Journal: Frontiers in Immunology

Article Title: A human 3D immune competent full-thickness skin model mimicking dermal dendritic cell activation

doi: 10.3389/fimmu.2023.1276151

Figure Lengend Snippet: Upon exposure to inflammatory stimuli such as LPS, or sensitizing chemicals such as 1 chloro-2,4-dinitrobenzene (DNCB) or nickel sulfate (NiSO 4 ), keratinocytes start to secrete inflammatory cytokines such as IL-1, TNF-α and IL-18. Subsequently cutaneous dendritic cells such as Langerhans cells and dermal dendritic cells become activated and start to phagocytose haptens or exogenous particles, which is accompanied by cell maturation and the upregulation of CD54 and CD86. Finally, DCs migrate to draining lymph nodes to present the processed antigen in order to activate CD4 + T cells. Created with BioRender.com .

Article Snippet: For staining the cells were incubated in Automacs Running Buffer with the following antibodies (1:50): REA Control (S)-VioGreen (Miltenyi, #130-113-444), REA Control (S)-PE (Miltenyi, #130-113-438), REA Control (S)-APC (Miltenyi, #130-113-434); REA Control (S)-PE-Vio770, (Miltenyi, #130-113-440); CD54-APC (Miltenyi, #130-121-342); CD86-APC (Miltenyi, #130-116-161), CD14-VioGreen (Miltenyi, #130-110-525), CD11c-APC (Miltenyi, #130-113-584) for 10 minutes in the dark.

Techniques:

Journal: Frontiers in Immunology

Article Title: A human 3D immune competent full-thickness skin model mimicking dermal dendritic cell activation

doi: 10.3389/fimmu.2023.1276151

Figure Lengend Snippet: Surface marker expression of CD54 and CD86 (depicted as fold of induction of the percentage of all positive cells) after (A) pre-treatment of THP-1-derived iDCs and (B) topical treatment of the immune competent skin model with dexamethasone for 1 h, followed by NiSO 4 treatment for 23 h. Results were depicted as fold of induction compared to the solvent control [0.3% DMSO]. (C–E) Cytokine secretion of iDCs after 1 h dexamethasone pre-treatment, followed by 23 h of NiSO 4 exposure. Error bars indicate the standard errors of the mean (n = 3 independent experiments for (A, C) and n=4 independent experiments for (B) with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).

Article Snippet: For staining the cells were incubated in Automacs Running Buffer with the following antibodies (1:50): REA Control (S)-VioGreen (Miltenyi, #130-113-444), REA Control (S)-PE (Miltenyi, #130-113-438), REA Control (S)-APC (Miltenyi, #130-113-434); REA Control (S)-PE-Vio770, (Miltenyi, #130-113-440); CD54-APC (Miltenyi, #130-121-342); CD86-APC (Miltenyi, #130-116-161), CD14-VioGreen (Miltenyi, #130-110-525), CD11c-APC (Miltenyi, #130-113-584) for 10 minutes in the dark.

Techniques: Marker, Expressing, Derivative Assay, Solvent, Control

Journal: Scientific Reports

Article Title: Shuang-Huang-Lian injection induces an immediate hypersensitivity reaction via C5a but not IgE

doi: 10.1038/s41598-018-21843-7

Figure Lengend Snippet: ( A ) SHLI elevates mouse serum tIgE levels 4–7 weeks after immunization (n = 8). The mice were injected weekly (i.p.) with aluminum adjuvant (100 μL/mouse) containing SHLI (200 μL/mouse) or ST (60 μg/mouse). The serum tIgE level was determined using a commercial ELISA kit. ( B ) SHLI causes an increase in mouse serum MMCP1 5 weeks after immunization (n = 8). The mice were injected weekly (i.p.) with aluminum adjuvant (100 μL/mouse) containing SHLI (200 μL/mouse) or ST (60 μg/mouse). The serum MMCP1 level was determined using a commercial ELISA kit. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs . NS. ( C,D ) Four constituents in SHLI increase THP-1 cells CD54 ( C ) and/or CD86 ( D ) levels. THP-1 cells were treated with 4 constituents (50 μg/mL) for 24 h at 37 °C and then stained with anti-human CD54-APC or anti-human CD86-FITC antibodies for 15 min at 25 °C. The expression of CD54 and CD86 on THP-1 cells was assayed by flow cytometry. Isotype control and unstained cells were used as NC. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs . the NC. ( E ) The serum MMCP1 concentration does not increased in the mice passively sensitized by SHLI antiserum. Balb/c mice were primed (i.v.) with NC serum (500 μL/mouse) or ST antiserum (10 μL/mouse) or SHLI antiserum (500 μL/mouse). Twenty-four hours later, the mice were challenged (i.v.) with ST (200 μg/mouse) or SHLI (200 μL/mouse). The NC mice were treated with an equal volume of NS. Four hours later, the whole blood was obtained and MMCP1 concentration in the serum was measured using an ELISA. *** P < 0.001.

