pe anti mouse trem2  (R&D Systems)

Journal: bioRxiv

Article Title: Spinal Cord Microglia Exhibit Impaired Repair Responses to Myelin Damage

doi: 10.64898/2026.02.23.707565

Figure Lengend Snippet: (A, C). Representative immunofluorescence images of the medial corpus callosum in the brain and the dorsal region in the lumbar spinal cord stained with antibodies for MerTK and TREM2. (B, D) Mean intensities graphed to depict the comparison between brain and spinal cord. Mean intensities were obtained using Fiji Image J software. Error bars represent standard error of the mean. Statistical analysis was performed using a 3-way ANOVA with Tukey’s post-hoc analysis, n = 5 – 15 where each sample represents a different animal, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001.

Article Snippet: The resulting cells were incubated in an FC block solution (Tonbo 70-0161, 1:100) for 10 minutes and then stained in an antibody mix for 30 minutes which consisted of CD45 violetFluor 450 (Tonbo 75-0451, 1:75), CD11b PerCP-Cyanine5.5 (Tonbo 65-0112, 1:75), Brilliant Violet 605 anti-mouse CD16 (Biolegend 158027, 1:75), FITC anti-mouse CD68 (Biolegend 137006, 1:75), APC/Fire 810 anti-mouse CX3CR1 (Biolegend 149054, 1:75), APC anti-mouse F4/80 (Biolegend 123116, 1:75), APC/Fire 750 anti-mouse CD73 (Biolegend 127221, 1:75), PE anti-mouse TREM2 (R&D Systems FAB17291P, 1:75), and viability dye (Tonbo 13-0871, 1:1000).

Techniques: Immunofluorescence, Staining, Comparison, Software

Journal: Clinical and Translational Medicine

Article Title: Comprehensive tumour‐immune profiling reveals TREM2 + tumour‐associated macrophages facilitating lymph node metastasis in head and neck squamous cell carcinoma

doi: 10.1002/ctm2.70604

Figure Lengend Snippet: TREM2 + TAMs co‐infiltrate with CD8 + Tex. (A) Correlation matrix of infiltration proportions between cell types, crosses denote p > .05 by Spearman test. (B) UMAP visualisation of TAM subsets. (C) Heatmap displaying top DEGs across TAM subsets. (D) Correlation matrix of infiltration proportions among TAM and T cell subsets, * p < .05, ** p < .01 by Spearman test. (E) Correlation of infiltration between TREM2 + TAMs and CD8 + Tex in scRNA‐seq and microarray datasets by Spearman test. (F) Proportions of TAM subsets in LN metastasis‐positive and ‐negative tumour tissues. (G) Infiltration analyses in scRNA‐seq and microarray datasets showing abundance of TREM2 + TAMs in LN metastasis‐positive and ‐negative tumour tissues, * p < .05, ** p < .01 by Mann–Whitney U test. (H) Distribution of TAM subsets across tissue types. (I) Representative mIHC images showing co‐infiltration of CD8 + Tex and TREM2 + TAMs in LN metastasis‐positive and ‐negative tumour tissues. Scale bar, 50 µm; insets, 20 µm. (J) Proportion of TREM2 + TAMs among total TAMs in LN metastasis‐positive and ‐negative tumours in mIHC. Error bars show the mean ± SD, ** p < .01 by Two‐way ANOVA test. (K) Correlation of infiltration levels between TREM2 + TAMs and CD8 + Tex in mIHC by Spearman test. (L) FACS of TAM subsets from HNSCC tissues. (M) and (N) Flow cytometric analysis of exhaustion markers on CD8 + T cells stimulated with TAM subsets. Error bars show the mean ± SD, ** p < .01 by Two‐way ANOVA test. (O) Western blotting analysis of BHLHE40 expression in CD8+ T cells co‐cultured with TAM subsets. (P) qRT‐PCR result showing relative BHLHE40 mRNA expression in CD8 + T cells transfected with si‐NC or si‐BHLHE4. Error bars show the mean ± SD, ** p < .01 by Two‐way ANOVA test. (Q) Immunofluorescence showing the expression of exhaustion markers on CD8 + T cells with or without BHLHE40 silencing co‐cultured with TAM subsets. Scale bar, 5 µm. (R) and (S) Flow cytometric analysis of exhaustion markers expression on CD8 + T cells with or without BHLHE40 silencing co‐cultured with TAM subsets. Error bars show the mean ± SD, ** p < .01 by Two‐way ANOVA test. ANOVA, Analysis of Variance; DEGs, differentially expressed genes; FACS, Fluorescence‐Activated Cell Sorting; HNSCC, head and neck squamous cell carcinoma; LN, lymph node; mIHC, multiplex immunohistochemistry; mRNA, messenger RNA; qRT‐PCR, quantitative real‐time PCR; scRNA‐seq, single‐cell RNA sequencing; SD, standard deviation; si‐NC, small interfering RNA negative control; TAMs, tumour‐associated macrophages; Tex, exhausted T cells; UMAP, uniform manifold approximation and projection.

