Journal: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 1. Influence of effector cell preparation, choice of effector cell, and E/T ratio on the measure- ment of trastuzumab-mediated ADCC using LDH ADCC assay. a–d. Influence of cryopreservation and the recovery cultivation time (4 h to overnight in RPMI-1640 with 10% FBS) on the recovery rate (a), viability of recovered PBMCs (b), the rate of NK cell isolation from the PBMCs (c), and the viability of isolated NK cells (d). Data were analyzed with GraphPad Prism using one-way ANOVA mixed-effects statistical analysis based on matched rows and recovery time as repeated measure with Tukey’s post hoc test. Overall p values for Figure 1a–d are <0.0001, 0.0126, 0.2553 (no statistical significance among three groups), and 0.0185, respectively; * p < 0.05 and **** p < 0.0001 (e) ADCC activity of trastuzumab with NK cells from immediately thawed PBMCs or overnight recovered PBMCs as effector cells with E/T ratio 20:1 measured after treatment for 5 h, 24 h, or 48 h. (f) ADCC activity of trastuzumab with immediately thawed or overnight recovered PBMCs as effector cells with E/T ratio 20:1 after treatment for 24 h. Data were analyzed with GraphPad Prism using two-way ANOVA with Šídák’s multiple comparisons test. Overall p value < 0.0001 for unrecovered compared to overnight recovered PBMCs. For (e,f), SKBR-3 cells were treated with trastuzumab (0–1 µg/mL) and NK cells, and killing of SKBR-3 was measured using the LDH ADCC assay. NC stands for the no-cell or media-only control, E stands for the effector-cell-only control, and T stands for the target-cell-only control. Data were analyzed with GraphPad Prism using two-way ANOVA, and p values are <0.0001. (g) Comparing Emax and EC50 values of LDH ADCC activity between different

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Cell Isolation, Isolation, Activity Assay, Control

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 2. Influence of the recovery culturing time and method on LDH ADCC assay and cellular expression of granulation proteins. (a) Comparison of ADCC activity of trastuzumab with NK cells isolated from PBMCs recovered for different times 0 h (NR), 4 h, or 24 h. Data were analyzed with GraphPad Prism using two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001; (b) Comparison of trastuzumab induced ADCC activity using NK cells that underwent overnight recovery after isolation from thawed cryopreserved PBMCs (R-iso for “recovered after isolation”), and NK cells isolated from the overnight recovered PBMCs (R-PB). Data were analyzed with GraphPad Prism using two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001. SKBR-3 cells were treated with trastuzumab (0–0.01 µg/mL) and NK cells with E/T ratio 5:1 for 24 h, and killing of SKBR-3 was measured with LDH ADCC assay; (c) Representative antiperforin, antigranzyme B, and Anti GAPDH immunoblot in NK cells isolated from unrecovered (NR) PBMCs or from PBMCs after recovery cultivation for different time points (4 h, 24 h) with E/T ratio 5:1. Lanes 1–3, 4–6, and 7–9 show expression levels for NK cells after incubation for 24 h hours with media only, with SKBR-3 only, and with SKBR-3 and trastuzumab, respectively. All lanes were run in the same gel, and images were

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Expressing, Comparison, Activity Assay, Isolation, Western Blot, Incubation

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 3. Influence of the recovery culturing time and method on the extracellular release of gran- ulation proteins and cytokines secretion. (a–d) Comparison of the secretion levels of perforin (a), granzyme B (b), TNFα (c), and IFNγ (d) using ELISA in the 24 h cell culture media of the target cell SKBR-3 (T), the effector cells NK (E), coculture of SKBR-3 and NK (E+T), and trastuzumab-treated coculture of SKBR-3 and NK (Tras) with E/T ratio 5:1. Data in this figure were analyzed with Graph- Pad Prism using one-way ANOVA analysis with Tukey’s post hoc test (**** p < 0.0001). * Fresh NK cells were prepared from a different donor; thus, they were not included in statistical analysis.

