high-dimensional flow cytometry  (GraphPad Software Inc)

Journal: Cancer discovery

Article Title: Human colon cancer-derived Clostridioides difficile strains drive colonic tumorigenesis in mice

doi: 10.1158/2159-8290.CD-21-1273

Figure Lengend Snippet: (A,B) Variation in colonic tumorigenic potential in GF ApcMin/+ mice at 12-14 wk post-inoculation (p.i.) by (A) individual biofilm-positive tumor (BF+T) or (B) biofilm-negative tumor (BF−T) CRC mucosal samples from 6 patients. N = 6-8 mice per group, from 2 independent experiments. (C) 16S rRNA amplicon sequencing relative abundances of the top 20 species in the 6 patient tumor slurry inocula and 12-14 wk p.i. stools from mice gavaged with the tumor slurries. (D) A consortium of 30 bacterial isolates derived from GF mice gavaged with the 3728T slurry recapitulated colonic tumorigenesis of the original 3728T slurry. N = 5-11 mice per group. (E-F) Colonic tumor counts (E) and representative methylene blue staining (F) of colonic tumors (arrows) at 12 wk p.i. in a vancomycin/gentamicin model (see Methods) of chronic colonization with non-toxigenic (TcdA−TcdB−) or toxigenic (TcdA+TcdB+) C. difficile strains. N = 5-18 mice per group, from two independent experiments. (G-H) Colonic tumor counts (G) and representative methylene blue staining (H) of colonic tumors (arrows) from GF ApcMin/+ mice gavaged with the 3728T isolates with or without the CIm_3728T or CIm_3752T C. difficile strains. N = 6-8 mice per group. (I) Colonic tumors induced in GF ApcMin/+ mice gavaged with either the 3979T slurry or the 3979T slurry with C. difficile strain CIm_3728T at 10 wk p.i. Cross represents a mouse death. N = 7-8 mice per group. Statistical significance was calculated via Kruskal-Wallis followed by Mann-Whitney tests. Mann-Whitney p-values are shown.

Article Snippet: Tumorigenesis, high-dimensional flow cytometry, and IHC statistics were performed in GraphPad Prism version 9.1.

Techniques: Amplification, Sequencing, Derivative Assay, Staining, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: In vitro characterization of anti-GARP antibody PIIO-1. (A) GARP expression on human regulatory T cells and platelets was evaluated by flow cytometry after staining with PIIO-1 at 10 µg/mL. (B) 293 FT cell line was transfected with empty vector (EV), human GARP (hGARP)-expressing vector only or co-transfected with hGARP and latent TGFβ1 expression vectors. GARP expression on indicated cell line was detected by flow cytometry after staining with PIIO-1 at 10 µg/mL. (C) Human GARP sequence was replaced by murine GARP according to the schematic diagram to generate the chimeric constructs of human and murine GARP that were tagged with HA (hemagglutinin) epitope. Transfection efficiency was determined using anti-HA antibody. All constructs were transfected into 293 FT cells. (D) Crystal structure of the GARP (green)-LTGFβ (gray) complex (PDB DOI: 10.2210/pdb6GFF/pdb). The region of PIIO-1 recognition is orange and the residues interacting with LTGFβ are cyan. LTGFβ occludes approximately 30% of the potential antibody binding site and may sterically or allosterically restrict access of the antibody to GARP in the LTGFβ-complexed state. Modeling was carried out using Pymol. (E) Jurkat cell line, made to overexpress hGARP, was incubated with LTGFβ1 along with mIgG1 or PIIO-1 at indicated concentration for 30 min at 37℃. Human LAP expression was detected by flow cytometry. All data are representative of 2–6 independent experiments. GARP, Glycoprotein-A repetitions predominant; LAP, latency-associated peptide.

Article Snippet: Antibody staining and high dimensional spectral flow cytometry analysis (Cytek) were performed as detailed previously.

