Journal: Cancer Research

Article Title: Spleen Tyrosine Kinase Is Overexpressed and Represents a Potential Therapeutic Target in Chronic Lymphocytic Leukemia

doi: 10.1158/0008-5472.can-08-4252

Figure Lengend Snippet: Figure 4. Apoptosis induction correlates with mutational status, ZAP70, and SYK protein expression. A, CLL cells (n = 38) were treated with SYKII (4 and 10 Amol/L) or R406 (4 Amol/L) for 48 h, and viability was assessed by flow cytometry using Annexin-V/7AAD staining. Columns, mean % of vehicle control; bars, SE. ***, P < 0.0001 (one-way ANOVA with Bonferroni correction). B, CLL cell viability (relative to DMSO) was assessed after 48 h treatment with 4 Amol/L SYKII (top) or with 4 Amol/L R406 (bottom) in mutated (n = 28) versus unmutated (n = 10) CLL samples (left), and in ZAP70+ (n = 22) versus ZAP70

Article Snippet: Tyrosine kinase activity assay (Sigma Aldrich) was performed in duplicates using human recombinant SYK and ZAP70 (R&D Systems).

Techniques: Expressing, Flow Cytometry, Staining, Control

Journal: Cancer Research

Article Title: Spleen Tyrosine Kinase Is Overexpressed and Represents a Potential Therapeutic Target in Chronic Lymphocytic Leukemia

doi: 10.1158/0008-5472.can-08-4252

Figure Lengend Snippet: Figure 5. Validation of SYK inhibitor specificity and SYK gene silencing. A, protein tyrosine kinase activity assay performed with human recombinant SYK (rSYK) and ZAP70 (rZAP70) in the absence and presence of 4 Amol/L R406 or SYKII (columns, mean of five independent experiments; bars, SE). For relative activity absorption without inhibition was defined as 1. B, primary CLL cells (7 106, respectively) were transfected with control siRNA or SYK-specific siRNA. After 72 h of culture, viability and SYK expression were determined by flow cytometry (n = 7). SYK expression and viability of control siRNA-transfected cells was defined as 100%. C, after siRNA transfection and 72 h of culture, active Caspase 3 expression was determined by flow cytometry (n = 7). Columns, mean; bars, SE (A–C). *, P < 0.05; **, P < 0.01. D, cells treated as described under C were analyzed for p-Akt by flow cytometry. A representative histogram is shown (n = 3).

Article Snippet: Tyrosine kinase activity assay (Sigma Aldrich) was performed in duplicates using human recombinant SYK and ZAP70 (R&D Systems).

Techniques: Biomarker Discovery, Kinase Assay, Recombinant, Activity Assay, Inhibition, Transfection, Control, Expressing, Flow Cytometry

Journal: Oncology Letters

Article Title: ZAP70 interaction with 13 mRNAs as a potential immunotherapeutic target for endometrial cancer

doi: 10.3892/ol.2023.13799

Figure Lengend Snippet: Primer sequences used for reverse transcription-quantitative PCR.

Article Snippet: Cells were fixed on coverslips, washed, permeabilized, sealed, and incubated with rabbit anti-ZAP70 polyclonal antibody (1:200; E-AB-19063; Elabscience Biotechnology, Inc.).

Techniques: Sequencing

Journal: Oncology Letters

Article Title: ZAP70 interaction with 13 mRNAs as a potential immunotherapeutic target for endometrial cancer

doi: 10.3892/ol.2023.13799

Figure Lengend Snippet: GO and KEGG enrichment analysis of 108 hub genes and identification of 14 key genes. (A) GO enrichment analysis of the hub genes. (B) KEGG pathway enrichment analysis of the hub genes. (C) Protein-protein interaction analysis found genes that interacted with each other. (D) Cox proportional hazards model was used to screen key genes. (E) Gene-gene correlation analysis. (F) Correlation between key genes and ESTIMATEScore. (G) Correlation between key genes and ssGSEA. GO, Gene Ontology; HR, hazard ratio; KEGG, Kyoto Encyclopedia of Genes ad Genomes; ZAP70, ζ-chain-associated protein kinase 70; ssGSEA, single sample Gene Set Enrichment Analysis.