Article Snippet: Anti-human CD54-allophycocyanin (APC) antibody and its REA control-APC were from Miltenyi Biotec. (Bergisch Gladbach, German).

Techniques: Injection, Adjuvant, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Flow Cytometry, Control, Concentration Assay

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: Single-cell characterization of cervical cancer and normal cervical tissues. A Left: UMAP projection of single-cell transcriptomes from cervical cancer (CC) and normal cervical tissues (NCT), colored by eight major cell types. Right: Dot plot displaying the average expression of established marker genes across annotated cell clusters. B Bar plot depicting the proportional distribution of cell types across samples. C Fibroblast abundance is significantly elevated in CC compared to normal tissue. D Left: Representative immunohistochemical (IHC) staining of α-SMA in CC (n = 10) and NCT (n = 6). Right: Quantification of α-SMA IHC intensity. E Kaplan–Meier survival analysis comparing patients with high versus low ACTA2 expression in the GEPIA2-CESC cohort. F Left: UMAP visualization of fibroblast subpopulations in tumor and normal tissues. Right: Dot plot showing mean expression of fibroblast subtype-defining markers. G Violin plots illustrating COL1A1 and COL1A2 expression across fibroblast subsets. H Volcano plot highlighting differentially expressed genes in fibroblasts between malignant and normal tissues. I Violin plot comparing CD54 expression levels in tumor versus normal tissue fibroblasts. J Aggregate CD54 expression in fibroblast subclusters, with iCAFs exhibiting predominant overexpression. Data in D (right panel) are presented as mean ± SD; statistical significance was determined by Student’s t-test. Survival analyses in E were performed using the log-rank test. *: P < 0.05, ***: P < 0.001

Article Snippet: In direct co-culture experiments, CD54 + iCAFs were pre-incubated with a neutralizing anti-human CD54 antibody (Proteintech) or an IgG isotype control.

Techniques: Expressing, Marker, Immunohistochemical staining, Immunohistochemistry, Over Expression

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: CD54⁺ iCAFs promote migration and predict poor prognosis in cervical cancer. A Multiplex immunofluorescence staining shows co-localization of CD54 and α-SMA in cervical cancer (CC) and normal cervical tissues (NCT) (Scale bar: 100 μm). B Flow cytometry analysis of isolated CD54⁺ iCAF subtypes from CC. C Transwell migration assay of HeLa cells co-cultured with CD54-knockdown or CD54-overexpression iCAFs and their corresponding control fibroblasts for 48 h. D Quantification of migrated HeLa cells from three independent experiments. E Left: Representative immunohistochemical staining of CD54 in CC stromal tissue (n = 105) and normal cervical stroma (n = 19). Right: Quantitative analysis of stromal CD54 IHC staining intensity. F Left: Representative IHC images of high and low stromal CD54 expression in CC tissues. Right: Kaplan–Meier survival analysis of patients stratified by high (n = 39) and low (n = 66) stromal CD54 IHC scores. G Kaplan–Meier survival analysis comparing CC patients with high vs. low CD54⁺ iCAF infiltration (GEPIA2-CESC dataset). Data in A (right panel), D, and E (right panel) are presented as mean ± SD; statistical significance was determined by Student’s t-test. Survival analyses in F and G were performed using the log-rank test. **: P < 0.01, ***: P < 0.001

Article Snippet: In direct co-culture experiments, CD54 + iCAFs were pre-incubated with a neutralizing anti-human CD54 antibody (Proteintech) or an IgG isotype control.