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 4. The recovery culturing downregulates expression of CD16 and CD56 on NK cells. (a) Represen- tative immunoblot of CD16A (FcγRIIIa) in the whole cell lysates of NK cells isolated from unrecovered

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: Expressing, Western Blot, Isolation

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 5. Phenotyping by flow cytometry indicates activation of NK cells by upregulation of CD69 during the recovery cultivation. (a) Flow cytometry analysis of surface CD69 expression in NK cells

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: Flow Cytometry, Activation Assay, Expressing

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 6. Choice of target cell lines in the LDH ADCC assay and ADCC reporter assay. (a) Represen- tative immunoblots using lysates from HER2-positive cell lines; (b) Quantitation of the immunoblot; (c) Dose-dependent curves of trastuzumab-mediated LDH ADCC assay using SKBR-3, BT-474, and NCI-N87 cells. Target cells were treated with trastuzumab (0–1 µg/mL) and NK cells with E/T ratio 5:1 for 24 h and killing of SKBR-3 was measured with LDH ADCC assay; (c) Dose-dependent curves of trastuzumab-mediated ADCC activity using ADCC reporter assay in SKBR-3, BT-474 and NCI-N87 cells. Target cells were treated with trastuzumab (0–5 µg/mL) and Promega ADCC reporter cells with E/T ratio 6:1 and luminescence was measured after 6 h.

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Reporter Assay, Western Blot, Quantitation Assay, Activity Assay

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 7. Influence of assay buffer, target cell plating method, and treatment time on the measurement of ADCC of trastuzumab. (a) Luminescence fold changes from the LDH ADCC assay using RPMI-1640 with 1% BSA or 1% FBS. Data were analyzed with GraphPad Prism using two-way ANOVA with Šídák’s multiple comparisons test. p value < 0.0001 for 1% BSA vs. 1% FBS; (b) Luminescence fold changes using the LDH ADCC assay performed with either overnight or same-day culture of SKBR-3 target cells plated with 1% BSA or overnight culture of SKBR-3 in 10% FBS. Data were analyzed with GraphPad Prism using two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001. SKBR-3 cells were treated with trastuzumab (0–1 µg/mL) and NK cells with E/T ratio 5:1, and the killing of SKBR-3 was measured with the LDH ADCC assay, where “E” stands for effector-cell-only control.

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Control

Journal: Theranostics

Article Title: Mesenchymal stromal cells and alpha-1 antitrypsin have a strong synergy in modulating inflammation and its resolution.

doi: 10.7150/thno.83942

Figure Lengend Snippet: Figure 1. MSCs synergized with A1AT to modulate inflammation in Raw 264.7 macrophages. Cells were stimulated with 100 ng/mL LPS plus 10 ng/mL IFNγ and treated with 0.5 mg/mL A1AT or MSCs (MSC/MΦ = 1/10) or their combination. Dexamethasone (Dex, 1 µg/mL) was used as a benchmark. Pro-inflammatory mouse cytokine IL6 (A), TNFα (B), and anti-inflammatory mouse cytokine IL10 (C) were measured via ELISA. The IL6/IL10 (D) and TNFα/IL10 ratio (E) was also shown. *:p < 0.05, **:p < 0.01, ***:p < 0.001.

Article Snippet: P4 MSCs were characterized with the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), including antibodies for positive markers CD90, CD73, CD105, and negative markers CD45, CD34, CD11b, CD79A, HLA-DR, as well as the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) including antibodies for positive markers CD44, CD106, CD146, and CD166.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Mesenchymal stromal cells and alpha-1 antitrypsin have a strong synergy in modulating inflammation and its resolution.

doi: 10.7150/thno.83942

Figure Lengend Snippet: Figure 2. MSCs synergized with A1AT to modulate inflammation in primary human PBMCs. Cells were stimulated with 100 ng/mL LPS + anti-CD3/CD28 antibodies (positive) and treated with 0.5 mg/mL A1AT or MSCs (MSC/PBMC = 1/10) or their combination for 24 hs. Dexamethasone (Dex, 1 µg/mL) was used as a benchmark. PBMCs without activation and treatment were used as a negative control. Pro-inflammatory human cytokine IL6 (A), TNFα (B), and anti-inflammatory human cytokine IL10 (C) were measured via ELISA. The IL6/IL10 (D) and TNFα/IL10 ratio (E) was also shown. *:p < 0.05, **:p < 0.01, ***:p < 0.001.

