Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD3 , REA1151 , 50 , 130-120-267 , FITC (APC) , Miltenyi Biotec.

Techniques: Imaging

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Single-Cell Survey Reveals Heterogeneity of cDC2s with Two Subsets Delineated by Expression of T-Bet (A) Representative contour plot showing gating strategy for splenic DCs in Tbx21 RFP-Cre mice. DCs defined as Lin(CD3,CD19,CD49b,Siglec-F) – Ly6C – CD64 – CD11c + MHCII + . (B) Frequency of T-bet + cDC2s across tissues. Each circle represents one mouse. In the peripheral and mesenteric LN (PLN and MLN), migratory DCs were defined as MHCII hi CD11c int and resident DCs as MHCII int CD11c hi . Error bars represent mean ± SEM. (C) Analysis of RFP + and YFP + splenic cDC2s from Tbx21 RFP-CreERT2 Rosa26 YFP mice, 3 days post tamoxifen gavage. (D) Percent RFP + and YFP + of cDC2 cells. Percent RFP + of YFP + cDC2s at indicated time points post tamoxifen gavage (right). Error bars represent mean ± SEM; n = 3–4 mice per time point. (E) t-SNE embedding of 4,464 DCs. Colors indicate unsupervised clustering by Phenograph (left panel) or classification based on expression of canonical markers (right panel). (F) Expression of canonical DC markers across the transcriptionally defined DC clusters from (E). (G) Proportion of T-bet (RFP + ) cells in each cell cluster identified in (D). (H) Violin plot showing expression of the cell-cycle signature across the DC clusters from (E). (I) Similarity of bulk T-bet – cDC2s, T-bet + cDC2, and cDC1 transcriptomes to the reference single-cell DC clusters (E). Colors represent the correlation coefficient between the cell population identified in the row label and the DC cluster identified by the column label. See also and .

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Single-Cell Survey Reveals Heterogeneity of cDC2s, Related to Figure 1 A. Representative histogram showing expression of T-bet (RFP) in splenic cells from Tbx21 RFP-cre mice. (B). Expression of T-bet in CD11b + XCR1 + DCs from the intestinal lamina propria. Data representative of > 5 independent experiments, with at least 3 mice per experiment. (C). Expression of T-bet in splenic myeloid cells. Cells were defined as: (i) Ly-6C hi monocytes (Lin – Ly6C + Ly6G – CD11b + CX3CR1 + ); neutrophils (Lin – Ly6C + Ly6G + ); macrophages (Lin – CD64 + Ly6C – ). Lineages (Lin) were defined as: CD3e, CD90.2, CD19, CD49b and Siglec F. Each circle represents an individual mouse, error bars represent mean ± SEM. (D). Left: Gating strategy for single-cell sorting. DCs were defined as Lin(CD3, CD19, CD90) – Ly6C – CD64 – CD11c + MHCII + . Two populations were sampled: RFP + DCs and RFP – DCs (encompassing XCR1 + cDC1s, CD11b + RFP – and CD11b – XCR1 – DCs). Right: Post-sort purity of RFP + and RFP – cells. Contaminating population of Ly6C + cells identifiable on post-sort purity (lower panel). (E). Similarity of splenic CD11c + MHCII + cells to reference myeloid cells (ImmGen Consortium) Colors represent the Pearson correlation between the mean gene expression from the dendritic cell cluster in the rows and the bulk reference transcriptome in the columns. (F). Top 20 positive and negative gene loadings of PC1 for T-bet + cDC2 clusters after cell-cycle correction (left panel). Scatterplot of PC1 and PC2 for T-bet + cDC2 clusters after cell-cycle correction (right panel).

