Journal: Kidney International Reports

Article Title: Impact of Induction Therapy on Circulating T Follicular Helper Cells and Subsequent Donor-Specific Antibody Formation After Kidney Transplant

doi: 10.1016/j.ekir.2018.11.020

Figure Lengend Snippet: Identification of circulating T follicular helper (cT FH ) cell cluster distribution by t-distributed Stochastic Neighbor Embedding (tSNE) reveal that thymoglobulin-induced kidney transplant (KTx) patients who developed donor-specific anti-HLA antibodies (DSA) display elevated effector memory (EM) programmed cell death 1 (PD-1) int/hi Th1-T FH phenotypes. We applied the tSNE algorithm to allow for an unsupervised analysis of cT FH cell compartment. (a) Three representative subjects from each group (exception, basiliximab DSA + [ n = 2]), for a total of 14 samples, were normalized to 500 cT FH cells by down sampling (FlowJo) and concatenated into 1 file to create a consensus tSNE map. Different marker (PD-1, CD62L, and CXCR3) fluorescence intensity was identified by color gradient. (b) Clusters with a high concentration of cells with similar phenotypes are highlighted by gating on deconcatenated tSNE for all groups. Basi, basiliximab; HC, healthy control; thymo, thymoglobulin.

Article Snippet: In addition, we applied t-distributed Stochastic Neighbor Embedding algorithm (FlowJo) and integrated the fluorescence intensity of phenotypic markers into a consensus map to visualize the overall differences of cT FH cell subset composition measured by cell cluster density.

Techniques: Sampling, Marker, Fluorescence, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Differential Pathogenic Th17 Profile in Mesenteric Lymph Nodes of Crohn's Disease and Ulcerative Colitis Patients

doi: 10.3389/fimmu.2019.01177

Figure Lengend Snippet: Predominance of CCR6 + CXCR3-CD4 + T cells in mLNs of CD when compared to UC patients. (A) CD3 + T cells isolated from mLNs of CD and UC patients were concatenated for t-SNE analysis. Feature plots of the indicated antigens (left panels). Frequencies of CD4 + and memory CD45RA − T cells (right panels). (B) Representative dot plots of CCR6 + CXCR3 − , CCR6 + CXCR3 + and CCR6 − CXCR3 + CD4 + subsets, (C) their expression of RORγ and Tbet, and (D) frequencies of indicated Th subsets. (E) Representative dot plots and frequencies of T EM (CD62L low ) and T CM (CD62L high ) among Th subsets. Unpaired t -test (+), Friedmann test followed by Dunn's test (*) and repeated measures one-way ANOVA followed by Tukey's test (#). * P < 0.05, ** ++ ## P < 0.01, and *** P < 0.001, **** P < 0.0001.

Article Snippet: FCS Express 6 (DeNovo Software) or t -SNE ( t -Distributed Stochastic Neighbor Embedding) plugin available in FlowJo version 10.5.3 (FlowJo, LLC) ( ) were used for data analysis.

Techniques: Isolation, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: M1 hot tumor-associated macrophages boost tissue-resident memory T cells infiltration and survival in human lung cancer

doi: 10.1136/jitc-2020-000778

Figure Lengend Snippet: Tumor-associated macrophages (TAMs) are enriched for both M2 and M1 features. (A) Scheme of the study. (B) Heatmap showing normalized expression of 1038 differentially expressed genes (DEG) (FDR ≤0.05, log 2 fold-change (FC) ≥ |1|, basemean ≥10, n=74). (C) t-SNE analysis shows clustering of TAMs and paired non-tumor-associated macrophages (NTAMs), selecting for the 3000 hypervariable genes. (D) Top dysregulated canonical pathways based on the expression of 1038 DEGs. Statistical values displayed as –log 10 (p value). Purple columns indicate pathways related to M1 functions and red columns pathways related to M2 functions. Statistical significance baseline (dotted line) corresponds to a p value of 0.05. (E) Heatmaps showing expression of selected genes from M2 typical antitumor functions in TAMs versus NTAMs. (F) gene set enrichment analysis (GSEA) of various gene sets in the transcriptome of TAMs versus that of all NTAMs, presented as the running enrichment score for the gene set list of genes ranked to degree of over-representation. P values, Kolmogorov-Smirnov test. (G) Predicted upstream regulators (cytokines and growth factors) related to gene expression changes observed in (B). Purple columns indicate upstream regulators related to M1 functions and red columns to M2 functions. Statistical significance baseline (dotted line) corresponds to a p value of 0.05. Data are from n=41 donors (n=40 TAMs, n=34 NTAMs; n=33 paired, n=66 samples in total). Related to .

Article Snippet: Visualizations including PCA, gene set enrichment analysis (GSEA), t-distributed Stochastic Neighbor Embedding (t-SNE), hierarchical clustering and heatmaps were performed using R V.3.3.2 and Qlucore (V.3.2).