Article Snippet: P4 MSCs were characterized with the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), including antibodies for positive markers CD90, CD73, CD105, and negative markers CD45, CD34, CD11b, CD79A, HLA-DR, as well as the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) including antibodies for positive markers CD44, CD106, CD146, and CD166.

Techniques: Activation Assay, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Mesenchymal stromal cells and alpha-1 antitrypsin have a strong synergy in modulating inflammation and its resolution.

doi: 10.7150/thno.83942

Figure Lengend Snippet: Figure 3. MSCs and A1AT combination treatment reduced neutrophil ROS and NETs production. HL-60 cells derived neutrophils were stimulated with 100 nM PMA and treated with 0.5 mg/mL A1AT or MSCs (MSC/neutrophil = 1/10) or their combination for 4 hs. Reactive oxygen species (ROS) (A-B) and neutrophil extracellular traps (NETs) production (C-D) were analyzed. *:p < 0.05, **:p < 0.01, ***:p < 0.001.

Article Snippet: P4 MSCs were characterized with the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), including antibodies for positive markers CD90, CD73, CD105, and negative markers CD45, CD34, CD11b, CD79A, HLA-DR, as well as the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) including antibodies for positive markers CD44, CD106, CD146, and CD166.

Techniques: Derivative Assay

Journal: Theranostics

Article Title: Mesenchymal stromal cells and alpha-1 antitrypsin have a strong synergy in modulating inflammation and its resolution.

doi: 10.7150/thno.83942

Figure Lengend Snippet: Figure 4. MSC and A1AT combination treatment enhanced phagocytosis in THP-1 derived macrophages (A-D) and HL-60 cells derived neutrophils (E-H). Macrophages were stimulated with 100 ng/mL LPS plus 10 ng/mL IFNγ for 24 hs. Neutrophils were stimulated with 100 nM PMA for 4 hs. Cells were treated with 0.5 mg/mL A1AT or MSCs

Article Snippet: P4 MSCs were characterized with the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), including antibodies for positive markers CD90, CD73, CD105, and negative markers CD45, CD34, CD11b, CD79A, HLA-DR, as well as the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) including antibodies for positive markers CD44, CD106, CD146, and CD166.

Techniques: Derivative Assay

Journal: Theranostics

Article Title: Mesenchymal stromal cells and alpha-1 antitrypsin have a strong synergy in modulating inflammation and its resolution.

doi: 10.7150/thno.83942

Figure Lengend Snippet: Figure 5. MSCs synergized with A1AT to improve survival rate and reduce lung injury in mice. (A) Illustration of the model. (B) The survival rate and (C) body weight development. N = 6. (D) H&E staining and (E) lung injury scores. The lung injury scores were calculated based on the five criteria shown in (D). *:p < 0.05, **:p < 0.01, ***:p < 0.001.

Article Snippet: P4 MSCs were characterized with the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), including antibodies for positive markers CD90, CD73, CD105, and negative markers CD45, CD34, CD11b, CD79A, HLA-DR, as well as the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) including antibodies for positive markers CD44, CD106, CD146, and CD166.

Techniques: Staining

Journal: Theranostics

Article Title: Mesenchymal stromal cells and alpha-1 antitrypsin have a strong synergy in modulating inflammation and its resolution.

doi: 10.7150/thno.83942

Figure Lengend Snippet: Figure 6. MSCs and A1AT synergized in reducing total protein (A) and pro-inflammatory cytokines while increasing anti-inflammatory cytokine IL10 (B-F) in BALF. *:p < 0.05, **:p < 0.01, ***:p < 0.001.