Article Snippet: Cells were fixed on coverslips, washed, permeabilized, sealed, and incubated with rabbit anti-ZAP70 polyclonal antibody (1:200; E-AB-19063; Elabscience Biotechnology, Inc.).

Techniques:

Journal: Oncology Letters

Article Title: ZAP70 interaction with 13 mRNAs as a potential immunotherapeutic target for endometrial cancer

doi: 10.3892/ol.2023.13799

Figure Lengend Snippet: Kaplan-Meier survival curve analysis was used to determine the effects of 14 key genes on survival time of patients with uterine corpus endometrial cancer. Kaplan-Meier survival curve analysis of (A) ZAP70; (B) LCK; (C) FOXP3; (D) TIGIT; (E) CTLA4; (F) ICOS; (G) CD5; (H) IL2RG; (I) PDCD1; (J) TNFRSF4; (K) CD27; (L) CRR7; (M) GZMB; (N) CXCL9. HR, hazard ratio; ZAP70, ζ-chain-associated protein kinase 70.

Article Snippet: Cells were fixed on coverslips, washed, permeabilized, sealed, and incubated with rabbit anti-ZAP70 polyclonal antibody (1:200; E-AB-19063; Elabscience Biotechnology, Inc.).

Techniques:

Journal: Oncology Letters

Article Title: ZAP70 interaction with 13 mRNAs as a potential immunotherapeutic target for endometrial cancer

doi: 10.3892/ol.2023.13799

Figure Lengend Snippet: In vivo and in vitro validation of ZAP70 expression and its potential role in prognosis. (A) ZAP70 mRNA expression was detected in tissues by reverse transcription-quantitative PCR. (B) ZAP70 protein expression was detected in tissues by immunohistochemistry. Image intake was presented through the microscope (cat. no. 703548; Nikon Corporation). (C) Localization of ZAP70 in cells was detected using immunofluorescence (400×). Image intake was presented through the microscope (cat. no. 703548; Nikon Corporation). Differences in ZAP70 between (D) healthy and tumoral tissues, (E) FIGO clinical stages I/II and stages III/IV, and (F) living and deceased patients. IHC, immunohistochemistry; OS, overall survival; TPM, transcripts per kilobase million; ZAP70, ζ-chain-associated protein kinase 70. *P<0.05 and ***P<0.001.

Article Snippet: Cells were fixed on coverslips, washed, permeabilized, sealed, and incubated with rabbit anti-ZAP70 polyclonal antibody (1:200; E-AB-19063; Elabscience Biotechnology, Inc.).

Techniques: In Vivo, In Vitro, Biomarker Discovery, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunohistochemistry, Microscopy, Immunofluorescence

Journal: Oncology Letters

Article Title: ZAP70 interaction with 13 mRNAs as a potential immunotherapeutic target for endometrial cancer

doi: 10.3892/ol.2023.13799

Figure Lengend Snippet: Correlation analysis between the expressions of ZAP70 and 16 known immune checkpoints in patients with uterine corpus endometrial cancer. Correlation analysis of ZAP70 and (A) PDCD1; (B) CD274; (C) PDCD1LG2; (D) CTLA4; (E) CD80; (F) CD86; (G) LAG3; (H) HAVCR2; (I) TIGIT; (J) TNFRSF4; (K) TNFRSF8; (L) TNFRSF9; (M) ICOS; (N) CD40; (O) CD27; (P) CD70 expression in TCGA-UCEC samples. ZAP70, ζ-chain-associated protein kinase 70.

Article Snippet: Cells were fixed on coverslips, washed, permeabilized, sealed, and incubated with rabbit anti-ZAP70 polyclonal antibody (1:200; E-AB-19063; Elabscience Biotechnology, Inc.).