Techniques: Expressing, Gene Expression

Journal: Journal for Immunotherapy of Cancer

Article Title: M1 hot tumor-associated macrophages boost tissue-resident memory T cells infiltration and survival in human lung cancer

doi: 10.1136/jitc-2020-000778

Figure Lengend Snippet: M1 hot tumor-associatedmacrophages (TAMs) are associated with robust T-cell responses in tumor. (A) Heatmap showing stratification of patient TAMs (n=40) into M1 cold , M1 intermediate and M1 hot , based on their expression of M1 markers chemokine (C-X-C motif) ligand ( CXCL)9 , CXCL10 , CXCL11 , CXCL12 , STAT1 , FAM26F . Expression of M2 markers matrix metallopeptidase 12 ( MMP12 ), WNT5A , IL10 , ADORA3 , IL4I1 , CD209 is also shown. Expression of M1 (B) and M2 (C) genes in non-tumor-associatedmacrophages (NTAMs), M1 hot TAMs and M1 cold TAMs (categorized by expression of CXCL9 in log 2 normalized counts, by RNA-sequencing (RNA-seq)). Statistical significance by one-way analysis of variance. (D) Heatmap showing the differential gene expression (DEG) of 222, obtained by DESeq2 analysis between M1 hot TAMs (top 25% percentile of CXCL9 expression by RNA-seq) versus M1 cold TAMs (bottom 25% percentile) represented by Row-Z-score across samples (FDR ≤0.05, log 2 fold-change (FC) ≥ |1|, basemean ≥10, n=20). Right margin, genes encoding M1 protumor functions and lipid uptake functions are indicated. Tumor subtype, cancer stage, gender and smoking history of each group were compared in . (E) Top dysregulated canonical pathways based on the expression of 222 DEGs. Statistical values displayed as –log 10 (p value). Purple columns indicate pathways related to M1 functions. Statistical significance baseline (dotted line) corresponds to a p value of 0.05. (F) Box and whisker graph indicates CD8 + T-cell infiltration in non-small cell lungcancer (NSCLC) tumors with M1 hot TAMs (n=10) versus infiltration in NSCLC tumors with M1 cold TAMs (n=10). Statistical significance by Mann-Whitney U test. (G) Gene set enrichment analysis (GSEA) of M1 and M2 gene sets in the transcriptome of TAMs from NSCLC with TIL high TAMs versus that of TIL low , presented as the running enrichment score. Statistical significance by Kolmogorov-Smirnov test. Statistical significance expressed as ns for non-significant (p>0.05), *p≤0.05, ***p≤0.001 and ****p≤0.00001. Related to .

Article Snippet: Visualizations including PCA, gene set enrichment analysis (GSEA), t-distributed Stochastic Neighbor Embedding (t-SNE), hierarchical clustering and heatmaps were performed using R V.3.3.2 and Qlucore (V.3.2).

Techniques: Expressing, RNA Sequencing, Gene Expression, Whisker Assay, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: M1 hot tumor-associated macrophages boost tissue-resident memory T cells infiltration and survival in human lung cancer

doi: 10.1136/jitc-2020-000778

Figure Lengend Snippet: M1 hot tumor-associatedmacrophages (TAMs) support tissue-resident memory T cells (T RM ) cell maintenance by allowing uptake of fatty acids. (A) Frequency of CD103 high , CD103 intermediate and CD103 low tumors among those categorized by chemokine (C-X-C motif) ligand 9 ( CXCL9 ) expression in TAMs (M1 hot , M1 intermediate , M1 cold ; numbers of patients above each bar). (B) Heatmap showing expression of genes related to Tissue Resident Memory features (T RM phenotype) in CD8 + T cells isolated from the same tumors from patients with non-small cell lungcancer (NSCLC) were M1 hot TAMs and M1 cold TAMs were isolated and studied. (C) Gene set enrichment analysis (GSEA) of 246 gene sets in the transcriptome of CD8 + T cells from patients with NSCLC with M1 hot TAMs versus those with M1 cold TAMs, presented as the running enrichment score. Statistical significance by Kolmogorov-Smirnov test. (D) Expression of FABP3, FABP4 and FABP5 in non-tumor-associatedmacrophages (NTAMs), M1 hot TAMs and M1 cold TAMs (log 2 normalized counts). Statistical significance by ordinary one-way analysis of variance. (E) Macrophages derived from blood (BDM) treated with interfern (INF)-γ/lipopolysaccharides (LPS) (M1 hot -like BDM, red) or interleukin (IL)-4 (M1 cold -like BDM, blue) cultured with bodipy lipid probe for 30 min (n=7). Lipid uptake measured by flow cytometry and expressed as mean fluorescence intensity. Wilcoxon test, two-tailed. *P≤0.05 (F) M1 hot -like BDM (red) or M1 cold -like BDM (blue) were co-cultured with CD3 + cells and bodipy lipid probe for 30 min (n=6). Uptake of lipid represented as Mean fluorescence intensities (MFI) in T RM (CD8 + CD103 + ), CD8 + (CD8 + CD103 - ) and CD3 + (CD8 - CD103 - ) cells was determined by flow cytometry. Wilcoxon test, two-tailed. *P≤0.05. ns, non-significant for p value >0.05, *p≤0.05, **p≤0.01 and ****p≤0.00001. Related to .

Article Snippet: Visualizations including PCA, gene set enrichment analysis (GSEA), t-distributed Stochastic Neighbor Embedding (t-SNE), hierarchical clustering and heatmaps were performed using R V.3.3.2 and Qlucore (V.3.2).

Techniques: Expressing, Isolation, Derivative Assay, Cell Culture, Flow Cytometry, Fluorescence, Two Tailed Test