Journal: Cells

Article Title: Alterations in Circulating Progenitor Cell Composition in Rheumatoid Arthritis

doi: 10.3390/cells15080726

Figure Lengend Snippet: Peripheral blood cell clusters and their proportions. ( a ) Cells were gated for size with the forward-light scatter versus granularity/complexity side scatter (FSC vs. SSC) and debris, doublets, and dead cells were excluded. Coloured rectangles highlight four cellular quadrants: CD45 − CD31 − (blue), CD45 + CD31 − (red), CD45 + CD31 + (orange), and CD31 + CD45 − (green). ( b ) Average cell frequency in HC (n = 9) and RA (n = 12). Pairwise statistical Chi-test. Cell frequencies of ( c ) CD45 + CD31 − , ( d ) CD45 + CD31 + ( e ) CD45 − CD31 − and ( f ) CD31 + CD45 − cells. Mean and standard deviation (SD); unpaired t -test ( c , d ) and Mann-Whitney test ( e , f ). Unsupervised dimensionality reduction analysis of the measured cell surface proteins in the concatenated file containing healthy controls (HC) (n = 9) and rheumatoid arthritis (RA) (n = 12) with a total of 260.000 life cells is visualised in ( g ) Uniform Manifold Approximation and Projection (UMAP) and ( h ) Triplet-based Manifold Approximation (triMAP) plots. ( i ) Distribution of the populations according to CD45 and CD31 expression. Populations from the Flow Self-Organizing Map (FlowSOM) algorithm are coloured and labelled with numbers from 1 to 20. ( j ) Mean proportions of each coloured and numbered population within the HC (n = 9) and RA (n = 12) cohort. Pairwise statistical Chi-test. ( k ) Heatmap from the FlowSOM cluster analysis shows the expression levels of the markers with intensity levels for each coloured and name-coded cell population (green/yellow panel) and the intensity of each population for the HC (n = 9) and RA (n = 12) (orange/yellow).

Article Snippet: CD45 , FITC-A , 130-113-117 , Miltenyi Biotec , 1/150 , B2.

Techniques: Standard Deviation, MANN-WHITNEY, Expressing

Journal: Cells

Article Title: Alterations in Circulating Progenitor Cell Composition in Rheumatoid Arthritis

doi: 10.3390/cells15080726

Figure Lengend Snippet: CD45 + CD31 int cells were reduced in RA patients. ( a ) CD45/CD31 dot plot showing populations 12, 4 and 13. Pop 4 expressed high levels of ( b ) CD133 and ( c ) CD34 (red dots). Bar graphs show a significantly reduced frequency in the percentage of ( d ) Pop 12 and ( e ) Pop 4 in RA patients (n = 12) when compared to HC (n = 9). Mean and SD; Mann–Whitney and t -test, respectively. ( f ) Heat map showing log fold change differences in PECAM1 expression in peripheral mononuclear cells (PBMCs) (RA n = 5, PsA n = 6) and in immune cells within synovial tissues (RA n = 13, PsA n = 12). MAST test, * p < 0.05, *** p < 0.001. Mean fluorescence intensity (MFI) of CD31 expression on ( g ) naïve CD4 T cells, ( h ) central memory (TCM), ( i ) effector memory (TEM), and ( j ) T cells expressing CD45RA (TEMRA). n = 9 per group, all Mann–Whitney, except in G, the t -test was used. All dot plots show mean and SD.

Article Snippet: CD45 , FITC-A , 130-113-117 , Miltenyi Biotec , 1/150 , B2.

Techniques: MANN-WHITNEY, Expressing, Fluorescence

Journal: Cells

Article Title: Alterations in Circulating Progenitor Cell Composition in Rheumatoid Arthritis

doi: 10.3390/cells15080726

Figure Lengend Snippet: CD235a + and CD45 − CD31 − stromal cells circulating in peripheral blood. ( a ) CD45/CD31 dot plot showing populations 16, 17, 18, 19 and 20. ( b ) UMAP shows the size of each cluster, and ( c ) TriMAP shows the relative cluster location. CD45/CD31 plots highlighting the expression of ( d ) CD235a, ( e ) CD133, ( f ) CD34 and ( g ) CD105. The intensity scale indicates high expression of the marker in red. Frequency of ( h ) Pop 20, ( i ) Pop 17, and ( j ) Pop 18. Mann-Whitney test. Dot plots showing the expression of ( k ) CD34/CD133, ( l ) CD271/ CD105, and CD90 with ( m ) CD105, ( n ) CD271, and ( o ) PDPN in Pop 16 and Pop 19. Frequency of ( p ) Pop 16 and ( q ) Pop 19. t -test for Pop 16; Mann–Whitney test for Pop 19. All dot plots show mean and SD, n = 12 RA and 9 HC.