Article Snippet: The following antibodies were used: CD8 (BioLegend, 344704), LAG‐3 (BD Biosciences, 565716), PD‐1 (BD Biosciences, 563245), and TIM‐3 (BD Biosciences, 565562) for CD8 + T cells; TREM2 (R&D Systems, FAB17291P) and SPP1 (eBioscience, 3002684) for TREM2 + TAMs.

Techniques: Microarray, MANN-WHITNEY, Western Blot, Expressing, Cell Culture, Quantitative RT-PCR, Transfection, Immunofluorescence, Fluorescence, FACS, Multiplex Assay, Immunohistochemistry, Real-time Polymerase Chain Reaction, RNA Sequencing, Standard Deviation, Small Interfering RNA, Negative Control

Journal: Clinical and Translational Medicine

Article Title: Comprehensive tumour‐immune profiling reveals TREM2 + tumour‐associated macrophages facilitating lymph node metastasis in head and neck squamous cell carcinoma

doi: 10.1002/ctm2.70604

Figure Lengend Snippet: ETV5 contributes to the differentiation of TREM2 + TAMs. (A) Trajectory of TAM subsets by scTour. (B) TAM subsets on Diffusion Map, coloured by subsets. (C) Trajectory of TAM subsets by Monocle3 on Diffusion Map. (D) Differentiation lineage of TAMs by Slingshot on Diffusion Map, D–G, and J are based on Slingshot results. (E) Distribution of TAM subsets along pseudotime. (F) Heatmap showing differentiation‐related genes in TAMs. (G) Expression of main TAM subset markers along pseudotime. (H) Dot plot displaying the top TF regulons with highest RSSs of TAM subsets. (I) t‐SNE plot based on AUC scores of TF regulons. The top‐left panel shows cell subsets, the remaining panels highlight cells that activate each regulon (AUC > threshold). (J) Expression of TREM2 + TAM‐specific TFs along pseudotime. (K) Spearman correlation between the scores of TREM2 + TAM‐specific TF regulons and the proportion of CD8 + Tex. (L) Regulon scores of each TREM2 + TAM‐specific TF between LN metastasis‐positive and ‐negative groups, ns not significant, * p < .05, ** p < .01 by Mann–Whitney U test. (M) Kaplan–Meier survival curves generated for ETV5 regulon scores from microarray datasets. The samples were divided into high‐ and low‐score groups (optimal cutoff), using the log‐rank test. (N) GO enrichment of the ETV5 regulon gene set, the top 14 pathways were shown. (O) GSEA results comparing ETV5 regulon gene set in TREM2 + TAMs and other TAM subsets. (P) Western blotting analysis of ETV5 expression in TAM subsets. AUC, Area under the curve; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; LN, lymph node; ns, not significant; RSS, Regulon Specificity Score; scTour, single‐cell trajectory inference using optimal transport; TAMs, tumour‐associated macrophages; Tex, exhausted T cells; TF, transcription factor; t‐SNE, t‐distributed Stochastic Neighbour Embedding.

Article Snippet: The following antibodies were used: CD8 (BioLegend, 344704), LAG‐3 (BD Biosciences, 565716), PD‐1 (BD Biosciences, 563245), and TIM‐3 (BD Biosciences, 565562) for CD8 + T cells; TREM2 (R&D Systems, FAB17291P) and SPP1 (eBioscience, 3002684) for TREM2 + TAMs.