Techniques: Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells

doi: 10.1007/s00018-022-04657-9

Figure Lengend Snippet: TCR/CD3 crosslinking in T cells promotes the association of cyclophilin A and ZAP70. A Jurkat T cells were stimulated by TCR/CD3 crosslinking (using OKT3 mAbs) for 1 min, followed by cell lysis and immunoprecipitation, as described in Materials and Methods. Whole-cell lysates and CypA immunoprecipitates were then subjected to SDS-PAGE under reducing conditions and immunoblotting with anti-pTyr mAbs. Presence of ZAP70 and CypA on the same membrane was determined by membrane stripping and reblotting with the indicated Abs. B Jurkat T cells co-transfected with GST-CypA- and Myc-ZAP70-encoding eukaryotic expression vectors were stimulated with OKT3 mAbs for 1 min and cell lysates were subjected to immunoprecipitation using anti-GST mAbs. Samples were immunoblotted sequentially with anti-Myc mAbs and anti-GST mAbs. Membrane staining with Ponceau S monitored the equal loading of proteins in all lanes. C Jurkat T cells were stimulated with OKT3 mAbs for the indicated time intervals and lysates were then subjected to CypA immunoprecipitation. Samples were immunoblotted sequentially with ZAP70-, CypA-, and β-actin-specific mAbs. Membrane staining with Ponceau S determined the equal loading of proteins in all lanes, and densitometry analysis determined the relative amount of ZAP70 that coimmunoprecipitated with CypA. D, E C57BL/6 J mouse spleen (D) and thymus (E) cells were stimulated with 2C11 (anti-CD3ε) mAbs for 1 min and cell lysates were then subjected to immunoprecipitation using anti-CypA mAbs. Samples were immunoblotted sequentially with anti-ZAP70 and anti-CypA mAbs. Membrane staining with Ponceau S monitored equal loading of proteins in all lanes. F–I C57BL/6 J mouse spleen and thymus cells were stimulated with 2C11 (anti-CD3ε) mAbs as before, fixed, permeabilized and incubated with rabbit anti-CypA and mouse anti-ZAP70 mAbs. After washing, the cells were immunostained with Alexa Fluor 488-conjugated anti-rabbit- and Alexa Fluor 546-conjugated anti-mouse-IgG and counterstained with DAPI. The cells were analyzed using a confocal laser microscope (scale bar equals 2 μm) (F, G). CypA-ZAP70 colocalization was quantified using the ImageJ plugin, and Pearson’s coefficient values (**P) for resting and stimulated cells are indicated on the right side of each panel. H, I A graph representing Mander’s coefficient values, M1 (red overlap with green) and M2 (green overlap with red). Results are representative of three independent experiments. Representative graphs show mean ± SD (n = 9). Molecular weight markers (in kDa) are indicated on the left of each panel of immunoblot. Arrows mark the position of the indicated protein bands. IB, immunoblot; IgH, Ig heavy chain; IgL, Ig light chain; IP, immunoprecipitates; WCL, whole-cell lysates

Article Snippet: GST-tagged human recombinant ZAP70 was from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Lysis, Immunoprecipitation, SDS Page, Western Blot, Membrane, Stripping Membranes, Transfection, Expressing, Staining, Incubation, Microscopy, Molecular Weight