Article Snippet: CD45 , FITC-A , 130-113-117 , Miltenyi Biotec , 1/150 , B2.

Techniques: Expressing, Marker, MANN-WHITNEY

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: ABHD16A promotes ILC3 aggregation and IL22 release in the immune microenvironment. A, A total of 5 × 10 6 MFC-luc or Abhd16a -knockdown MFC-luc cells were injected into the epidermis of the greater curvature of the stomach in the 615 mice. Tumor growth was monitored weekly through bioluminescence imaging from the first day of MFC cell inoculation. Scale bar, 2.000e+4 – 5.000e + 5 p/s/cm 2 /sr. B and C, Representative images of the excised stomachs ( B ) and the orthotopic tumor volume ( C ) on day 28 after gastric cancer cells were implanted orthotopically ( n = 9 per group). D, H&E images of the stomach, peritoneum, liver, intestine, and lung on day 42 after MFC or Abhd16a -knockdown MFC cells were implanted into 615 mice. Scale bar, 400 μm. E–G, Representative images ( E ), volume ( F ), and weight ( G ) of control and Abhd16a -knockdown subcutaneous tumors ( n = 5 per group). H, IHC images of PD-L1 in control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues. Scale bar, 500 μm. I, Detection of mouse cytokines in the TIF of orthotopic gastric cancer tissues by proteome profiler mouse XL cytokine array. J, Flow cytometry gating strategy and frequencies of ILC3s and Th17/Th22 cells out of CD45 + cells in control and Abhd16a -knockdown gastric cancer tissues in 615 mice. K, Flow cytometry gating strategy and frequencies of ILC3s out of ILCs in control and Abhd16a -knockdown gastric cancer tissues in Rag1 −/− mice. ***, P < 0.001.

Article Snippet: FITC anti-human CD3 (Thermo Fisher Scientific, cat. #11-0038-42, RRID: AB_2043831, 5 μL/1 × 10 6 cells), PE anti-human CD127 (BioLegend, cat. #351304, RRID: AB_10720185, 5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-human CD294 (BioLegend, cat. #350112, RRID: AB_2562468, 5 μL/1 × 10 6 cells), APC anti-human CD117 (BioLegend, cat. #313206, RRID: AB_314985, 5 μL/1 × 10 6 cells), FITC anti-human CD19 (eBioscience, cat. #11-0199-42, RRID: AB_10669461, 5 μL/1 × 10 6 cells), FITC anti-human CD14 (eBioscience, cat. #11-0149-42, RRID: AB_10597597, 5 μL/1 × 10 6 cells), BV650 anti-human CD45 (eBioscience, cat. #416-0459-42, RRID: AB_2925684, 5 μL/1 × 10 6 cells), iFluor 647 anti-Ki67 (HUABIO, cat. #HA720163F, RRID: AB_3072100, 1 μL/1 × 10 6 cells), PE/Cyanine7 anti-human CD274 (Elabscience, cat. #E-AB-F1133H, 5 μL/1 × 10 6 cells), and PE anti-human CD273 (Elabscience, cat. #E-AB-F1175D, 5 μL/1 × 10 6 cells).