Techniques: Migration, Multiplex Assay, Immunofluorescence, Staining, Flow Cytometry, Isolation, Transwell Migration Assay, Cell Culture, Knockdown, Over Expression, Control, Immunohistochemical staining, Immunohistochemistry, Expressing

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: Interaction between fibroblasts and mononuclear phagocytes in cervical cancer. A Heatmap displaying Spearman correlation coefficients between cell types, with clustering analysis revealing the shortest Euclidean distance between mononuclear phagocytes (MPs) and fibroblasts. B CellChat analysis demonstrating robust fibroblast–MP interactions at both Counts and Weight levels. C UMAP projection of re-clustered MP subsets following dimensionality reduction. D Dot plot depicting average expression of marker genes across MP subpopulations. E CellChat analysis identified iCAFs as the fibroblast subtype with the strongest interaction (Counts and Weight) with macrophages. F Ligand–receptor analysis reveals ITGAL as the top predicted receptor for CD54 (ligand). G Multiplex immunofluorescence staining confirmed the presence of ITGAL + macrophage clusters co-expressing the M2 marker CD163 in CC tissues (Scale bar, 100 μm). H Multiplex immunofluorescence revealed spatial co-localization of CD54 + iCAFs and ITGAL + macrophages in cervical cancer tissues (Scale bar, 100 μm)

Article Snippet: In direct co-culture experiments, CD54 + iCAFs were pre-incubated with a neutralizing anti-human CD54 antibody (Proteintech) or an IgG isotype control.

Techniques: Expressing, Marker, Multiplex Assay, Immunofluorescence, Staining

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: Co-localization of CD54 + iCAFs and ITGAL + macrophages revealed by spatial transcriptomics. A Left: Hematoxylin and eosin (H&E) staining of spatial transcriptomic sections from two representative cervical cancer patients (Scale bar: 500 μm). Right: Spatial feature plots showing signature scores of CD54⁺ iCAFs, ITGAL⁺ macrophages, and their co-localization regions in matched tissue sections. B Distribution of signature scores for CD54⁺ iCAFs, ITGAL⁺ macrophages, and ITGAL⁻ macrophages across all spatial clusters. CD54⁺ iCAFs show strong co-localization with ITGAL⁺ macrophages, but not with ITGAL⁻ macrophages. C Pearson correlation analysis between signature scores of CD54⁺ iCAFs and ITGAL⁺ macrophages within co-localized spatial clusters across six patients. D GO analysis of biological processes significantly enriched in regions where CD54⁺ iCAFs and ITGAL⁺ macrophages co-localize. E Top eight KEGG pathways enriched among predicted target genes derived from co-localized CD54⁺ iCAF and ITGAL⁺ macrophage clusters

Article Snippet: In direct co-culture experiments, CD54 + iCAFs were pre-incubated with a neutralizing anti-human CD54 antibody (Proteintech) or an IgG isotype control.

Techniques: Staining, Derivative Assay

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: CD54⁺ iCAFs promote monocyte migration and M2-like polarization through CCL2 secretion. A Left: Transwell migration assay of THP-1 cells co-cultured with CD54⁺ iCAFs or normal fibroblasts (NFs) for 48 h. Right: Quantification of migrated cells (n = 3 independent experiments). Scale bar: 100 μm. B-C THP-1 cells co-cultured with CD54⁺ iCAFs show upregulated expression of M2-like macrophage biomarkers and cytokines. D CD54⁺ iCAFs were transfected with CD54-targeting siRNA (si-CD54) or negative control siRNA (si-NC). Volcano plot of differentially expressed genes (DEGs) identified by RNA-seq analysis between si-CD54 and si-NC groups. E CD54 + iCAFs were transfected with si-CD54 or si-NC. Then, CD54 and CCL2 protein levels were assessed by Western blot. Left: Representative images. Right: Quantification from n = 3 independent experiments. F ELISA quantification of CCL2 secretion from CD54⁺ iCAFs and NFs. G Left: THP-1 cell migration in response to recombinant CCL2 treatment (50 ng/ml, 48 h) or PBS (Control group). Right: Quantification of migrated cells. All quantitative data are presented as mean ± SD. Statistical significance was determined using Student’s t-test for panels A (right), B, C, E (right), F, and G (right). *: P < 0.05, **: P < 0.01, ***: P < 0.001; ns, not significant

Article Snippet: In direct co-culture experiments, CD54 + iCAFs were pre-incubated with a neutralizing anti-human CD54 antibody (Proteintech) or an IgG isotype control.

