 -deficient DCs is dependent on the recognition of tumor-derived DNA and independent of prolonged cell survival of vaccinated DCs. (A–C) B16F10 mice were treated with ACT + WT or Aim2 −/− DC-gp100 and intratumoral (i.t.) administration of DNase I or PBS. On day 20 after PMEL transfer, tissues were harvested. (A) Therapy regimen scheme. (B) Tumor growth over time (left; n = 9). Sample photo of B16F10 tumor on day 20 after PMELs transfer (right). Scale bar, 10 mm. (C) Flow cytometry analysis of the numbers of PMELs, CD8 + T cells, and CD4 + T cells among 10 4 live singlet cells, percentage of FoxP3 + cells in CD4 + T cells, and PMEL/T reg cell ratio in the tumor ( n = 9). (D) Experimental scheme for analyzing DC vaccine infiltration in the tumor, TdLN, and spleen. B16F10-bearing CD45.1 congenic B6 mice were treated with ACT using 1.0 × 10 6 PMELs (CD45.2) + 1.0 × 10 6 WT or Aim2 −/− DC-gp100 (CD45.2), and tissues were harvested on day 10 ( n = 7) and day 20 ( n = 8) after PMELs transfer. (E) Representative contour plot for CD45.2 + Thy1.1 − CD11c + MHC-II + DC-gp100 (DC vaccine) present at the tumor, TdLN, and spleen on day 20 after PMELs transfer. (F) The absolute number of vaccinated DCs present in the tumor, TdLN, and spleen on days 10 ( n = 7) and 20 ( n = 8) after PMELs transfer. Data are shown as mean ± SEM and are pooled from four (B and C) or three (F) independent experiments or are representative of three independent experiments (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-way ANOVA with Tukey’s multiple-comparisons test (B), one-way ANOVA with Tukey’s multiple-comparisons test (C), or Mann–Whitney test (F).

Article Snippet: Nonspecific binding was blocked in 1% goat serum for 30 min at room temperature, and sections were incubated with the following primary Abs diluted in PBS with Tween 20: rabbit anti-AIM2 polyclonal Ab (eBioscience; 1:800) and mouse anti-CD11c mAb (2F1C10; Proteintech; 1:200), mouse anti-CD141 mAb (141C01; Invitrogen; 1:50), or mouse anti-CD1c mAb (OTI2F4; Abcam; 1:150) overnight at 4°C.

Techniques: Derivative Assay, Flow Cytometry, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: Activin A and CCR2 regulate macrophage function in testicular fibrosis caused by experimental autoimmune orchitis

doi: 10.1007/s00018-022-04632-4

Figure Lengend Snippet: List of antibodies and isotype control antibodies used for flow cytometric analysis

Article Snippet: Recombinant monoclonal anti-CD11c PE-Vio770 REAfinityTM , Miltenyi Biotec , 130-110-840 , REA754 , 1:50.

Techniques: Control, Clone Assay, Recombinant