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells

doi: 10.1007/s00018-022-04657-9

Figure Lengend Snippet: TCR/CD3 stimulation promotes CypA binding to the ZAP70 interdomain B region and recruitment of CypA to the phospho-CD3ζ-associated ZAP70. A Resting and OKT3-stimulated Jurkat T cell lysates were subjected to pulldown assay using bead-immobilized GST proteins that are fused to full-length or truncated products of ZAP70 (10 μg/group), as described in material and methods. Samples were subjected to SDS-PAGE under reducing conditions, followed by protein electrotransfer to a membrane. The membrane was immunoreacted with anti-CypA mAbs and stained with Ponceau S in order to visualize the GST-immobilized fusion proteins. B Jurkat T cells and ZAP70-deficient P116 T cells were stimulated with OKT3 mAbs for 1 min followed by cell lysis and immunoprecipitation using anti-pCD3ζ mAbs. Whole-cell lysates and pCD3ζ immunoprecipitates were then divided into two groups and subsequently subjected to SDS-PAGE under reducing conditions on an 8% and 12% polyacrylamide gels. The 8% gel-transferred nitrocellulose membrane was sequentially immunoblotted with ZAP70-, Lck-, and β-actin-specific mAbs, and the 12% gel-transferred nitrocellulose membrane was sequentially immunoblotted with CypA-, pCD3ζ-, and β-actin-, and pTyr-specific mAbs. Ponceau S staining of the membranes determined equal loading of proteins in all lanes. C–F Jurkat and P116 T cells were stimulated with OKT3 mAbs for 1 min. After fixation and permeabilization, the cells were incubated with rabbit anti-CypA and mouse anti-pCD3ζ mAbs, followed by immunostaining with Alexa Fluor 488-conjugated anti-rabbit and Alexa Fluor 546-conjugated anti-mouse Ig Abs, and counterstained with DAPI. Cells were analyzed using a confocal laser microscope (C, D scale bar equals 2 μm) and comparative colocalization of CypA and pCD3ζ in Jurkat vs. P116 cells was quantified using the ImageJ plugin, JACoP. Mander’s coefficient values for M1 (red overlap with green) and M2 (green overlap with red) (E) and Pearson’s coefficient values (F) are indicated. Results are representative of three independent experiments. Representative graphs show mean ± SD (n = 9). Molecular weight markers (in kDa) are indicated on the left of each panel of immunoblot. Arrows mark the position of the indicated protein bands. IB, immunoblot; IgH, Ig heavy chain; IgL, Ig light chain; IP, immunoprecipitates; WCL, whole-cell lysates

Article Snippet: GST-tagged human recombinant ZAP70 was from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Binding Assay, SDS Page, Electrotransfer, Membrane, Staining, Lysis, Immunoprecipitation, Incubation, Immunostaining, Microscopy, Molecular Weight, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells

doi: 10.1007/s00018-022-04657-9

Figure Lengend Snippet: ZAP70-bound CypA recruits to the TCR and stimulation by peptide-loaded MHC antigens on the surface of APC induces CypA-ZAP70 colocalization at the IS. A–D Jurkat T cells were stimulated with OKT3 mAbs for 1 min or left untreated. Fixed and permeabilized cells were then incubated with rabbit CypA-, mouse ZAP70-, and goat CD3ε-specific Abs, followed by immunostaining with Alexa Fluor 488-conjugated anti-rabbit-, Alexa Fluor 633-conjugated anti-mouse-, and Alexa Fluor 546-conjugated anti-goat-Ig Abs and counterstained with DAPI. The cells were analyzed using a confocal laser microscope (A, B; scale bar equals 2 μm). The extent of colocalization of CypA and either ZAP70 or CD3ε was determined using the ImageJ plugin, JACoP. Pearson’s coefficient values (C) and Mander’s coefficient values (± SD) M1 and M2 for the respective protein colocalization (D) are indicated (n = 9). E Conjugate formation between Jurkat T cells, clone CH7C17, and APCs (B cell line, LG2) was performed as described in Materials and Methods. Following antigen (HA peptide 307–319, or an inactive HA peptide, HA K316E) pre-pulsing and coincubation for 5 min, T cells and APCs were fixed, permeabilized, and analyzed by confocal microscopy (scale bar equals 2 μm). Immunofluorescence staining was performed using primary Abs; mouse anti-ZAP70, and rabbit anti-CypA, followed by Alexa Fluor 633-conjugated phalloidin and secondary Abs, Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 546-conjugated anti-rabbit IgG. CytoPainter stain was detected at 405 nm wavelength. Results are representative of three independent experiments

