Journal: OncoImmunology

Article Title: TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

doi: 10.1080/2162402x.2023.2246322

Figure Lengend Snippet: Figure 2. Expression of GalBP by TEX. a. Venn diagram depicting the percentage of 23 selected proteins revealed by mass spectrometry analysis that were expressed or contained by sEVs isolated from MDA-MB-231 and MCF10-A cells and in plasma of healthy volunteers and TNBC patients. Gal3BP as part of the 43.5% proteins expressed of the 23 selected proteins, was found in the samples from the TNBC groups. b. Bars showing the percentage of CD9 expression and CD9+/Gal3BP+ identified on sEVs isolated from normal breast cells (EX) or from TNBC cells (TEX). Results are expressed as mean±SD (n = 5); p values are shown in the figure (two-tailed t test). c. Representative dot blot showing double stained CD9+/Gal3BP+ in TEX vs EX by FACS analysis.

Article Snippet: The CD45 inhibitor (CAS 345,630-40-2, 2.9 μM; Calbiochem) is a PTPase CD45 inhibitor and a CD45 blocking peptide.19 Cells were transferred to FACS tubes and stained with antiCD3-PC5.5 (Beckman Coulter), anti-Gal3-PE (R&D Systems), anti-mouse Gal3BP (Novus Biologicals) and mouse antihuman FITC (Jackson ImmunoResearch).

Techniques: Expressing, Mass Spectrometry, Isolation, Clinical Proteomics, Two Tailed Test, Dot Blot, Staining

Journal: OncoImmunology

Article Title: TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

doi: 10.1080/2162402x.2023.2246322

Figure Lengend Snippet: Figure 3. Formation of a complex of Gal3BP with Gal3. a. Gal3 secretion by MDA-MB-231, MCF10-A, ER+ MCF-7, and Her2+ BT474 cells. Results are expressed as mean ±SD (n = 3) (two-tailed t test). b. An assay was developed to evaluate the physical interactions between Gal3BP and Gal3. c. Bars showing a linear curve shape, depending on the sample dilution. Significantly higher physical interactions between Gal3BP and Gal3 derived from MDA-MB-231 and plasma of TNBC patients cells compared to MCF10-A or plasma of healthy women in triplicate (p < 0.007). Results are expressed as mean±SD (two-tailed t-test). d. Percentage of Gal3BP protein in TEX and EX immunoprecipitated with either anti-Gal3BP or anti-Gal3 antibodies (n = 3). Results are expressed as mean±SD; p values are shown in the figure (two-tailed t test). e. Percentage of Gal3 protein in TEX or EX immunoprecipitated with either anti Gal3BP or anti Gal3 antibodies (n = 3, mean±SD) (2-tailed t test). f. Western blot analysis showing expression of Gal3 only in TEX immunoprecipitated with anti-Gal3BP. Gal3BP expression was found in EX or TEX immunoprecipitated with anti-Ga3BP. Immunoprecipitation with anti-Gal3, resulted in higher Gal3BP expression in TEX than in EX. g. Immunofluorescence of PBMCs treated with EX. CD3 (purple) expressing Gal3BP (green)/Gal3 (red) complex in EX. Scale bar = 50 μm. H. Immunofluorescence of PBMCs treated with TEX. Triple staining of Gal3BP/Gal3 complex on CD3 cells (yellow). Pictures taken under confocal microscope, nucleus stained with DAPI (blue), Scale bar = 50 μm.

Article Snippet: The CD45 inhibitor (CAS 345,630-40-2, 2.9 μM; Calbiochem) is a PTPase CD45 inhibitor and a CD45 blocking peptide.19 Cells were transferred to FACS tubes and stained with antiCD3-PC5.5 (Beckman Coulter), anti-Gal3-PE (R&D Systems), anti-mouse Gal3BP (Novus Biologicals) and mouse antihuman FITC (Jackson ImmunoResearch).

