Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Semiquantitative evaluation of protein expression using proteome profiler arrays. Representative protein profiler arrays of defects treated with hADM, hADM + BMC, or hADM + BMC-CD8 after three days are shown in ( A ). The differentially expressed proteins are marked by a red square. Protein expression was analyzed denitometrically and spot density was normalized to control spots located in the upper left, upper right, and lower left corners. Heatmap generated on basis of protein expression is shown in ( B ). Heatmap contains data from differentially pretreated hADM recovered on D3 and 7. Proteins newly secreted on D7 are marked in red. Color code indicates fold change. The pie charts ( C ) show the number of secreted proteins from membranes that covered the bone defect for 3 (upper pie chart) and 7 days (lower pie chart). The categories inflammation (red), anti-inflammation (blue) and regeneration (orange) are shown. Bi- or multifunctional proteins were assigned to the respective categories, so that the sum of the fractions is higher than the total number of proteins detected. There was a general increase in the number of expressed proteins from D3 to D7. Detected proteins and their main biological functions is listed in .

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Expressing, Control, Generated

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Protein profiler analysis of conditioned media from defects treated with hADM, hADM + BMC, or hADM + BMC-CD8 after three days. Protein levels are normalized to the intensity of the control spots (=100%). Only proteins with significant differences ( p < 0.05) or trends ( p < 0.1) are shown. Data are presented as boxplots (median, minimum, maximum; n = 3 per group). * indicates explorative significance ( p < 0.05) versus the respective comparison group.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Control, Comparison

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Protein profiler analysis of conditioned media from defects treated with hADM, hADM + BMC, or hADM + BMC-CD8 after 7 days . Protein levels are normalized to the intensity of the control spots (=100%). Only proteins with significant differences ( p < 0.05) or trends ( p < 0.1) are shown and presented in alphabetical order. Data are presented as boxplots (median, minimum, maximum; n = 3 per group). * indicates explorative significance ( p < 0.05) versus the respective comparison groups.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Control, Comparison

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Direct comparison of protein profiler data obtained after 3 and 7 days in defects treated with hADM (white), hADM + BMC (light gray), or hADM + BMC-CD8 (dark gray). Values are normalized to the density of the control spots (=100%). Only proteins with significant differences ( p < 0.05) or trends ( p < 0.1) are shown and presented in alphabetical order. Data are presented as boxplots (median, minimum, maximum; n = 3 per group). * indicates explorative significance ( p < 0.05) versus the respective comparison group.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Comparison, Control

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Osteogenic gene expression (A–D) and ALP protein expression (E) in MSC after cultivation in osteogenic differentiation medium (ODM) supplemented with CM. CM was generated from hADM (white), hADM seeded with BMC (light gray), or hADM seeded with CD8-depleted BMC (BMC-CD8, dark gray) wrapped around the bone defect for 3 or 7 days, then explanted and incubated in medium for 24 h. MSC were cultured for 3 weeks in ODM, with 10% CM added during the first 3 days, followed by analysis of gene expression relative to the Gapdh housekeeping gene ( A – D ) or ALP measurement ( E ). Control MSC (salmon) were cultured in expansion medium (M, only in E ) or in ODM, both without CM. # = p < 0.05 vs. ODM; * = p < 0.05 vs. indicated group. All significances are exploratory.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Gene Expression, Expressing, Generated, Incubation, Cell Culture, Control

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Influence of CM on expression of M1 ( Cd80, Nos2 ) and M2 ( Arg1, Socs1 ) marker genes in macrophages differentiated into M0 (A–D), M1 (E,F), or M2 (G,H). CM was generated from hADM (white), hADM seeded with BMC (light gray), or hADM seeded with CD8-depleted BMC (BMC-CD8, dark gray) wrapped around the bone defect for 3 or 7 days, then explanted and incubated in medium for 24 h. Primary rat monocytes from bone marrow aspirates were cultured for 7 days, with 10% CM added during the first 3 days. Monocytes cultured in differentiation media without CM served as controls (salmon). # = p < 0.05 vs. control; * = p < 0.05 vs. indicated group. All significances are exploratory.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Expressing, Marker, Generated, Incubation, Cell Culture, Control

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Influence of CM on expression of CCL2 and IL-1RA genes in macrophages differentiated into M0 (A,B), M1 (C,D), or M2 (E,F). CM was generated from hADM (white), hADM seeded with BMC (light gray), or hADM seeded with CD8-depleted BMC (BMC-CD8, dark gray) wrapped around the bone defect for 3 or 7 days, then explanted and incubated in medium for 24 h. Primary rat monocytes from bone marrow aspirates were cultured for 7 days, with 10% CM added during the first 3 days. Monocytes cultured in differentiation media without CM served as controls (salmon). # = p < 0.05 vs. control; * = p < 0.05 vs. indicated group. All significances are exploratory.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Expressing, Generated, Incubation, Cell Culture, Control

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD8 , REA734 , 50 , 130-110-677 , FITC , Miltenyi Biotec.