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing, FACS, Gene Expression

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Environmental Cues Drive Distinct DC2 Differentiation Pathways within the Spleen, Related to Figure 5 (A). Gating strategy for the identification of DC progenitors in the bone marrow (BM) (B). Palantir pseudo-time analysis of differentiation potential and branch probabilities from the Siglec-H + pre-DC state to T-bet + cDC2 and T-bet – cDC2 terminal states. (C). Plots showing Palantir differentiation potential (y axis) along Palantir pseudo-time (x axis) for Siglec-H + DC and T-bet + cDC2s (top) or Siglec-H + DC and T-bet – cDC2 clusters (bottom) (D). Plots showing the top two diffusion component embeddings for Siglec-H + DC and T-bet + cDC2 clusters (top) or Siglec-H + DC and Tbet – cDC2 clusters (bottom). Black arrow indicates Siglec-H + DC cluster cells adjacent to cells from the proliferative T-bet + cDC2 clusters 6 and 8. (E). Top panel: plots showing probability of each cell being within 20 nearest neighbors of randomly sampled shortest paths from the Siglec-H + DC to the indicated end points. Middle panel: plots showing the proportion of cells belonging to Siglec-H + DC, T-bet + cDC2, or T-bet – cDC2 from 20 nearest neighbors of randomly sampled shortest paths. Bottom: plots showing diffusion distance step sizes for each step along the indicated shortest paths (bottom panel). Colors illustrate cluster membership. (F). Graph showing AUC (x axis) for genes differentially expressed between Siglec-H + DC cluster (cluster 11) and all other cDC2 clusters. EMD on the y axis. Dashed lines represents μ EMD ± 3σ EMD . (G). Gating strategy for FACS-isolation of MHCII + ILC3s: Lin = CD3, CD19, CD49b, Siglec-F. (H). Heatmap reports scaled expression of 3550 differentially expressed genes (log 2 FC > 1, FDR < 0.01) between ILC3s and Rorγt fm cDC2s. Selected genes listed to the right. (I). Representative flow cytometric analysis of phenotypes of splenic progeny from Tbx21 RFP-cre CD45.2 + Ly6C − CD64 – MHCII + CD11c + Siglec-H + pre-DCs adoptively transferred into sub-lethally irradiated CD45.1 recipient mice 7 days earlier (data from one experiment with n = 3). J. Sort purified T-bet + or T-bet – cDC2 were cultured for 24hrs in the presence of LPS, CpG, TNF-α or IFN−γ. Representative overlay histogram showing the expression of RFP(T-bet) at 24hrs. Data representative of 2 (TNF-α) or 4 (all other cytokines/TLR agonists) independent experiments, n = 2-3.

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Diffusion-based Assay, Isolation, Expressing, Irradiation, Purification, Cell Culture

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Human DC Heterogeneity, Related to Figure 7 (A). Violin plots showing expression distribution of mouse DC subset marker genes across human peripheral blood DC and monocyte clusters identified in Villani et al. (2017) . (B). Representative flow cytometric analysis of mouse peripheral blood cDC2s showing absence of T-bet (RFP) + cDC2s. (C). Gating strategy for FACS-isolation of human spleen DCs for scRNA-seq. DCs were defined as live, LIN(CD3,CD56,CD19) − CD14 – CD11C + HLA-DR + . (D). Representative flow cytometry analysis of human spleen cDC2s gated as Lin(CD3,CD56,CD19) – CD14 – CD11c + HLA-DR + CD123 – XCR1 – CLEC4A + cells. Left panel: cell surface expression of CD1c and CLEC10A by cDC2s. Right panel: overlay of CLEC10A + and CLEC10A – cDC2s distinguished by differential expression of CLEC4A and FcεR1a. Summary bar graphs show frequency of CD1C + CLEC10A + and CD1C + CLEC10A – cDC2s as a percentage of cDC2s (n = 4 individuals). (E). t -SNE embedding of 9,315 FACS-isolated CD45 + immune cells from two melanoma tumors. Colors indicate unsupervised clustering by Phenograph (left panel) or classification based on expression of canonical markers and correlations with bulk RNA-seq data (right panel). Each dot represents an individual cell. (F). Pearson correlations between cluster centroids in (F) and bulk RNA-seq data from purified immune populations ( Jeffrey et al., 2006 , Novershtern et al., 2011 ) (G). t-SNE map of 2,122 myeloid cells identified in (F). Colors indicate patient sample (left) or unsupervised clustering by Phenograph (right panel). Each dot represents an individual cell. (H). Heatmap of normalized, log transformed and MAGIC imputed expression of top 20 differentially expressed genes, defined by the highest earth mover’s distance (EMD), per Phenograph cluster in E. The colored bar at the top of the heatmap shows assignment of cells to clusters labeled in F, right panel. (I). t-SNE map of human melanoma myeloid cells (H) colored by imputed expression of labeled genes.