Article Snippet: GST-tagged human recombinant ZAP70 was from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Incubation, Immunostaining, Microscopy, Confocal Microscopy, Immunofluorescence, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells

doi: 10.1007/s00018-022-04657-9

Figure Lengend Snippet: CypA downregulates the catalytic activity of ZAP70. A–F C57BL/6 J mice-derived spleen and thymus cells were stimulated with 2C11 mAbs for 1 min. Subsequently, cell lysates were subjected to ZAP70 immunoprecipitation and ZAP70-containing beads were incubated in the presence of the indicated concentrations of enzymatically active recombinant human CypA (rCypA) for 10 min at 37 °C (A, C), or with 4 μg/ml of rCypA for indicated time intervals (B, D). Samples were then subjected to a ZAP70 kinase assay in the presence of [γ-32P]-ATP, as described in Materials and Methods. Samples were resolved by SDS-PAGE under reducing conditions, transferred to nitrocellulose membranes, and developed by autoradiography and immunoblotting using anti-ZAP70 and anti-CypA mAbs. Ponceau S staining of the membrane determined equal loading of proteins in all lanes. pZAP70 and ZAP70 protein band signals were quantified using the ImageJ software, and the relative amounts of pZAP70/ZAP70 in Jurkat cells, and mouse spleen and thymus cells (± SD)) were presented in a graph (E, F). G E6.1 Jurkat T cells and CypA-deficient Jurkat T cells were stimulated with OKT3 mAbs for 1 min. Cell lysates were then subjected to a ZAP70 kinase assay in the presence of [γ-32P]-ATP, as previously described. Ponceau S staining monitored equal loading of proteins in all lanes. pZAP70 and ZAP70 protein band signals were quantified using the ImageJ software, and the relative amounts of pZAP70/ZAP70 in E6.1 vs CypA-deficient Jurkat cells were presented in a bar graph (H). Graph values show mean ± SEM, *p < 0.05, using Student’s unpaired t test. Molecular weight markers (in kDa) are indicated on the left and arrows mark positions of the indicated protein bands. Results are representative of three independent experiments. IB, immunoblot; IgH, Ig heavy chain

Article Snippet: GST-tagged human recombinant ZAP70 was from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Activity Assay, Derivative Assay, Immunoprecipitation, Incubation, Recombinant, Kinase Assay, SDS Page, Autoradiography, Western Blot, Staining, Membrane, Software, Molecular Weight

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells

doi: 10.1007/s00018-022-04657-9

Figure Lengend Snippet: CypA ablation or its inhibition by cyclosporin A annuls the effect of CypA on ZAP70 activity. A C57BL/6 J-derived spleen cells were treated with CsA and stimulated with 2C11 mAbs for 1 min. Whole cell lysates and CypA immunoprecipitates were resolved by SDS-PAGE under reducing conditions followed by sequential immunoblotting with ZAP70-, CypA-, and β-actin-specific mAbs. Membrane staining with Ponceau S determined equal loading of proteins in all lanes. B CsA-treated or untreated C57BL/6 J spleen cells were left unstimulated or stimulated using 2C11 mAbs for 1 min. Fixed and permeabilized cells were then incubated with rabbit anti-CypA and mouse anti-ZAP70 mAbs, followed by immunostaining with Alexa Fluor 488-conjugated anti-rabbit and Alexa Fluor 546-conjugated anti-mouse Ig Abs and counterstained with DAPI. The cells were analyzed using a confocal laser microscope. Scale bar equals 2 μm. C–D Quantification of the extent of colocalization of CypA and ZAP70 was performed using the ImageJ plugin, JACoP. Mander’s coefficient values for M1 (red overlap with green) and M2 (green overlap with red) (± SEM) (C) and Pearson’s coefficient values (D) are indicated (n = 9). E C57BL/6 J-derived spleen cells were stimulated as before and cell lysates were subjected to ZAP70 immunoprecipitation. The ZAP70-containing beads were incubated in the presence or absence of enzymatically active recombinant human CypA (rCypA; 4 μg/100 μl) plus the indicated concentrations of CsA, for 10 min at 37 °C. Samples were then subjected to a radioactive ZAP70 kinase assay in the presence of [γ-32P]-ATP followed by SDS-PAGE under reducing conditions and protein electroblotting onto nitrocellulose membranes. The membranes were developed by autoradiography and immunoblotting using anti-ZAP70 and anti-CypA mAbs. Ponceau S staining of the nitrocellulose membrane monitored the equal usage of ZAP70 mAbs. F–G Jurkat T cells that were left untreated or treated with CsA (F), and CypA-deficient Jurkat T cells (G) were stimulated with OKT3 mAbs for indicated time intervals. Cell lysates were then subjected to LAT immunoprecipitation and samples of whole-cell lysates and immunoprecipitates were subjected to SDS-PAGE on 10% acrylamide gels under reducing conditions followed by sequential immunoblotting using anti-p-Tyr (F, G), anti-LAT (F, G), and anti-CypA (G) mAbs. Membrane staining with Ponceau S determined the equal loading of proteins in all lanes. pTyr-LAT and LAT protein band signals were quantified using the ImageJ software and the relative amounts of pTyr-LAT/LAT (± SD) were presented in a bar graph. H Jurkat T cells were nucleofected with ROZA-XL or ROZA-XL-YF plasmids. 48 h post-transfection, the cells were left untreated or treated with CsA, and subsequently stimulated with OKT3 mAbs for indicated time intervals or left unstimulated. Cells were then analyzed by FACS at CFP excitation wavelength (405 nm) and simultaneous emission was detected in the CFP and YFP emission spectra. FRET data analysis was performed using FlowJo v10.7 software and data (± SEM) were presented in a bar graph. Data are representative of three independent experiments. *p < 0.05 and **p < 0.01, using Student’s unpaired t test. Molecular weight markers (in kDa) are indicated on the left of each panel of immunoblot. Arrows mark the position of the indicated protein bands. IB, immunoblot; IgH, Ig heavy chain; IP, immunoprecipitates; WCL, whole-cell lysates

Article Snippet: GST-tagged human recombinant ZAP70 was from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Inhibition, Activity Assay, Derivative Assay, SDS Page, Western Blot, Membrane, Staining, Incubation, Immunostaining, Microscopy, Immunoprecipitation, Recombinant, Kinase Assay, Autoradiography, Software, Transfection, Molecular Weight

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells

doi: 10.1007/s00018-022-04657-9

Figure Lengend Snippet: A schematic model of ZAP70 regulation by CypA. Resting T lymphocytes (A) can be activated by the engagement of their antigen receptors (TCR) with a peptide antigen (Ag) presented on MHC molecules on the surface of an antigen-presenting cell (B). CD4- or CD8-associated Lck is then recruited to the liganded TCR (C) and phosphorylates the immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic tails of the CD3 chains, predominantly CD3ζ. The phosphorylated ITAMs serve as docking sites for the SH2 domains of the cytosolic, inactive, ZAP70 tyrosine kinase (D). Lck-mediated phosphorylation of ZAP70 (E) upregulates the catalytic activity of ZAP70 which then undergoes autophosphorylation and transphosphorylate TCR downstream effector molecules, including LAT and SLP76. Cyclophilin A (CypA) binding to phosphorylated ZAP70 (G) inhibits ZAP70 catalytic activity, either by steric hindrance of ATP and/or substrate binding, or by modulation of its conformation, leading to downregulation of phosphorylation of downstream substrates (F). Cyclosporin A (CsA) interaction with CypA reverses the effect of CypA on ZAP70 (H), either by reducing the affinity of CypA to ZAP70 and outcompeting CypA binding to ZAP70 or by inhibition of the CypA catalytic activity and reversing the effect of CypA on ZAP70 enzymatic activity

Article Snippet: GST-tagged human recombinant ZAP70 was from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Activation Assay, Phospho-proteomics, Activity Assay, Binding Assay, Inhibition

Journal: Cancer Research Communications

Article Title: Collagen-Bearing Exosomes from Breast Cancer–Associated Fibroblasts Promote T-cell Dysfunction

doi: 10.1158/2767-9764.CRC-25-0259

Figure Lengend Snippet: Exosomal ECM from CAFs attenuates T-cell proliferation. A, Mean difference plot showing the log fold change and average abundance of each protein from CAF exosomes. Proteins with fold changes >1 are highlighted [red, up after proteinase K (PK) or blue, down after PK treatment]. Top 10 labeled. B, Gene set enrichment analysis identified proteins associated with ECM organization in CAF exosomes. C, Western blot of COL1A1 and COL5A2 proteins in cells (top) and in exosomes (bottom) of normal fibroblasts (NF) from the MFP of naïve animals and CAFs from the MCaP murine cell line. D, Western blot of COL1A1 and COL5A2 proteins in cells (top) and in exosomes (bottom) of untransformed normal human fibroblasts (NF) and CAFs from luminal breast cancer. E and F, Protein content based on BCA assay ( E ) and extracellular vesicle composition ( F ) of fractions 1–12 collected by iodixanol density gradient centrifugation. G, Western blot of fractions from ultracentrifugation, probed with antibodies against COL1A1, COL5A2, exosome markers Alix and CD63, and the serum protein albumin. H, Western blot of COL1A1 (top) and COL5A2 (bottom) in MCaP CAFs with empty vector (vector) or targeted sgRNA (sg) vectors. I, Exosomes from MCaP CAFs with empty vector or targeted sgRNA constructs were added to CFSE-labeled T cells along with anti-CD3/CD28 antibodies. CFSE dilution in T cells was measured by flow cytometry 72 hours later. J, Western blot of CAF exosomes after collagenase I digestion for 1 hour at indicated concentrations. K, CAF exosomes after digestion with collagenase I at indicated concentration were added to CFSE-labeled T cells along with anti-CD3/CD28 antibodies. CFSE dilution was measured by flow cytometry 72 hours later. Significance was tested using one-way ANOVA with Tukey honestly significant difference post hoc test ( I and K ). L, Naïve CD8 + T cells isolated from Balb/c mice were activated with anti-CD3/CD28 antibodies and nucleofected 72 hours later with Cas9 and sgRNAs targeting SHP2, DDR1, or LAIR-1, or with mock nucleofection (mock). Knockdown efficiency was assessed by Western blot. M, A subset of T cells from each cohort described in ( L ) was labeled with CFSE and subsequently stimulated with CAF-derived exosomes and anti-CD3/CD28 antibodies. Proliferation was assessed by flow cytometry 72 hours later. CFSE-labeled T cells stimulated with anti-CD3/CD28 antibodies alone served as positive controls (Stim.), whereas unstimulated cells (“Unstim.”) served as negative controls. Significance was tested using one-way ANOVA with Tukey honestly significant difference post hoc test.

Article Snippet: The following primary antibodies were purchased from Proteintech: Alix, cat. #67715-1-Ig, RRID: AB_2882905; anti-calnexin, cat. #10427-2-AP, RRID: AB_2069033; anti-ZAP-70, cat. #60200-1-Ig, RRID: AB_10944567; anti–PD-L1 (cat. #66248-1-Ig, RRID: AB_2756526; and anti–SLP-76, cat. #12728-1-AP, RRID: AB_2136718).

Techniques: Labeling, Western Blot, BIA-KA, Gradient Centrifugation, Plasmid Preparation, Construct, Flow Cytometry, Concentration Assay, Isolation, Knockdown, Derivative Assay