Techniques: Knockdown, Injection, Imaging, Control, Flow Cytometry

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: High expression of ABHD16A mediates the cumulative release of LysoPS into the TME. A, mIF images of the orthotopic gastric cancer (GC) tissues stained for PD-L1, RORC, and CD3. Yellow, PD-L1 + tumor cells; red, RORC + ILC3s; green, CD3 + cells. Scale bars, 100 μm (left) and 25 μm (right). B and C, Flow cytometry gating strategy and frequencies of ILC3s ( B ) and proportions of ILC3s in total CD45 + cells ( C ) isolated from control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues ( n = 3 per group). D and E, Flow cytometry gating strategy and frequencies of ILC3s ( D ) and proportions of ILC3s in total CD45 + cells ( E ) derived from the peripheral blood of healthy controls (HC) and patients with gastric cancer ( n = 8 per group). F and G, LC-MS/MS analysis of 18:0 and 18:1 LysoPS in the control and Abhd16a -knockdown orthotopic ( F ) and subcutaneous ( G ) gastric cancer tissues ( n = 3 per group). H–J, ELISA was used to measure levels of LysoPS in gastric cancer cell supernatant ( H ), TIF ( I ), and in vitro tumor culture supernatant ( J ) of control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tumors ( n = 3 per group). K, LysoPS levels in the serum of healthy controls and patients with gastric cancer detected by ELISA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: FITC anti-human CD3 (Thermo Fisher Scientific, cat. #11-0038-42, RRID: AB_2043831, 5 μL/1 × 10 6 cells), PE anti-human CD127 (BioLegend, cat. #351304, RRID: AB_10720185, 5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-human CD294 (BioLegend, cat. #350112, RRID: AB_2562468, 5 μL/1 × 10 6 cells), APC anti-human CD117 (BioLegend, cat. #313206, RRID: AB_314985, 5 μL/1 × 10 6 cells), FITC anti-human CD19 (eBioscience, cat. #11-0199-42, RRID: AB_10669461, 5 μL/1 × 10 6 cells), FITC anti-human CD14 (eBioscience, cat. #11-0149-42, RRID: AB_10597597, 5 μL/1 × 10 6 cells), BV650 anti-human CD45 (eBioscience, cat. #416-0459-42, RRID: AB_2925684, 5 μL/1 × 10 6 cells), iFluor 647 anti-Ki67 (HUABIO, cat. #HA720163F, RRID: AB_3072100, 1 μL/1 × 10 6 cells), PE/Cyanine7 anti-human CD274 (Elabscience, cat. #E-AB-F1133H, 5 μL/1 × 10 6 cells), and PE anti-human CD273 (Elabscience, cat. #E-AB-F1175D, 5 μL/1 × 10 6 cells).

Techniques: Expressing, Staining, Flow Cytometry, Isolation, Control, Knockdown, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: LysoPS enhances the proliferation and activation of ILC3s in the TME. A, Flow cytometry was performed to detect the frequencies of Ki67 + MNK3 under treatment with LysoPS (10 μmol/L) for 24 hours. B and C, Flow cytometry gating strategy and frequencies of ILC3s ( B ) and proportions of ILC3s in total CD45 + cells ( C ) in the isolated human PBMCs treated with exogenous LysoPS (10 μmol/L) for 24 hours ( n = 4 per group). D, Numbers of ILC3s treated with LysoPS. Cell counts were recorded daily for 5 days ( n = 3 per group). E, ELISA analysis of IL22 in the supernatant of MNK3 cells stimulated with incremental concentrations (0, 5, 10, and 20 μmol/L) of LysoPS ( n = 3 per group). F, Frequencies of IL22 + ILC3s out of total ILC3s isolated from control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues ( n = 3 per group). G and H, ELISA analysis of IL22 in the TIF of control and Abhd16a -knockdown orthotopic and subcutaneous gastric cancer tissues ( G ) and IL22 in the serum of healthy controls (HC) and patients with gastric cancer ( H ). I, Correlation analysis between IL22 and LysoPS in the serum of patients with gastric cancer. J, The levels of LysoPS and IL22 in the serum of patients with PNI − and PIN + gastric cancer measured by ELISA. K, ROC analysis of the diagnostic value of LysoPS and IL22 for gastric cancer. **, P < 0.01; ***, P < 0.001.