Techniques: Migration, Transwell Migration Assay, Cell Culture, Expressing, Transfection, Negative Control, RNA Sequencing, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Control

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: CD54⁺ iCAF-driven macrophage reprogramming promotes CXCL8 expression and tumor progression in vivo. A THP-1-derived macrophages, transfected with control siRNA (Control group) or ITGAL-targeting siRNA (Treatment group), were co-cultured with CD54⁺ iCAFs for 48 h and subsequently subjected to RNA-seq analysis. Volcano plot shows differentially expressed genes (DEGs) between control and treatment groups. B Left: Representative flow cytometry plots of CD54⁺ iCAF abundance. Middle: Quantitative analysis of CXCL8⁺ cell frequencies in CD54⁺ iCAF-high tumors (n = 3). Right: quantitative analysis of CXCL8 expression in CD54 + iCAF-high versus CD54 + iCAF-low (Displaying in Supplementary Fig. 5F) tumors. C Flow cytometric quantification of CXCL8-producing immune cell subsets in cervical cancer tissues. D Multiplex immunohistochemistry images showing macrophage-specific CXCL8 expression in cervical cancer tissue (Scale bar: 100 μm). E NIH/3T3 fibroblasts transfected with CD54 overexpression plasmid (OE-CD54) or empty vector control (OE-NC). CD54 and CCL2 secretion levels were quantified by ELISA. F Representative tumor images from C57BL/6 mice co-injected with TC-1 cells and NIH/3T3 fibroblasts expressing CD54 or empty vector (Control). G Tumor growth curves measured every 3 days (n = 3 mice/group). H-I Flow cytometry analysis of CD206⁺ macrophage infiltration in tumors from (F). I (Right): Quantification of CD206⁺ macrophage proportions. J Serum MIP-2 levels measured by ELISA in experimental groups from (F). All quantitative data are presented as mean ± SD. Statistical significance was determined using Student’s t-test for panels B (right), E, G(right), I (right), and J. *: P < 0.05, **: P < 0.001, ***: P <0.001

Article Snippet: In direct co-culture experiments, CD54 + iCAFs were pre-incubated with a neutralizing anti-human CD54 antibody (Proteintech) or an IgG isotype control.

Techniques: Expressing, In Vivo, Derivative Assay, Transfection, Control, Cell Culture, RNA Sequencing, Flow Cytometry, Multiplex Assay, Immunohistochemistry, Over Expression, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Injection

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: CXCL8 correlates with CD8⁺ T cell exclusion and promotes PD-L1 expression on macrophages through cell-contact and soluble-factor dependent mechanisms. A CIBERSORT analysis showing an inverse correlation between CXCL8 mRNA levels and CD8⁺ T cell infiltration. B Representative immunohistochemistry (IHC) images showing CD8⁺ T cell density in high- versus low-CXCL8 expressing tumors (Scale bar: 100 μm). C Negative correlation between protein levels of CXCL8 and PD-L1 based on IHC scoring (Pearson correlation). D Left: Flow cytometry plots of PD-L1⁺ cells. Right: Quantification of PD-L1 expression in high- versus low-CXCL8 tumors (n = 3 per group). E Representative IHC staining confirming macrophage-specific PD-L1 expression (Scale bar: 100 μm). F Left: Gating strategy for identifying PD-L1⁺ cells. Right: Quantification of PD-L1 expression across immune cell subtypes, showing macrophage dominance (n = 6). G Left: Flow cytometry profiles of PD-L1 expression. Right: PD-L1 levels in high- versus low-CXCL8 tumors. H Flow cytometry analysis of CXCL8 and PD-L1 expression on CD68+ macrophages co-cultured with CD54⁺ iCAFs under direct contact or Transwell conditions, with normal fibroblasts (NFs) and macrophage-only cultures as controls. The bar graph shows geometric mean fluorescence intensity from three independent experiments. Corresponding representative flow cytometry plots are shown in Supplementary Fig. 6D. I CXCL8 and PD-L1 expression on macrophages after co-culture with CD54⁺ iCAFs and treatment with IgG control, anti-CD54, or anti-ITGAL blocking antibodies. See Supplementary Fig. 6E for flow plots. J CXCL8 and PD-L1 expression on macrophages transfected with control siRNA (si-NC) or ITGAL-targeting siRNA (si-ITGAL), with or without CD54 + iCAF co-culture. Corresponding representative flow cytometry plots are shown in Supplementary Fig. 6F. K CXCL8 and PD-L1 expression on macrophages co-cultured with CD54⁺ iCAFs and treated with IgG control or anti-CCL2 neutralizing antibody. Representative flow plots are provided in Supplementary Fig. 6G. Data are presented as mean ± SD. Statistical tests used: two-tailed Student’s t-test (D, right; G, right; K); one-way ANOVA with Tukey's multiple comparisons test (F, right; H, I, J). **: P < 0.01, ***: P < 0.001; ns, not significant