Article Snippet: P4 MSCs were characterized with the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), including antibodies for positive markers CD90, CD73, CD105, and negative markers CD45, CD34, CD11b, CD79A, HLA-DR, as well as the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) including antibodies for positive markers CD44, CD106, CD146, and CD166.

Techniques:

Journal: Theranostics

Article Title: Mesenchymal stromal cells and alpha-1 antitrypsin have a strong synergy in modulating inflammation and its resolution.

doi: 10.7150/thno.83942

Figure Lengend Snippet: Figure 8. MSCs synergized with A1AT to reduce total cell (A-B), macrophage (C), and neutrophil number (D) in BALF. The M1/M2 macrophage ratio was reduced by all treatments (E). *:p < 0.05, **:p < 0.01, ***:p < 0.001. (F-G) MSCs synergized with A1AT to reduce cell death as identified via TUNEL staining.

Article Snippet: P4 MSCs were characterized with the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), including antibodies for positive markers CD90, CD73, CD105, and negative markers CD45, CD34, CD11b, CD79A, HLA-DR, as well as the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) including antibodies for positive markers CD44, CD106, CD146, and CD166.

Techniques: TUNEL Assay, Staining

Journal: bioRxiv

Article Title: Lymph node resident memory T cells retain effector capabilities by evading lung resident memory dysfunction

doi: 10.64898/2026.02.03.703569

Figure Lengend Snippet: (A) Naive CD90.1+ OT-I T cells were transferred into naive B6 mice before intranasal infection with influenza expressing SIINFEKL (PR8-OVA). Expression of CD69 and CD103 was measured 21 days post infection on extravascular cells. (B) Quantification of CD69 + CD103 + OT-I T cells. (C) Naïve CD90.1 + OT-I Nur77-GFP T cells were transferred into mice one day before intranasal PR8-OVA infection. Nur77-GFP expression was evaluated 3-14 days later by extravascular OT-I T cells. (D)Experimental setup for (E-G). Flow cytometry plot (E) of CD90.1 + OT-I T cells isolated from indicated locations. (F-G) Longitudinal labeling of cells and quantification. (H) Experimental setup for (I-J). (I) Labeling after intravenous and intraperitoneal injection of OT-I T cells or naïve endogenous T cells. (J) Expression of CD69 and CD103. Naïve= CD8 + CD90.1 - CD44 low . Each point represents a mouse and N=2-5 mice per experiment. Data are representative (A) or are pooled from at least two experiments (n=2–4/group), except for day 113 in (B), which reflect a single experiment.

Article Snippet: Measurements were obtained in Flow Cytometry (FACS) Staining Buffer (Rockland, MB-086-0500) at room temperature for about 30 minutes per sample and channels were flushed with buffer in between samples.

Techniques: Infection, Expressing, Flow Cytometry, Isolation, Labeling, Injection

Journal: bioRxiv

Article Title: Lymph node resident memory T cells retain effector capabilities by evading lung resident memory dysfunction

doi: 10.64898/2026.02.03.703569

Figure Lengend Snippet: (A)Chromatin accessibility with gene expression (right) and linked chromatin peaks (bottom) at the Gzmb locus. (B) Protein expression of GranzymeB as measured by flow cytometry. (C-D) Same as (A-B) but for the Pdcd1 locus. Protein expression on OT-I T cells is 49 days post infection where each dot represents one mouse. (J and L) Data are from one experiment and representative of at least two experiments with N=3. (E-F) Surface expression of PD-1 and TIM3 at 7 days post infection. (G) Transcription factor motif footprint analysis from dataset. (H) Inferred eRegulons identified by SCENIC+ across memory T cells after influenza infection. (I-K) Legend (left) for network plots illustrating inferred transcription factor relationships to target genes. Transcription factor and gene relationship is denoted by color of line represents activation (red) or repression (blue).

Article Snippet: Measurements were obtained in Flow Cytometry (FACS) Staining Buffer (Rockland, MB-086-0500) at room temperature for about 30 minutes per sample and channels were flushed with buffer in between samples.

Techniques: Gene Expression, Expressing, Flow Cytometry, Infection, Activation Assay