Techniques: Diffusion-based Assay, Expressing, MANN-WHITNEY, Generated, Microarray, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Comprehensive tumour‐immune profiling reveals TREM2 + tumour‐associated macrophages facilitating lymph node metastasis in head and neck squamous cell carcinoma

doi: 10.1002/ctm2.70604

Figure Lengend Snippet: TREM2 + TAMs crosstalk with CD8 + Tex through SPP1–CD44 signalling pathway. (A) Circle plot depicting the crosstalk among TAMs and T cell subsets. Width indicates the weight of the interaction strength. (B) Circle plot depicting the crosstalk among the CD8 + Tex and TAM subsets. Width indicates the weight of the interaction strength. (C) Interaction frequency of each subset as senders (source) and receivers (target). (D) Chord diagram showing all the ligand–receptor interactions between TREM2 + TAMs and CD8 + Tex, coloured by interaction strength. (E) Interaction strength between pairwise cell subsets, coloured by strength score. (F) Heatmap illustrating pathways involved in the interaction between TREM2 + TAMs and CD8 + Tex, outgoing signalling (left), and incoming signalling (right), coloured by interaction strength. (G)–(I) Heatmap showing the interaction probability of signalling pathways among subsets (top), and chord diagram displaying the interaction strength of corresponding ligand–receptor pairs among subsets (bottom). (J) Violin plot showing the expression level of ligands in TAM subsets. (K) UMAP plot visualising the expression density of TREM2 and SPP1 in TAMs. (L) and (M) Flow cytometric analysis showing SPP1 expression in TAM subsets ( n = 4). Error bars show the mean ± SD, ** p < .01 by Two‐way ANOVA test. (N) Correlation between the expression level of SPP1 with the proportion of TREM2 + TAM/TAMs (left) and the proportion of CD8 + Tex/CD8 + T cells (right) across samples by Spearman test. (O) Correlations between the expression level of SPP1 with ETV5 (left) and BHLHE40 (right) across samples by Spearman test. (P) Correlations between the expression level of SPP1 with the proportion of CD8 + Tex in microarray dataset using MCP‐counter deconvolution by Spearman test. ANOVA, Analysis of Variance; SD, standard deviation; TAMs, tumour‐associated macrophages; Tex, exhausted T cells; UMAP, uniform manifold approximation and projection; ns, not significant.

Article Snippet: The following antibodies were used: CD8 (BioLegend, 344704), LAG‐3 (BD Biosciences, 565716), PD‐1 (BD Biosciences, 563245), and TIM‐3 (BD Biosciences, 565562) for CD8 + T cells; TREM2 (R&D Systems, FAB17291P) and SPP1 (eBioscience, 3002684) for TREM2 + TAMs.

Techniques: Expressing, Microarray, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Comprehensive tumour‐immune profiling reveals TREM2 + tumour‐associated macrophages facilitating lymph node metastasis in head and neck squamous cell carcinoma

doi: 10.1002/ctm2.70604

Figure Lengend Snippet: TREM2 + TAMs promote CD8 + T cell exhaustion through SPP1–CD44–BHLHE axis. (A) Representative mIHC images showing co‐infiltration of CD8 + Tex and SPP1 + TREM2 + TAMs in LN metastasis‐positive and ‐negative tumour tissues. Scale bar, 100 µm; insets, 50 µm. (B) Expression percentage of TREM2 and SPP1 in LN metastasis‐positive and ‐negative tumours in mIHC, * p < .05 by Two‐way ANOVA test. (C) Correlation of expression percentage between TREM2 and SPP1 in mIHC by Spearman test. (D) Proportion of TREM2 + SPP1 + TAM in total TAMs in LN metastasis‐positive and ‐negative tumours in mIHC, * p < .05 by Two‐way ANOVA test. (E) Correlation of TREM2 + SPP1 + TAM and CD8 + Tex infiltration proportion in mIHC by Spearman test. (F) co‐IP followed by Western blotting analysis demonstrating the interaction between SPP1 and CD44 in CD8 + T cells co‐cultured with TREM2 + TAMs. (G) Western blotting analysis for the binding of recombinant CD44 and His‐tagged SPP1 in co‐IP assay. (H) qRT–PCR result showing relative CD44 mRNA expression in CD8 + T cells transfected with si‐NC or si‐CD44. Error bars show the mean ± SD, ** p < .01 by Two‐way ANOVA test. (I) Immunofluorescence showing the co‐localisation of SPP1 and CD44 on CD8 + T cells with or without CD44 silencing. Scale bar: 5 µm. (J) Representative mIHC images of SPP1 and CD44 co‐location on CD8 + T cell membranes in LN metastasis‐positive and ‐negative tumour tissues. Scale bar: 100 µm; inset scale bar: 50 µm. (K) Quantification of CD44 + SPP1 + CD8 + T cells across LN metastasis‐positive ( n = 7) and ‐negative ( n = 7) tumours by mIHC. Error bars show the mean ± SD, ** p < .01 by Two‐way ANOVA test. (L) Western blotting analysis of BHLHE40 expression in CD8 + T cells co‐cultured with TAM subsets with or without anti‐SPP1 or anti‐CD44 neutralising antibodies. (M) Western blotting analysis of BHLHE40 expression in CD8 + T cells stimulated with rSPP1, with or without CD44 knockdown. (N) and (O) Flow cytometric analysis of exhaustion markers on CD8 + T cells co‐cultured with TAM subsets with or without anti‐SPP1 or anti‐CD44 antibodies. Error bars show the mean ± SD, ** p < .01 by Two‐way ANOVA test. (P) and (Q) Flow cytometric analysis showing exhaustion markers on CD8 + T cells treated with rSPP1, with or without CD44 or BHLHE40 knockdown. Error bars show the mean ± SD, ** p < .01 by Two‐way ANOVA test. (R) Immunofluorescence showing the expression of exhaustion markers on CD8 + T cells treated with rSPP1, with or without CD44 or BHLHE40 knockdown. Scale bar: 5 µm. ANOVA, Analysis of Variance; SD, standard deviation; si‐NC, small interfering RNA negative control; TAMs, tumour‐associated macrophages; Tex, exhausted T cells.

