cd3 magnetic beads  (Miltenyi Biotec)

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