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing, Marker, Isolation, Flow Cytometry, Quantitative Proteomics, RNA Sequencing, Purification, Transformation Assay, Labeling

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Recombinant, Staining, Multiplex Assay, Cell Isolation, Gene Expression, Software

Journal: Molecular Cancer Research

Article Title: Neoadjuvant Chemotherapy Induces IL34 Signaling and Promotes Chemoresistance via Tumor-Associated Macrophage Polarization in Esophageal Squamous Cell Carcinoma

doi: 10.1158/1541-7786.mcr-20-0917

Figure Lengend Snippet: Figure 1. Increased infiltration and polarized differentiation of TAMs by NAC in ESCC. A, Schematic illustration of the experimental design. B, Representative IHC images of CD163 expression in ESCC specimens from patients who received NAC [NAC (þ)] and did not receive NAC [NAC ()]. Scale bars, 100 mm; original magnification 200. Pre-, Pre-NAC; Post-, Post-NAC. C, Comparison of the number of CD163þ TAMs between biopsy and surgically resected ESCC specimens in NAC () (n ¼ 12) and NAC (þ) (n ¼ 33). D, Comparison of the number of CD163þ TAMs between NAC () (n ¼ 12) and NAC (þ) (n ¼ 33) in biopsy and surgically resected ESCC specimens. E, Comparison of the intensities of CD163 expression between biopsy and surgically resected ESCC specimens in NAC () (n ¼ 12) and NAC (þ) (n ¼ 33). F, Comparison of the intensities of CD163 expression between NAC () (n ¼ 12) and NAC (þ) (n ¼ 33) in biopsy and surgically resected ESCC specimens. G, Representative IHC images of the expression of CD163, CD3, CD8, CD105, E-cadherin, and vimentin in surgically resected ESCC specimens from patients who received NAC [NAC (þ)]. Scale bars, 100 mm; original magnification 200. Post-, Post-NAC. H, Correlations between the number of CD163þ cells and the number of CD3þ TILs, the number of CD8þ TILs, CD105þ microvessel counts, E-cadherin staining score, or Vimentin staining score in surgically resected ESCC specimens of NAC (þ) (n ¼ 33). , P < 0.01; , P < 0.001; , P < 0.0001.

Article Snippet: Reagents used in this study were acquired from the indicated suppliers: 5-FU, KU-55933, and VE-821 (Merck Sigma-Aldrich); cisplatin (FUJIFILM Wako Pure Chemical Corporation); antihuman IL34 antibody [1D12] (ab101443), anti-M-CSF antibody [EP1179Y] (ab52864; Abcam); Novocastra Liquid Mouse Monoclonal Antibody CD163 (NCL-L-CD163; Leica Biosystems); anti-human CD3 antibody (IR503), anti-human CD8 (M7103), and E-cadherin mAb (NCH-38; Dako/Agilent Technology); anti-human CD105 antibody (28117-1-AP; Proteintech); vimentin mAb (SP20; Nichirei Bioscience); antibody APC/Cyanine7 anti-human CD14 [HCD14] (325619), PE anti-human CD163 [GHI/61] (333605; BioLegend); 7-AAD (559925; BD Biosciences); PLX-3397 [PLX; HY-16749], BLZ945 [BLZ; HY-12768] (MedChemExpress).

Techniques: Expressing, Comparison, Staining

Journal: Nature Communications

Article Title: PI3Kγ signaling controls trafficking of CD8 + T cells between lymphoid and non-lymphoid organs and drives hypertension in a murine model

doi: 10.1038/s41467-025-61009-4

Figure Lengend Snippet: a Schematics of the experiment: CD8 + T lymphocytes were purified from total splenocytes from PI3Kγ +/+ , PI3Kγ −/− , and PI3Kγ KD/KD mice receiving angiotensin II or vehicle, and were plated on wells coated with anti-CD3 and anti-CD28 antibodies. PIP3 production was analyzed by immunocytochemistry, and Akt phosphorylation at Thr308 and Ser473 sites by western blot. b , c Immunocytochemistry representative images ( b ) of PIP3 (green) expression on CD8 + T cells (red); DAPI (blue) was used to identify nuclei. Images were acquired by confocal microscopy at 40X magnification (scale bar = 20 µm). c Quantitative analysis of the % of PIP3 + CD8 + T cells from the spleen of n = 3 PI3Kγ +/+ Veh, n = 3 PI3Kγ +/+ AngII, n = 3 PI3Kγ −/− Veh, n = 3 PI3Kγ −/− AngII, n = 3 PI3Kγ KD/KD Veh and n = 3 PI3Kγ KD/KD AngII mice. Representative western blot images ( d , f ) and quantitative analysis ( e, g ) of Akt phosphorylation of Thr308 ( d , e ) and Ser473 ( f , g ) in CD8 + T cells isolated from the spleen of n = 6 PI3Kγ +/+ Veh, n = 6 PI3Kγ +/+ AngII, n = 5 PI3Kγ −/− Veh, n = 5 PI3Kγ −/− AngII, n = 5 PI3Kγ KD/KD Veh and n = 5 PI3Kγ KD/KD AngII mice. Quantitative analysis ( e , g ) as fold changes of pThr308 Akt ( e ) and pSer473 ( g ), normalized over β-actin levels, are shown for angiotensin II treated mice as compared to vehicle controls. All data are expressed as mean ± SEM. Statistical analysis was performed by Two-way ANOVA and Tukey’s correction for multiple comparisons in c , e , g . N represents biologically independent samples. Source data are provided as a file. Schematic in a is Created in BioRender. Perrotta, M. (2025) https://BioRender.com/1qw2qst .