Techniques: In Vitro, Expressing, Flow Cytometry, Staining, Transfection, Plasmid Preparation, Sequencing, Construct, Binding Assay, Incubation, Concentration Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: PIIO-1 monotherapy modulates CD8 + T cells in the TME and confers protection against cancer in hLRRC32 KI mice. (A) 1×10 5 MB-49 cells were injected s.c. on the right flank of hLRRC32 KI male mice. PIIO-1 or isotype control (ISO) was administered (200 µg/mouse, i.p.) every 3 days for a total of four treatments starting on day 4. Shown is one representative tumor growth curve. (B) 1×10 5 MB-49 cells were injected s.c. on the right flank of hLRRC32 KI male mice. PIIO-1 was delivered (200 µg/mouse, i.p.) on days 6 and 9. Tumors were collected on day 10 and tumor-infiltrating leucocytes were stained and analyzed by flow cytometry. Frequency of CD8 + T cells as a proportion of live CD45 + lymphocytes is shown (left). A similar experiment was performed wherein mice were treated with PIIO-1 for a total of 6 treatments starting on day 6. CD8 + TILs were quantified on day 22 (right). (C) Frequency of total Tregs (CD25 + Foxp3 + ) (left) and CTLA4 + VISTA + Tregs (right) in tumor-infiltrating CD4 + T cells in the indicated treatment groups. (D) Differential expression analysis of cluster frequency of CD8 + TILs between ISO and PIIO-1 treated TILs. UMAP dimension reduction of tumor-infiltrating CD8 + T cells from B after staining with 33 markers and high dimensional spectral flow cytometry analysis. Data shown is gated on live CD45 + CD3 + CD8 + T cells, subsampled on 5000 cells per sample. Unsupervised clustering analysis was done using FlowSOM algorithm with an elbow method approach for cluster number determination. (E) Heatmap of D showing the relative expression levels of indicated markers by each cluster. (A–E) n=4–6 mice per group. (F) Differential expression analysis of cytokine production by CD8 + TILs between ISO and PIIO-1 treated tumors. 1×10 5 MB-49 cells were injected s.c. on the right flank of hLRRC32 KI male mice. PIIO-1 or IOS was administered every 3 days for a total of 4 treatments starting on day 5. Tumors were collected on day 17. Intracellular stain for 17 cytokine panel was done, followed by spectral flow cytometry and analysis of CD45 + CD3 + CD8 + T cells. (G) Cytokine level in panel (F) indicated by heatmap showing expression intensity of cytokines by each CD8 + T cell cluster. Tumor curve analysis was performed using repeated measures two-way analysis of variance. Cluster differences were measured by two-tailed Student’s t test. Data are represented as mean±SEM. *P<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Antibody staining and high dimensional spectral flow cytometry analysis (Cytek) were performed as detailed previously.

Techniques: Injection, Control, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: PIIO-1 attenuates canonical TGFβ signaling pathway in tumor-infiltrating immune cells and rejuvenates antitumor immunity in hLRRC32 KI mice. (A) 1×10 5 MB-49 cells were injected s.c. on the right flank of hLRRC32 KI male mice. Humanized PIIO-1 (200 µg/mouse, i.p.) were administered on days 18 and 20. Tumors were collected on day 21. TILs were isolated and stained for intracellular pSMAD2/3 with indicated cell linage markers, followed by flow cytometry analysis. (B) Quantification of panel A. (C–E) 1×10 5 MB-49 cells were injected s.c. on the right flank of hLRRC32 KI male mice. Humanized PIIO-1 (200 µg/mouse, i.p.) was delivered on days 6 and 9. Tumors were collected on day 10. Single cell suspension and RNA isolation were prepared, and then subjected to bulk RNA sequencing. (C) Volcano plot of transcript expression. Differential gene expression was shown in red (up) or blue (down). Representative transcripts such as Ccl3, Ccl9, Cxcl14, Cxcl15 , Il6 and Tnfrsf25 were indicated. (D) GSEA of differential expression genes between tumors treated with PBS and PIIO-1. (E) Comparison of TILs between PBS and PIIO-1 treated tumors based on deconvolution of bulk RNA sequencing data. Data were performed using two-tailed Student’s t-test, data in (B) represented mean±SEM. *p<0.05, **p<0.01.

Article Snippet: Antibody staining and high dimensional spectral flow cytometry analysis (Cytek) were performed as detailed previously.

Techniques: Injection, Isolation, Staining, Flow Cytometry, Suspension, RNA Sequencing Assay, Expressing, Comparison, Two Tailed Test