Journal: International Journal of Molecular Sciences

Article Title: Proinflammatory Cytokine Preconditioning Enhances the Therapeutic Potency of Different Types of MSCs in Inflammation

doi: 10.3390/ijms27094090

Figure Lengend Snippet: The effects of naïve and Cytomix-stimulated MSCs on T cell proliferation. The gating strategy is shown in . ( A – C ) BM-MSCs, iMSCs WT and iMSCs B2M KO inhibition on the expansion of CD3+, CD3+CD4+ and CD3+CD8+ T cells via CM. Cytomix enhanced the three types of MSCs’ inhibition on the EI of T cells. ( D – F ) All types of MSCs significantly inhibited the division of CD3+, CD3+CD4+ and CD3+CD8+ T cells when co-cultured with PBMCs, in a dose-dependent manner. ( G – I ) All of the MSCs, naïve or Cytomix stimulated, significantly reduced the EI of CD3+, CD3+CD4+ or CD3+CD8+ T cells, at the lowest ratio of MSCs, with iMSCs WT both naïve and Cytomix-stimulated presenting the strongest inhibition. ( J ) Co-culturing all types of MSCs, naïve or Cytomix-stimulated, increased the production of G-CSF in the media, with a dose-dependent effect. ( K ) All types of MSCs significantly decreased the level of IFN-γ at the ratios of 1:20 and 1:10, while the three types of naïve MSCs significantly decreased it at 1:50. ( L ) Naïve and Cytomix-stimulated iMSC WTs significantly increased the level of TNF-α at the ratios of 1:50, 1:20 and 1:10, while Cytomix-stimulated iMSC B2M KO significantly increased it at the ratio of 1:50 and 1:20. ( M ) Naïve BM-MSCs significantly increased the level of IL-10 production at the ratios of 1:20 and 1:10. Representative results of four independent experiments are shown. Results are shown as mean ± SD ( n = 3 in CM; n = 4 in co-culture, each group), and analysed by two-way ANOVA with Šídák’s multiple comparisons test to compare naïve vs. Cytomix in each type of MSCs (no significance in all of the settings), or Tukey’s multiple comparisons test to compare different ratios of MSCs to PBMCs in each MSC group, or Dunnett’s multiple comparisons test to compare different MSC groups vs. vehicle in the setting of the same ratio. */**/***/**** represent comparisons of each type of MSC vs. vehicle; #/## represent comparisons of different ratios of each cell type with different levels of p < 0.05/0.01. EI: expansion index; DP: division percentage.

Article Snippet: The PBMCs were stained with anti-human CD3-APC-Vio770, CD4-APC and CD8-PE-Vio770 antibodies (Cat# 130-113-136, 130-113-222, 130-110-680; Miltenyi Biotec) for 30 min on ice in the dark.

Techniques: Inhibition, Cell Culture, Co-Culture Assay

Journal: Cell Reports Medicine

Article Title: Single-cell analysis highlights the significance of malignant cell IFN/MHC-II for immunotherapy response in head and neck squamous cell carcinoma

doi: 10.1016/j.xcrm.2026.102715

Figure Lengend Snippet: Malignant cell-specific expression of MHC-II is IFN-γ dependent but dispensable for anti-PD1-mediated tumor control (A) Post-treatment multiplex immunofluorescence (mIF) PhenoCycler images of formalin-fixed paraffin-embedded-stained tissues show representative areas of high HLA-DR expression on malignant cells from a representative tumor (OCSCC3). Top left image shows whole-slide view, while other panels represent magnified fields of view from inset, showing (from top left to bottom right) malignant cells highlighted by TP63 and panCK, HLA-DR expression, HLA-DR expression within malignant cells, assigned cell boundaries following Instanseg cell segmentation and HLA-DR +/− classification based on centered log-ratio threshold, staining for T cell and macrophages (CD14 and CD16A combined in blue, CD8 in green, CD3 in red, and FOXP3 in white), post-classification cell-type assignment, and DNA stain for all cells. (B) Line plots show percent of HLA-DR-positive malignant cells by mIF, pre- and post-treatment. Lines connect paired samples from individual patients ( n = 4). (C) Bar plots show relative count of seven neighboring cell types within a 30 μm radius of malignant cells that are HLA-DR+ (orange bars) and HLA-DR− (blue bars). Bars show counts relative to values for HLA-DR- malignant cells. Pink dots represent responders, while green dots represent non-responders. Error bars represent the SEM. Asterisks (∗ p < 0.05, ∗∗ p < 0.01) denote significance by unpaired one-sided t test, HLA-DR+ greater than HLA-DR−. HLA-DR+ malignant cells are surrounded by greater numbers of CD4 T cells ( p = 0.006) and macrophages ( p = 0.036) than HLA-DR− malignant cells. (D and E) (D) Schematic for (E–G). C57BL/6 mice received anti-IFN-γ or IgG control 1 week prior to tumor implantation and once weekly starting at tumor implantation. MOC1 tumor-bearing mice then received anti-PD1 or IgG control antibodies on days 3, 6, and 9 post-tumor implantation. Growth of tumors was tracked until day 30 post-implantation. Tumors were then harvested for bulk RNA-seq. (E) Growth of tumors from (D). Error bars denote standard error between mice within each treatment group. Asterisks (∗∗∗ p < 0.001) denote significance by t test at day 30. (F) Volcano plot shows genes with largest expression differences between tumor-bearing mice treated with anti-PD1 vs. anti-PD1 + anti-IFN-γ. Red dots highlight genes involved in MHC-II presentation. (G) Boxplot shows MHC-II expression score by bulk RNA-seq of MOC1 tumors from (D). Each dot represents one mouse. Asterisks (∗∗ p < 0.01, ∗∗∗ p < 0.001) denote significance by t test. (H and I) (H) Schematic for (I). WT or Ciita KO MOC1 cell lines were implanted into C57BL/6 mice. Tumor-bearing mice received anti-PD1 or IgG control antibodies on days 3, 6, and 9 post-tumor implantation. Growth of tumors was tracked until day 30 post-implantation. Tumors were then harvested for flow cytometry analysis. (I) Growth of tumors from (H). Error bars denote standard error between mice within each treatment group. Asterisks (∗ p < 0.05, ∗∗∗ p < 0.001) denote significance by t test.