Article Snippet: In the ex-vivo co-culture experiments of MRA and conditioned culture medium of CD8 + T lymphocytes, the cells were isolated from total splenocytes as described above and cultured 5 * 10 5 cells/well at a density of about 7 * 10 5 /cm 2 for 72 h at 37 °C, 5% CO 2 in TexMACS medium (Miltenyi Biotec, 130-097-196) with 10% Dialyzed FBS (Gibco 26400-044), 1% Penicillin-Streptomycin (Gibco, 15140-122), 55 μM β-mercaptoethanol (Gibco, 31350-010) in 48 well pre-coated with CD3ε antibody (pure-functional grade, Miltenyi Biotec, 130-092-973) and then stimulated with CD28 antibody (pure-functional grade, Miltenyi Biotec, 130-093-182).

Techniques: Purification, Immunocytochemistry, Phospho-proteomics, Western Blot, Expressing, Confocal Microscopy, Isolation

Journal: Nature Communications

Article Title: PI3Kγ signaling controls trafficking of CD8 + T cells between lymphoid and non-lymphoid organs and drives hypertension in a murine model

doi: 10.1038/s41467-025-61009-4

Figure Lengend Snippet: a Immunohistochemistry of CD3 + (magenta) and B220 + (green) lymphocytes to analyze the white pulp area in the spleen of PI3Kγ +/+ , PI3Kγ −/− and PI3Kγ KD/KD mice after in vivo administration of angiotensin II or vehicle as control. Nuclei are visualized in blue by DAPI. Images were acquired by confocal microscopy at 10X magnification (scale bar = 100 µm). b Quantitative analysis of the white pulp area marked by CD3 + T cells. n = 6 PI3Kγ +/+ Veh, n = 6 PI3Kγ +/+ AngII, n = 5 PI3Kγ −/− Veh, n = 7 PI3Kγ −/− AngII; n = 7 PI3Kγ KD/KD Veh and n = 5 PI3Kγ KD/KD AngII mice. c Analysis of the myogenic tone of mesenteric resistance arteries (MRA) dissected from naïve WT mice ex vivo co-cultured with CD8 + T cells purified from the spleen of PI3Kγ +/+ and PI3Kγ −/− mice stimulated in vivo for 3 days with angiotensin II or vehicle. n = 4 CD8 Veh ; n = 5 CD8 AngII-PI3Kγ+/+ and n = 5 CD8 AngII-PI3Kγ−/− . Representative plots of flow cytometry ( d ) and quantitative analysis ( e – h ) of total CD8+ T cells ( e ), naïve ( f ), effector ( g ) and resident memory ( h ) CD8+ T cells in kidneys dissected from PI3Kγ +/+ , PI3Kγ −/− and PI3Kγ KD/KD mice after in vivo administration of angiotensin II or vehicle for 28 days. n = 5 PI3Kγ +/+ Veh, n = 7 PI3Kγ +/+ AngII, n = 6 PI3Kγ −/− Veh, n = 8 PI3Kγ −/− AngII, n = 6 PI3Kγ KD/KD Veh and n = 7 PI3Kγ KD/KD AngII. All data are expressed as mean ± SEM. Statistical analysis was performed by Two-way ANOVA, followed by Tukey’s correction for multiple comparisons in b , e , f , g , h ; One-way ANOVA for repeated measures, followed by Tukey’s correction for multiple comparisons in c. N represents biologically independent samples. Source data are provided as a file. Schematics in a , c , d were created in BioRender. Perrotta M (2025) https://BioRender.com/1f77gjq , https://BioRender.com/fbmd4r3 , https://BioRender.com/g0be7ts .