Techniques: Imaging

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: scRNA-seq reveals differential immune infiltration in αOX40-treated tumors based on response (A) Schematic of the bilateral MC38 tumor model assessing αOX40 response. Humanized OX40 mice received three doses of αOX40, followed by resection of the left tumor for scRNA-seq and flow cytometry analysis. Contralateral tumor dynamics and survival were monitored longitudinally. (B) UMAP visualization of scRNA-seq data from immune cells in MC38-bearing mice following αOX40 treatment. Cells are color-coded by annotated cell type. (C) Heatmap depicting nine transcriptionally distinct immune cell subpopulations. (D) Pie chart shows the relative abundance of nine immune cell clusters in αOX40 responders and nonresponders. (E) Flow cytometry analysis of tumor-infiltrating immune cell frequencies in αOX40-treated MC38-bearing mice. Frequencies of CD4 + T cells, CD8 + T cells, and macrophages were quantified after the third αOX40 dose (control, n = 5 mice; mice with a robust therapeutic response named as responder, n = 4 mice; mice with minimal to no response named as nonresponder, n = 4 mice). Data represent mean ± SD from one of two independent experiments (E). Statistical significance was determined using one-way ANOVA with multiple comparisons. ∗∗ p < 0.01.

Article Snippet: CD8a (Ly-2) microbeads, mouse , Miltenyi Biotec , Cat# 130-117-044.

Techniques: Flow Cytometry, Control, Clinical Proteomics

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-expressing macrophages is associated with response to αOX40 therapy (A) UMAP of monocytes/macrophages subclusters from scRNA-seq data in αOX40-treated MC38-bearing mice. (B) Representative marker genes in the monocyte/macrophage subclusters. (C) Pie chart showing the proportional distribution of monocyte/macrophage subsets of responders and nonresponders. (D) QuSAGE pathway analysis demonstrated enrichment of innate immune and phagocytic signaling pathways in distinct monocyte/macrophage subsets. (E) UMAP showing Mac_C1 signature genes and a heatmap of immune-related gene expression across TAM subclusters ( Z score normalized). (F) Violin plots comparing Nos2 expression levels in Mac_C1 subset between responsive and nonresponsive. (G) Flow cytometry analysis shows the percentage of M1-like (F4/80 + NOS2 + ) and M2-like (F4/80 + CD206 + ) macrophages in tumor tissues of control ( n = 5 mice), nonresponders (with minimal to no response, n = 4 mice), and responders (with a robust therapeutic response, n = 4 mice). (H and I) Comparison of Nos2 expression levels in responders versus nonresponders pre- or post-αOX40 treatment. Bilateral-MC38-bearing mice were treated with αOX40, and tumors from one side were analyzed by RNA-seq prior to (H) or following αOX40 treatment (I). The Nos2 expression was analyzed from RNA-seq data (left) and validated by RT-qPCR (right) ( n = 5 biological replicates). (J) NOS2 expression in tumor biopsies post-treatment determined by RNA-seq. Patients with advanced solid tumors and >1 prior therapy received HFB301001 monotherapy. Tumor biopsy samples were obtained on day 8 of cycle 2 for subsequent RNA-seq analysis. NOS2 expression were compared between patients achieving stable disease (SD, n = 3) and those with progressive disease (PD, n = 3). (K) GO enrichment analysis of upregulated genes in Mac_C1 of responders. (L) Calreticulin expression was quantified by flow cytometry in different response groups following αOX40 treatment ( n = 3 mice per group). (M) NOS2 expression in BMDMs was analyzed by flow cytometry after stimulation with CD8 + T cell supernatant and MC38 lysate, combined with TLR inhibition and IFN-γ blockade ( n = 5 biological replicates). (N) Quantification of Nos2 expression in BMDM by RT-qPCR after 24-h stimulation with MPLA (TLR4 agonist, 100 ng/mL), IFN-γ (20 ng/mL), or both. Data normalized to Gapdh and presented as fold-change relative to unstimulated controls ( n = 4 biological replicates). Data are shown as means ± SD from one of two independent experiments (G, H, I, L, M, and N). Statistical significance was determined using one-way ANOVA with multiple comparisons (G, L, M, and N) or using an unpaired two-tailed t test (H, I, and J). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; VST, variance stabilized transformation; sup., supernatant; lys., tumor lysate; inh., inhibitor.

Article Snippet: CD8a (Ly-2) microbeads, mouse , Miltenyi Biotec , Cat# 130-117-044.

Techniques: Expressing, Marker, Protein-Protein interactions, Gene Expression, Flow Cytometry, Control, Clinical Proteomics, Comparison, RNA Sequencing, Quantitative RT-PCR, Inhibition, Two Tailed Test, Transformation Assay

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: The antitumor efficacy of the Combo therapy is contingent upon CD8 + T cells and macrophages (A) UMAP of scRNA-seq data from tumor-infiltrating immune cells in OX40-humanized MC38-bearing mice treated with MPLA, IFN-γ, αOX40, or Combo. Cells are color-coded by annotated cell type. (B) Bubble chart showing the top variable marker genes for identified immune cell types. (C) Pie chart shows the relative abundance of 11 immune cell clusters in control, αOX40, MPLA+IFN-γ, or Combo. (D) Macrophage frequency and absolute count in tumors of MC38-bearing mice after two and three treatment cycles with MPLA, IFN-γ, αOX40, or Combo, analyzed by flow cytometry ( n = 5 mice per group). (E) Schematic of CD8 + T cell depletion assay. (F) Tumor volume and survival were monitored. Kaplan-Meier survival analysis corresponding to the depletion study of CD8 + T cell ( n = 6 mice per group). (G) Schematic of macrophage depletion assay in early and late stage. (H and I) Tumor volume and survival were monitored. Kaplan-Meier survival analysis corresponding to the depletion study in (G) ( n = 6–10 mice per group). Data are shown as means ± SD from one of two independent experiments (D, F, H, and I). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (D). Log rank test was used (F, H, and I) for statistical comparison. n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: CD8a (Ly-2) microbeads, mouse , Miltenyi Biotec , Cat# 130-117-044.

Techniques: Marker, Control, Flow Cytometry, Depletion Assay, Comparison