Article Snippet: The single-cell suspension was sorted via magnetic column using either human CD45 magnetic MicroBeads (Miltenyi) or human CD3 magnetic MicroBeads (Miltenyi) according to manufacturer’s protocol for up to 10 7 total cells.

Techniques: Expressing, Control, Multiplex Assay, Immunofluorescence, Formalin-fixed Paraffin-Embedded, Staining, Tumor Implantation, RNA Sequencing, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Single-cell analysis highlights the significance of malignant cell IFN/MHC-II for immunotherapy response in head and neck squamous cell carcinoma

doi: 10.1016/j.xcrm.2026.102715

Figure Lengend Snippet: Malignant cell-specific IFN-γ signaling sustains MHC-II expression and correlates with a favorable immune microenvironment and therapeutic response under PD-1 blockade (A) Schematic for (B and C). WT or Ifngr1 KO MOC1 cells were implanted into C57BL/6 mice. Tumor-bearing mice received anti-PD1 or IgG control antibodies on days 3, 6, and 9 post-tumor implantation. Growth of tumors was tracked until day 30 post-implantation. Tumors were then harvested for flow cytometry analysis. (B) (Top) Representative flow cytometry plots for malignant cell-specific MHC-II (I-A/I-E) expression (gated: live cells/CD45 − /EpCAM + /I-A/I-E + ). Expression was determined by MFI. (Bottom) Boxplot shows malignant cell-specific MHC-II expression for each group from (A). Each dot represents one mouse. Asterisk (∗ p < 0.05) denotes significance by t test. (C) Growth of tumors from (A). Error bars denote standard error between mice within each treatment group. Asterisks (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001) denote significance by t test. (D) Schematic for (E–J). WT MOC1 tumors were implanted into C57BL/6 mice. Tumor-bearing mice received anti-PD1 or IgG control antibodies on days 3, 6, and 9 post-tumor implantation. Tumors were harvested for flow cytometry analysis on day 30 post-implantation. (E) Representative flow cytometry plots for MHC-II high and MHC-II low tumors from (D). (F) Scatterplot shows, for each mouse from (D), malignant cell-specific MHC-II MFI ( x axis) and the tumor mass ( y axis). Spearman correlation and p value are shown. Each dot represents one mouse. Data were pooled from 3 independent experiments. (G) Violin plot shows the distribution of MHC-II expression from (D). The top 8 tumors were classified as MHC-II high ; the bottom 8 tumors were classified as MHC-II low . (H) Boxplots show malignant cell-specific MHC-II expression and infiltrating immune cell types between MHC-II high and MHC-II low tumors. Values for each comparison were scaled by the average of MHC-II low tumors. T cells were gated live cells/CD45 + /CD3 + . Monocytes were gated live cells/CD45 + /CD3 − /CD11b + /Ly-6C + /Ly-6G − . Neutrophils were gated live cells/CD45 + /CD3 − /CD11b + /Ly-6G + /Ly-6C int . Macrophages were gated live cells/CD45 + /CD3 − /CD11b + /Ly-6G − /Ly-6C − /F4/80 + . Asterisks (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001) denote significance by t test. (I) Violin plot shows the distribution of tumor masses from (D). The top 5 tumors were classified as non-response; the middle 17 tumors were classified as response; the bottom 3 tumors were classified as exceptional response. (J) Boxplots show potential response markers (tumor-specific MHC-II, MHC-I, PD-L1, and myeloid MHC-II) across non-responding, responding, and exceptional-response tumors. Values for each comparison were scaled by the average expression in exceptional-response tumors. Asterisks (∗ p < 0.05, ∗∗∗ p < 0.001) denote significance by t test.

Article Snippet: The single-cell suspension was sorted via magnetic column using either human CD45 magnetic MicroBeads (Miltenyi) or human CD3 magnetic MicroBeads (Miltenyi) according to manufacturer’s protocol for up to 10 7 total cells.

Techniques: Expressing, Clinical Proteomics, Control, Tumor Implantation, Flow Cytometry, Comparison