Article Snippet: In the ex-vivo co-culture experiments of MRA and conditioned culture medium of CD8 + T lymphocytes, the cells were isolated from total splenocytes as described above and cultured 5 * 10 5 cells/well at a density of about 7 * 10 5 /cm 2 for 72 h at 37 °C, 5% CO 2 in TexMACS medium (Miltenyi Biotec, 130-097-196) with 10% Dialyzed FBS (Gibco 26400-044), 1% Penicillin-Streptomycin (Gibco, 15140-122), 55 μM β-mercaptoethanol (Gibco, 31350-010) in 48 well pre-coated with CD3ε antibody (pure-functional grade, Miltenyi Biotec, 130-092-973) and then stimulated with CD28 antibody (pure-functional grade, Miltenyi Biotec, 130-093-182).

Techniques: Immunohistochemistry, In Vivo, Control, Confocal Microscopy, Ex Vivo, Cell Culture, Purification, Flow Cytometry

Journal: Nature Communications

Article Title: PI3Kγ signaling controls trafficking of CD8 + T cells between lymphoid and non-lymphoid organs and drives hypertension in a murine model

doi: 10.1038/s41467-025-61009-4

Figure Lengend Snippet: a , b Immunocytochemistry representation ( b ) of PIP3 + (green) expression in CD8 + (red) T cells isolated from the spleen of PI3Kγ +/+ and PI3Kγ CX/CX mice and plated onto anti-CD3 and anti-CD28 coated wells. Nuclei are stained by DAPI and visualized in blue. b Quantitative analysis of the % of PIP3 + CD8 + T cells was performed in n = 3 PI3Kγ +/+ and n = 4 PI3Kγ CX/CX mice. Images were acquired by confocal microscopy at 40X magnification (scale bar = 20 µm). c – f Western blot analysis of Akt phosphorylation in Thr308 (c) and Ser473 ( e ) in CD8 + T cells isolated from the spleens of PI3Kγ +/+ and PI3Kγ CX/CX mice. Representative blot ( c , e ) and quantitative analysis of phosphorylated Akt at Thr308 ( d ) and Ser473 ( f ) as fold changes over β-actin levels, in n = 6 PI3Kγ +/+ and PI3Kγ CX/CX mice in d , and n = 8 PI3Kγ +/+ and PI3Kγ CX/CX mice in ( f ). Metabolic-flux analysis by the Seahorse XF Analyzer was used to measure the mitochondrial respiration and the aerobic glycolysis by the oxygen-consumption rate (OCR) ( g – j ) and extracellular acidification rate (ECAR) ( k – m ) under basal conditions and after drug-induced mitochondrial inhibition in CD8 + T cells purified from the spleen of PI3Kγ CX/CX and PI3Kγ +/+ mice. For g – m , n = 9 PI3Kγ +/+ and n = 8 PI3Kγ CX/CX animals were analyzed and measured in triplicates. All data are expressed as mean ± SEM. Statistical analysis was performed by unpaired Mann–Whitney test in b ; two-sided unpaired T -test in d , f , h , i , j , l , m . N represents biologically independent samples. Source data are provided as a file.

Article Snippet: In the ex-vivo co-culture experiments of MRA and conditioned culture medium of CD8 + T lymphocytes, the cells were isolated from total splenocytes as described above and cultured 5 * 10 5 cells/well at a density of about 7 * 10 5 /cm 2 for 72 h at 37 °C, 5% CO 2 in TexMACS medium (Miltenyi Biotec, 130-097-196) with 10% Dialyzed FBS (Gibco 26400-044), 1% Penicillin-Streptomycin (Gibco, 15140-122), 55 μM β-mercaptoethanol (Gibco, 31350-010) in 48 well pre-coated with CD3ε antibody (pure-functional grade, Miltenyi Biotec, 130-092-973) and then stimulated with CD28 antibody (pure-functional grade, Miltenyi Biotec, 130-093-182).

Techniques: Immunocytochemistry, Expressing, Isolation, Staining, Confocal Microscopy, Western Blot, Phospho-proteomics, Inhibition, Purification, MANN-WHITNEY