Article Snippet: FITC anti-human CD3 (Thermo Fisher Scientific, cat. #11-0038-42, RRID: AB_2043831, 5 μL/1 × 10 6 cells), PE anti-human CD127 (BioLegend, cat. #351304, RRID: AB_10720185, 5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-human CD294 (BioLegend, cat. #350112, RRID: AB_2562468, 5 μL/1 × 10 6 cells), APC anti-human CD117 (BioLegend, cat. #313206, RRID: AB_314985, 5 μL/1 × 10 6 cells), FITC anti-human CD19 (eBioscience, cat. #11-0199-42, RRID: AB_10669461, 5 μL/1 × 10 6 cells), FITC anti-human CD14 (eBioscience, cat. #11-0149-42, RRID: AB_10597597, 5 μL/1 × 10 6 cells), BV650 anti-human CD45 (eBioscience, cat. #416-0459-42, RRID: AB_2925684, 5 μL/1 × 10 6 cells), iFluor 647 anti-Ki67 (HUABIO, cat. #HA720163F, RRID: AB_3072100, 1 μL/1 × 10 6 cells), PE/Cyanine7 anti-human CD274 (Elabscience, cat. #E-AB-F1133H, 5 μL/1 × 10 6 cells), and PE anti-human CD273 (Elabscience, cat. #E-AB-F1175D, 5 μL/1 × 10 6 cells).

Techniques: Activation Assay, Flow Cytometry, Isolation, Enzyme-linked Immunosorbent Assay, Control, Knockdown, Diagnostic Assay

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: ABHD16A–LysoPS enhances ILC3 activation via the GPR34–STAT3–AKT signaling pathway. A, MNK3 cells were treated with or without LysoPS (10 μmol/L); the levels of IL22, pAKT, AKT, pSTAT3, and STAT3 were assessed by Western blotting. B, Western blotting was performed to check IL22, pAKT, AKT, pSTAT3, and STAT3 levels in MNK3 cells cocultured with MFC cells, Abhd16a -knockdown MFC cells, or Abhd16a -knockdown MFC cells with simultaneous supplementation of LysoPS (10 μmol/L). C, Western blotting was conducted to assess IL22, pAKT, AKT, pSTAT3, and STAT3 levels in MNK3, Gpr34 -knockdown MNK3, and perifosine-pretreated MNK3 cells, all under LysoPS supplementation (10 μmol/L). D, ELISA analysis of IL22 production in supernatants from MNK3 cells under the same treatments as in B . E, ELISA analysis of IL22 production in the supernatant from MNK3 cells under the same treatments as in C . F, ELISA analysis of IL22 production by ILC3s and ILC3s treated with LysoPS in the presence or absence of perifosine. G and H, LysoPS (2.5 mg/kg) was administered to Abhd16a -knockdown gastric cancer mice and control mice in the presence or absence of a GPR34 inhibitor (GPR34 receptor antagonist 2, 20 mg/kg) or AKT inhibitor (perifosine, 20 mg/kg). The representative living images on days 3, 10, and 17 ( G ) and tumor volume at the end of the experiment ( H ) are shown. Scale bar, 1.000e+4 – 1.000e + 5 p/s/cm 2 /sr; n = 5 per group. I–K, The proportions of ILC3s ( I ), pSTAT3 + ILC3s ( J ), and IL22 + ILC3s ( K ) in total CD45 + cells derived from orthotopic gastric cancer tissues. Mice treatments were the same as G and H . **, P < 0.01; ns, nonsignificant.

Article Snippet: FITC anti-human CD3 (Thermo Fisher Scientific, cat. #11-0038-42, RRID: AB_2043831, 5 μL/1 × 10 6 cells), PE anti-human CD127 (BioLegend, cat. #351304, RRID: AB_10720185, 5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-human CD294 (BioLegend, cat. #350112, RRID: AB_2562468, 5 μL/1 × 10 6 cells), APC anti-human CD117 (BioLegend, cat. #313206, RRID: AB_314985, 5 μL/1 × 10 6 cells), FITC anti-human CD19 (eBioscience, cat. #11-0199-42, RRID: AB_10669461, 5 μL/1 × 10 6 cells), FITC anti-human CD14 (eBioscience, cat. #11-0149-42, RRID: AB_10597597, 5 μL/1 × 10 6 cells), BV650 anti-human CD45 (eBioscience, cat. #416-0459-42, RRID: AB_2925684, 5 μL/1 × 10 6 cells), iFluor 647 anti-Ki67 (HUABIO, cat. #HA720163F, RRID: AB_3072100, 1 μL/1 × 10 6 cells), PE/Cyanine7 anti-human CD274 (Elabscience, cat. #E-AB-F1133H, 5 μL/1 × 10 6 cells), and PE anti-human CD273 (Elabscience, cat. #E-AB-F1175D, 5 μL/1 × 10 6 cells).

Techniques: Activation Assay, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay, Control, Derivative Assay