Article Snippet: The following antibodies were used: CD8 (BioLegend, 344704), LAG‐3 (BD Biosciences, 565716), PD‐1 (BD Biosciences, 563245), and TIM‐3 (BD Biosciences, 565562) for CD8 + T cells; TREM2 (R&D Systems, FAB17291P) and SPP1 (eBioscience, 3002684) for TREM2 + TAMs.

Techniques: Expressing, Co-Immunoprecipitation Assay, Western Blot, Cell Culture, Binding Assay, Recombinant, Quantitative RT-PCR, Transfection, Immunofluorescence, Knockdown, Standard Deviation, Small Interfering RNA, Negative Control

Journal: Clinical and Translational Medicine

Article Title: Comprehensive tumour‐immune profiling reveals TREM2 + tumour‐associated macrophages facilitating lymph node metastasis in head and neck squamous cell carcinoma

doi: 10.1002/ctm2.70604

Figure Lengend Snippet: Construction of an HNSCC prognostic model based on TREM2 + TAMs. (A) Kaplan–Meier survival curves based on TREM2 + TAM infiltration scores from three microarray datasets. The samples were stratified into high‐ and low‐score groups using the optimal cutoff by log‐rank test. (B) Kaplan–Meier survival curves based on SPP1 expression levels from three microarray datasets. The samples were divided into high‐ and low‐expression groups using the optimal cutoff, by log‐rank test. (C) Workflow diagram of prognostic model construction. (D) LASSO coefficient profiles of 119 genes in the training set ( GSE65858 ). (E) Ten‐fold cross‐validation identifying the optimal value of the penalty parameter (λ). (F) Relationship between error rate and the number of classification trees in RSF model. (G) Importance scores of the top 6 genes selected by the RSF model. (H) Calibration curves of 1‐year OS predictions in training set and validation sets. (I) Time‐dependent ROC curves and AUC values for 1‐, 3‐, and 5‐year OS predictions in both training and validation sets, using the Kaplan–Meier Method. (J) Heatmap of risk‐associated gene expression in training and validation sets. The samples were stratified into high‐ and low‐risk groups using the Youden index, by log‐rank test. (K) Kaplan–Meier survival curves based on risk scores in training and validation sets. The samples were divided into high‐ and low‐risk groups using the Youden cutoff, by log‐rank test. AUC, Area under the curve; HNSCC, head and neck squamous cell carcinoma; LASSO, least absolute shrinkage and selection operator; OS, overall survival; RSF, Random Survival Forest; TAMs, tumour‐associated macrophages.

Article Snippet: The following antibodies were used: CD8 (BioLegend, 344704), LAG‐3 (BD Biosciences, 565716), PD‐1 (BD Biosciences, 563245), and TIM‐3 (BD Biosciences, 565562) for CD8 + T cells; TREM2 (R&D Systems, FAB17291P) and SPP1 (eBioscience, 3002684) for TREM2 + TAMs.

Techniques: Microarray, Expressing, Biomarker Discovery, Gene Expression, Selection