Techniques: Two Tailed Test, Derivative Assay, Clinical Proteomics, Immunoprecipitation, Western Blot, Expressing, Immunofluorescence, Staining, Microscopy

Journal: OncoImmunology

Article Title: TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

doi: 10.1080/2162402x.2023.2246322

Figure Lengend Snippet: Figure 4. Gal3/Gal3BP complex binding to CD45 and inhibiting CD45 signaling pathway in TEX. A. Percentage of T cells displaying Gal3BP/Gal3 complex with and without CD45 blocking in EX- and TEX-treated cells (n = 3). Results are expressed as mean±SD (two-tailed t test). B. Representative dot blot of FACS analysis of CD3+ gated cells expressing Gal3BP/Gal3 complex. C. Percentage of Lck Y505, Lck Y394, ZAP-70 Y493 and VAV-1 Y174 phosphorylation in PBMCs treated with EX, TEX, or complex (Gal3BP and Gal3 recombinant proteins). D. Representative histogram showing phosphorylation of proteins in the CD45 pathway after treatment with EX (red) or TEX (blue).

Article Snippet: The CD45 inhibitor (CAS 345,630-40-2, 2.9 μM; Calbiochem) is a PTPase CD45 inhibitor and a CD45 blocking peptide.19 Cells were transferred to FACS tubes and stained with antiCD3-PC5.5 (Beckman Coulter), anti-Gal3-PE (R&D Systems), anti-mouse Gal3BP (Novus Biologicals) and mouse antihuman FITC (Jackson ImmunoResearch).

Techniques: Binding Assay, Blocking Assay, Two Tailed Test, Dot Blot, Expressing, Phospho-proteomics, Recombinant

Journal: OncoImmunology

Article Title: TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

doi: 10.1080/2162402x.2023.2246322

Figure Lengend Snippet: Figure 5. Effect of CD45 blocking on Gal3BP/Gal3 protein-induced immunosuppression. a. Percentage of Treg cells (CD4+/CD25+FOXP3+) in PBMCs treated with EX compared to TEX or Gal3BP/Gal3 complex ± CD45 blocking. Results are expressed as mean±SE (n = 4) (p value by ANOVA). b. Representative dot blot of FACS data showing percentage gated CD4 cells expressing CD25-APC and FOXP3-FITC (Treg cells) in EX- or TEX-treated cells. c. IL-10 and IL-35 were detected in supernatants of PBMCs treated with either TEX or EX from the different experiments. IL-10 secretion by cells. Results are expressed as mean±SE (n = 6) (p value by ANOVA). d. IL-35 secretion by cells. Results are expressed as mean±SE (n = 7); (p values by ANOVA). e. IFNγ secretion of lymphocytes incubated with TEX or TEX+GW4869 (an inhibitor of exosome secretion) and with/without CD45 blocking. f. Percentage of activated T and NK cells after incubation of lymphocytes with EX or TEX with/without CD45 blocking. Results are expressed as mean±SE (n = 4); p values by ANOVA). g. Representative dot blot of FACS data.

Article Snippet: The CD45 inhibitor (CAS 345,630-40-2, 2.9 μM; Calbiochem) is a PTPase CD45 inhibitor and a CD45 blocking peptide.19 Cells were transferred to FACS tubes and stained with antiCD3-PC5.5 (Beckman Coulter), anti-Gal3-PE (R&D Systems), anti-mouse Gal3BP (Novus Biologicals) and mouse antihuman FITC (Jackson ImmunoResearch).

Techniques: Blocking Assay, Dot Blot, Expressing, Incubation

Journal: OncoImmunology

Article Title: TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

doi: 10.1080/2162402x.2023.2246322

Figure Lengend Snippet: Figure 6. A new TNBC immunosupressive mechanism. Gal3BP/Gal3 complex secreted by TNBC cells induces immunosuppression through the CD45 receptor on T cells.

Article Snippet: The CD45 inhibitor (CAS 345,630-40-2, 2.9 μM; Calbiochem) is a PTPase CD45 inhibitor and a CD45 blocking peptide.19 Cells were transferred to FACS tubes and stained with antiCD3-PC5.5 (Beckman Coulter), anti-Gal3-PE (R&D Systems), anti-mouse Gal3BP (Novus Biologicals) and mouse antihuman FITC (Jackson ImmunoResearch).

Techniques: