Journal: Journal for immunotherapy of cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity.

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: Figure 2 In vitro characterization of anti-GARP antibody PIIO-1. (A) GARP expression on human regulatory T cells and platelets was evaluated by flow cytometry after staining with PIIO-1 at 10 µg/mL. (B) 293 FT cell line was transfected with empty vector (EV), human GARP (hGARP)-expressing vector only or co-transfected with hGARP and latent TGFβ1 expression vectors. GARP expression on indicated cell line was detected by flow cytometry after staining with PIIO-1 at 10 µg/mL. (C) Human GARP sequence was replaced by murine GARP according to the schematic diagram to generate the chimeric constructs of human and murine GARP that were tagged with HA (hemagglutinin) epitope. Transfection efficiency was determined using anti- HA antibody. All constructs were transfected into 293 FT cells. (D) Crystal structure of the GARP (green)-LTGFβ (gray) complex (PDB DOI: 10.2210/pdb6GFF/pdb). The region of PIIO-1 recognition is orange and the residues interacting with LTGFβ are cyan. LTGFβ occludes approximately 30% of the potential antibody binding site and may sterically or allosterically restrict access of the antibody to GARP in the LTGFβ-complexed state. Modeling was carried out using Pymol. (E) Jurkat cell line, made to overexpress hGARP, was incubated with LTGFβ1 along with mIgG1 or PIIO-1 at indicated concentration for 30 min at 37℃. Human LAP expression was detected by flow cytometry. All data are representative of 2–6 independent experiments. GARP, Glycoprotein-A repetitions predominant; LAP, latency-associated peptide.

Article Snippet: Generation of anti-human GARP (hGARP) antibodies The generation of anti- hGARP antibody has been described previously.19 BALB/c mice was immunized with recombinant human GARP (R&D Systems) in Freund’s complete adjuvant and followed by boosting with SP2/0hGARP cells for 2–3 times.

Techniques: In Vitro, Expressing, Flow Cytometry, Staining, Transfection, Plasmid Preparation, Sequencing, Construct, Binding Assay, Incubation, Concentration Assay

Journal: Journal for immunotherapy of cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity.

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: Figure 3 PIIO-1 enhanced antitumor efficacy of anti-PD-1 ICB in GARP+ TNBC. (A) Experimental scheme. BALB/c mice were injected with 1×105 4T1-hGARP cells in the mammary fat pad, followed by i.p. injection of 200 µg/mouse of PIIO-1 and/or 150 µg/mouse anti-PD-1 every 3 days. (B) Primary tumor growth curve. (C) Overall survival of mice. (D) Summary of the incidence of tumor-free mice among groups. (E) Lungs were collected and sectioned at experimental end point, then stained with H&E. Representative images from each group of mice are shown. Scale bar, 20 µm. The numbers of visible lung metastatic nodules are quantified. (F) Summary of the incidence of metastasis among groups. (G) Tumors were collected at end point and stained by IHC for pSMAD3, α-SMA. Representative images of tumor tissues from four groups of mice are shown (left). Scale bar, 50 µm. Quantification of the IHC staining is shown (right). (H) Sera from each mouse was collected at end point. Total and active TGFβ level in the sera were assessed by ELISA. (I) Mice with tumor regression following combination therapy were monitored for 300 days, then rechallenged with 5×105 wild-type 4T1 mammary tumor in contralateral mammary fat pad. Naive BALB/c mice without pre-exposure to tumor were used as control. Shown is the overall survival. Tumor curve analysis was performed using repeated measures 2-way analysis of variance. Overall survival is analyzed by log-rank (Mantel-Cox) test. (E, G) were analyzed by paired t-test according to the tumor collection time points. Other data were analyzed by two-tailed Student’s t-test. B, C were corrected for multiple testing using the Tukey procedure. All data are presented as mean±SEM. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001.

Article Snippet: Generation of anti-human GARP (hGARP) antibodies The generation of anti- hGARP antibody has been described previously.19 BALB/c mice was immunized with recombinant human GARP (R&D Systems) in Freund’s complete adjuvant and followed by boosting with SP2/0hGARP cells for 2–3 times.

Techniques: Injection, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Expression of GARP Is Increased in Tumor-Infiltrating Regulatory T Cells and Is Correlated to Clinicopathology of Lung Cancer Patients

doi: 10.3389/fimmu.2017.00138

Figure Lengend Snippet: Level of glycoprotein A repetitions predominant (GARP) expression on Tregs and Tconvs from lung cancer patients . Mononuclear cells from tumor tissues and peripheral bloods (PBs) from the indicated sources were stained for CD4, GARP, and intracellular Foxp3 and analyzed with FACS. (A,C) Proportion of GARP + Tregs in tumor tissues and PBs from lung cancer patients or healthy donors, respectively. Typical flow plots are shown. (B) Proportion of GARP + Tregs and mean fluorescence intensity (MFI) of GARP expression by Tregs and Tconvs in tumor tissues from lung cancer patients. Data shown are the summary from 39 lung cancer patients. (D) Proportion of GARP + Tregs and MFI of GARP expression by Tregs and Tconvs in PBs both from lung cancer patients and healthy donors. Data shown are the summary from 50 lung cancer patients and 10 healthy donors. (E) Proportion of Foxp3 + Tregs from lung cancer patients. Data shown are the summary from 39 lung cancer patients’ tumor tissues and 50 lung cancer patients’ PBs. (F) Proportion of GARP + Tregs and MFI of GARP expression from lung cancer patients. Data shown are the summary from 39 lung cancer patients’ tumor tissues and 50 lung cancer patients’ PBs. Results are expressed as median and interquartile range. P value shown is obtained from the comparison between the indicated groups by non-parametric t -test and Kruskal–Wallis test.

Article Snippet: The antibodies used for surface staining in this study included antihuman CD4-PerCP/Cy5.5 (BD Bioscience, San Diego, CA, USA, clone RPA-T4, cat num 560650), antihuman GARP-PE (Miltenyi Biotec, Germany, cat num 130-103-889), and antihuman LAP (TGF-β)-APC (R&D, USA, cat num FAB2463A).

Techniques: Expressing, Staining, Fluorescence, Comparison

Journal: Frontiers in Immunology

Article Title: Expression of GARP Is Increased in Tumor-Infiltrating Regulatory T Cells and Is Correlated to Clinicopathology of Lung Cancer Patients

doi: 10.3389/fimmu.2017.00138

Figure Lengend Snippet: Continued Different lung cancer cell lines induced glycoprotein A repetitions predominant (GARP) expression on Tregs from peripheral bloods (PBs) of lung cancer patients . CD4 + CD25 + Tregs were isolated from the PBs of lung cancer patients. After 72 h of TCR stimulation, these subsets were stained with CD4, GARP, latency-associated peptide (LAP), and intracellular Foxp3 and analyzed with FACS. (A) Typical FACS plots. Black histogram: Ab staining after TCR stimulation; gray-filled histogram: negative control (before TCR stimulation). Numbers in the plots indicate the proportion of positive cells after TCR stimulation. (B) Summary of proportion of GARP + and LAP + Tregs before and after TCR stimulation. Coculture assays were performed between lung cancer cell lines and CD4 + CD25 + Tregs, which were isolated from the PBs of lung cancer patients for 72 h. Tregs were stained for CD4, GARP, and intracellular Foxp3 and analyzed with FACS. (C) Typical FACS plots. Black histogram: Ab staining; gray-filled histogram: isotype control. Numbers in the plots indicate the proportion of positive cells. (D) Summary of the proportion of GARP + Tregs cocultured with different lung cancer cell lines. Results are expressed as mean ± SEM ( n = 3). P value shown is obtained from the comparison between the indicated groups by two-sample t -test.

Article Snippet: The antibodies used for surface staining in this study included antihuman CD4-PerCP/Cy5.5 (BD Bioscience, San Diego, CA, USA, clone RPA-T4, cat num 560650), antihuman GARP-PE (Miltenyi Biotec, Germany, cat num 130-103-889), and antihuman LAP (TGF-β)-APC (R&D, USA, cat num FAB2463A).

Techniques: Expressing, Isolation, Staining, Negative Control, Comparison

Journal: Frontiers in Immunology

Article Title: Expression of GARP Is Increased in Tumor-Infiltrating Regulatory T Cells and Is Correlated to Clinicopathology of Lung Cancer Patients

doi: 10.3389/fimmu.2017.00138

Figure Lengend Snippet: Level of glycoprotein A repetitions predominant (GARP) expression on Tregs by transwell coculture assays . Transwell coculture assays were performed between lung cancer cell lines (A549 and H520) and CD4 + CD25 + Tregs, which were isolated from peripheral bloods of lung cancer patients for 72 h. Tregs were stained for CD4, GARP, and intracellular Foxp3 and analyzed with FACS. (A) Typical FACS plots. Black histogram: Ab staining; gray-filled histogram: isotype control. Numbers in the plots indicate the proportion of positive cells. (B) Summary of the proportion of GARP + Tregs in Transwell and contact cocultured groups with A549 and H520, respectively. Results are expressed as mean ± SEM ( n = 3). P value shown is obtained from the comparison between the indicated groups by two-sample t -test.

Article Snippet: The antibodies used for surface staining in this study included antihuman CD4-PerCP/Cy5.5 (BD Bioscience, San Diego, CA, USA, clone RPA-T4, cat num 560650), antihuman GARP-PE (Miltenyi Biotec, Germany, cat num 130-103-889), and antihuman LAP (TGF-β)-APC (R&D, USA, cat num FAB2463A).

Techniques: Expressing, Isolation, Staining, Comparison

Journal: Frontiers in Immunology

Article Title: Expression of GARP Is Increased in Tumor-Infiltrating Regulatory T Cells and Is Correlated to Clinicopathology of Lung Cancer Patients

doi: 10.3389/fimmu.2017.00138

Figure Lengend Snippet: Cancer cell line supernatant induced glycoprotein A repetitions predominant (GARP) expression on Tregs . The supernatant of cancer cell lines (A549 and H520) was collected to culture CD4 + CD25 + Tregs, which were isolated from the peripheral bloods of lung cancer patients for 72 h. Tregs were stained with CD4, GARP, and intracellular Foxp3 and analyzed with FACS. (A) Typical FACS plots. Black histogram: Ab staining; gray-filled histogram: isotype control. Numbers in the plots indicate the proportion of positive cells. (B) Summary of the proportion of GARP + Tregs in supernatant cultured and contact cocultured groups with A549 and H520, respectively. Results are expressed as mean ± SEM ( n = 3). P value shown is obtained from the comparison between the indicated groups by two-sample t -test.

Article Snippet: The antibodies used for surface staining in this study included antihuman CD4-PerCP/Cy5.5 (BD Bioscience, San Diego, CA, USA, clone RPA-T4, cat num 560650), antihuman GARP-PE (Miltenyi Biotec, Germany, cat num 130-103-889), and antihuman LAP (TGF-β)-APC (R&D, USA, cat num FAB2463A).

Techniques: Expressing, Isolation, Staining, Cell Culture, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: GARP: a key receptor controlling FOXP3 in human regulatory T cells

doi: 10.1111/j.1582-4934.2009.00782.x

Figure Lengend Snippet: GARP is exclusively induced in CD4 + CD25 hi -derived T reg cells. (A) Human CD4 + CD25 hi T reg and CD4 + CD25 − T cells were sorted to a purity of >99% and analysed ex vivo (no stim.) and 24 hrs after stimulation using anti-CD3/IL-2 and anti-CD3/anti-CD28/IL-2. GARP signal intensity as determined by GenChip analysis and is represented normalized to the signal of RPS9. (B) Confirmation by real-time RT-PCR analysis of GARP (upper panel) and FOXP3 (lower panel) mRNA expression in independently sorted CD4 + CD25 hi T reg and CD4 + CD25 − T h cells analysed ex vivo (t 0h ), 12, 48 hrs and day 9 after stimulation with anti-CD3/anti-CD28 Dynalbeads at a ratio of 1:1 and IL-2. Relative mRNA expression of CD4 + CD25 − T h cells at time t 0 was arbitrarily set as 1. (C) The same CD4 + CD25 hi T reg cells as in (B) were tested for suppressor function of alloantigen-stimulated CD4 + CD25 − T h cells at a ratio of 1 to 1. Proliferation was assessed at day 3 by measuring incorporation of H 3 -thymidin (cpm). (D) Fourier shell correlation of refinement process and EOTEST are shown (left part). The values 1.4 and 1.8 correspond to the estimated resolution of 14 and 18 Å. The insert is an asymmetric triangular showing the distribution of the particles’ orientations. Representative gallery of projections of the single particles alternating with the corresponding class averages is shown on the right. (E) Surface display of the 3D reconstruction together with a ribbon representation of the model of human GARP. The asparagine residues of putative glycosylation sites are indicated as space-filling spheres coloured according to atom type (carbon light brown, oxygen red, nitrogen blue). (F) Western blot analysis of alloantigen-specific T reg and T h cells without or with retroviral overexpression of GARP, FOXP3, LGMN, LGALS3, and GFP under resting and activated conditions using anti-CD3/IL-2 stimulation for 3 days using anti-GARP specific mAb 50G10; anti-Tubulin served as loading control. (G) Detection of cell surface expression of wt GARP and mutant GARPΔPDZ expressed in 293 cells (left panel) using mAb 272G6 compared to WB detection in the same cells (insert) using mAb 50G10. (H) T reg and T h cells as in (F) were treated for 4 hrs with PMA (40 ng/ml) and ionomycine (0.5 μg/ml) to induce up-regulation of the early-induced gene CD83 (lower panel) and tested for surface expression of GARP (upper panel).

Article Snippet: Monoclonal antibodies against human glycoprotein-A repetitions predominant (GARP) were generated according to standard protocols ( http://www.sysy.com/mabservice.html ).

Techniques: Derivative Assay, Ex Vivo, Quantitative RT-PCR, Expressing, Glycoproteomics, Western Blot, Retroviral, Over Expression, Control, Mutagenesis

Journal: Journal of Cellular and Molecular Medicine

Article Title: GARP: a key receptor controlling FOXP3 in human regulatory T cells

doi: 10.1111/j.1582-4934.2009.00782.x

Figure Lengend Snippet: Ectopic expression of GARP in human alloantigen-specific T h cells induces sustained expression of FOXP3. (A) T h cells as in were analysed for FOXP3 and (B) CD25, CTLA4, LGALS3, CD27, CD33, GITR and CD83 expression by flow cytometry under resting conditions. (C) The same cells as in (A) were stimulated for 3 days with cognate antigen and 50 U/ml IL-2 and analysed for FOXP3 and LGALS3 protein expression. Gates for figures (A–C) were set according to non-stained control (CD25) or isotype control (CTLA4, LGALS3, FOXP3, CD33, CD83, GITR) represented as shaded histogram (A, B) or set as quadrant (C); percentage of positive cells is indicated. T reg cells were included for comparison (A–C).

Article Snippet: Monoclonal antibodies against human glycoprotein-A repetitions predominant (GARP) were generated according to standard protocols ( http://www.sysy.com/mabservice.html ).

Techniques: Expressing, Flow Cytometry, Staining, Control, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: GARP: a key receptor controlling FOXP3 in human regulatory T cells

doi: 10.1111/j.1582-4934.2009.00782.x

Figure Lengend Snippet: GARP induces T reg -signature of transcriptional control. (A) Resting and anti-CD3/IL-2 stimulated T cells as in and were analysed for mRNA expression of IL-2 by real-time RT-PCR. The individual fold change of relative mRNA expression of indicated T h and T reg cells were compared to T h GFP cells, arbitrarily set as 1, is indicated.; n.d. = not detected. (B) K-Means clusterization of significantly regulated genes in T reg , T h GARP and T h FOXP3 cells compared to T h GFP cells, all stimulated for 3 days with anti-CD3/IL-2. The heat map represent signal log ratios. Numbers correspond to clusters of Table S2. (C) Real-time RT-PCR analysis of KLF-2 expression in anti-CD3/IL-2 stimulated T h cells transduced with GARP, FOXP3 or GFP. Relative mRNA expression of T h GFP cells was arbitrarily set as 1 (upper panel). Real-time RT-PCR analysis of KLF-2 mRNA in T h cells transduced with LGALS3 and LGMN; T h cells transduced with GFP, GARP, and parental cells without transduction (T h GFP-) served as control (cell line T h B). Relative mRNA expression of T h GFP- cells was arbitrarily set as 1 (lower panel). (D) FOXP3 expressio5n in antigen-specific T h cells separated for different levels of CD33 expression (T h CD33+ and T h CD33− cells, respectively) by cell sorting following stimulation for 3 days using cognate antigen and IL-2. Isotype control = grey filled, FOXP3 = bold black. (E) The same cells as in (D) were analysed for cell surface CD83 and CD33 expression following stimulation with cognate antigen and IL-2 for 3 days. (F) A5 cells, transduced with GARP, mutant GARPΔPDZ or control lentiviral vector were analysed for GFP expression without (grey filled) and with 1 μg/ml Ionomycine stimulation for 2 (thin line) and 4 (thick line) hrs (upper panel);% GFP is represented. Lower panel represents the corresponding cell surface expression of GARP, detected with mAb 272G6 as in .

Article Snippet: Monoclonal antibodies against human glycoprotein-A repetitions predominant (GARP) were generated according to standard protocols ( http://www.sysy.com/mabservice.html ).

Techniques: Control, Expressing, Quantitative RT-PCR, Transduction, FACS, Mutagenesis, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: GARP: a key receptor controlling FOXP3 in human regulatory T cells

doi: 10.1111/j.1582-4934.2009.00782.x

Figure Lengend Snippet: Anergy and suppressor function induced by overexpression of GARP in human alloantigen-specific T h cells. (A) T reg cells and T h cells as in – were stimulated for proliferation using irradiated allogeneic EBV B cells (stim.); bkg. = background proliferation. Proliferation was assessed at day 3 by measuring incorporation of H -thymidin (cpm). (B) T reg and T h cells as in (A) were tested for suppressor function of alloantigen-stimulated T h GFP cells at a ratio of 1 to 1 either separated by a transwell membrane (no contact, upper panel) or without separation (cell contact, middle panel); lower panel represents induced T h GFP cell proliferation without the addition of a potential suppressor or control cell population. Proliferation was assessed at day 3. Similar results were obtained using antigen-specific T h cells as responder cells instead of T h GFP cells (Fig. S6B). (C) Single donor platelets as natural source of GARP + cells was tested for suppressor function of alloantigen-stimulated T h cells at indicated ratios as in (B); addition of T h and T reg cells as in (B) at a ratio of 1:1 were included as negative and positive control of suppressor function, respectively.

Article Snippet: Monoclonal antibodies against human glycoprotein-A repetitions predominant (GARP) were generated according to standard protocols ( http://www.sysy.com/mabservice.html ).

Techniques: Over Expression, Irradiation, Membrane, Control, Positive Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: GARP: a key receptor controlling FOXP3 in human regulatory T cells

doi: 10.1111/j.1582-4934.2009.00782.x

Figure Lengend Snippet: Positive feedback loop between GARP and FOXP3 in human T reg cells. (A) Real-time RT-PCR analysis of GARP and FOXP3 expression in human T reg cells, lentivirally transduced with siRNA constructs specific for FOXP3 (T reg siFOXP3 4 ), GARP (T reg siGARP 4 ) or non-specific control (T reg siGL4). Relative mRNA expression of T h cells was arbitrarily set as 1. (B) The same cells as in (A) were analysed for surface expression of CD83, CD27, and CD25 following antigen-specific stimulation with EBV B cells and IL-2. (C) Impairment of suppressor function of T reg siFOXP3 4 and T reg siGARP 1 cells of compared to T reg siGL4 cells was assessed in a suppressor assay at a cell ratio of 1:1 as described in . (D) Relative expression of GARP and FOXP3 mRNA in the indicated thymic T-cell subsets of normal donors (open symbols), assessed by TaqMan assay, normalized to the expression of β-actin, is represented; black symbols = mean of relative mRNA expression, rel. = relative, *= P < 0.002 by 2-sided Student’s t-test.

Article Snippet: Monoclonal antibodies against human glycoprotein-A repetitions predominant (GARP) were generated according to standard protocols ( http://www.sysy.com/mabservice.html ).

Techniques: Quantitative RT-PCR, Expressing, Transduction, Construct, Control, TaqMan Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: GARP: a key receptor controlling FOXP3 in human regulatory T cells

doi: 10.1111/j.1582-4934.2009.00782.x

Figure Lengend Snippet: Simplified model of the reprogramming or ‘transdifferentation’ of effector towards regulatory T cells via the GARP-FOXP3 positive feedback loop. The upper part illustrates the change in the ‘quality’ of TCR signalling outcome from effector (green) towards regulatory (green) TCR signalling. Thus, each TCR stimulation enhances the positive feedback indicated by the size of the feedback loop illustrated in the middle. For simplicity, other components of the regulatory network described, like LGALS3, LGMN, CD33, CD27 and CD83 or direct impairment of NFAT by GARP have been excluded. Identification of further components, fine tuning, timed-sequential expression and interconnectivity between the components of the regulatory network represents a major challenge for the molecular definition of the regulatory program.

Article Snippet: Monoclonal antibodies against human glycoprotein-A repetitions predominant (GARP) were generated according to standard protocols ( http://www.sysy.com/mabservice.html ).

Techniques: Expressing