Article Snippet: In direct co-culture experiments, CD54 + iCAFs were pre-incubated with a neutralizing anti-human CD54 antibody (Proteintech) or an IgG isotype control.

Techniques: Expressing, Immunohistochemistry, Flow Cytometry, Cell Culture, Fluorescence, Co-Culture Assay, Control, Blocking Assay, Transfection, Two Tailed Test

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: Therapeutic targeting of the CXCL8 pathway enhances anti-tumor immunity in cervical cancer. A Frequencies of PD-L1⁺ cells and PD-L1⁺ macrophages (gated on CD68⁺ cells) in primary cervical cancer tissues after ex vivo treatment with reparixin or DMSO control (n = 9 independent patient samples with sufficient cell yield for analysis). B-C Frequencies of CD45⁺CD3⁺CD8⁺ tumor-infiltrating lymphocytes (TILs) and percentages of cytokine-producing (TNF-α, IFN-γ) and cytolytic (CD107a, PRF1, GZMB) CD8⁺ TILs in patient tissues following reparixin or DMSO treatment (n = 6 patients with adequate cell numbers for full T cell immunophenotyping). D Experimental timeline: C57BL/6 mice were subcutaneously inoculated with TC-1 tumor cells (5 × 10⁵) and CD54-overexpressing NIH/3T3 fibroblasts (5 ×10 6 ) on day 0, followed by treatment with anti-PD-1 (αPD-1, every three days) and/or reparixin (50 µg/mouse, every two days) (n = 3 per group). E Left: Representative tumor images at endpoint. Right: Tumor growth curves. Treatments were initiated when tumor volumes reached approximately 50 mm³. F-G Functional profiles of CD8⁺ TILs showing frequencies of cells expressing effector cytokines (IFN-γ, TNF-α) and cytolytic markers (GZMB, CD107a) following treatment with αPD-L1, reparixin, or their combination. Data in A–C, E (right), F, and G are presented as mean ± SD. Statistical analyses: paired two-tailed Student’s t-test (reparixin vs. DMSO control for each patient); one-way ANOVA with Dunnett’s multiple comparisons test (E, right, F, G). *: P < 0.05, **: P < 0.01, ***: P < 0.001; ns, not significant

Article Snippet: In direct co-culture experiments, CD54 + iCAFs were pre-incubated with a neutralizing anti-human CD54 antibody (Proteintech) or an IgG isotype control.

Techniques: Ex Vivo, Control, Functional Assay, Expressing, Two Tailed Test

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: CD54 + iCAFs-ITGAL + macrophage drive immunosuppression through CXCL8-PD-L1 mechanisms, and targeting this axis with reparixin and PD-L1 represents a promising strategy to overcome immune resistance in cervical cancer. This image was created with the help of BioRender

Article Snippet: In direct co-culture experiments, CD54 + iCAFs were pre-incubated with a neutralizing anti-human CD54 antibody (Proteintech) or an IgG isotype control.

Techniques: