Journal: eLife

Article Title: Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion

doi: 10.7554/elife.59350

Figure Lengend Snippet: Figure 1. Downregulation of ICOSL upon MCMV infection of antigen-presenting cells (APCs). (A) Peritoneal macrophages (MF) were mock-infected or infected for 72 hr with MCMV-GFP at an moi of 5 and analyzed by flow cytometry for surface expression of ICOSL, MHC I, or CD62L using specific mAbs against each of these molecules. Gray histograms represent the expression on mock-infected cells, green histograms represent the expression on MCMV-infected (GFP+) cells, and blue histograms represent the expression on uninfected (GFP-) cells from the same culture. (B) Bone-marrow- derived macrophages (BMM), bone-marrow-derived dendritic cells (BMDC), IC-21, DC2.4, and SVEC4-10 cells were infected with MCMV-GFP at different moi (10, 20, 20, and 5, respectively) to obtain an infection of around 50% of the cell culture, and analyzed by flow cytometry for surface expression of ICOSL. Gray, green, and blue histograms, as indicated in A. (C) Same as in A, except that peritoneal macrophages were infected with MCMV-GFP at an moi of 10, or treated for 72 hr with the same amount of MCMV-GFP UV-inactivated. Gray and green as indicated in A, and red histograms represent the expression on MCMV-GFP UV-inactivated infected cells. (D) Peritoneal macrophages, BMDCs, and IC-21 cells were mock- infected (time 0) or infected with MCMV-GFP at an moi of 10, 40, and 20, respectively, and analyzed by flow cytometry for surface expression of ICOSL at the different time points after infection indicated. In A, B, C, and D, the isotype for each antibody was used as a negative control (dotted lines). Data are representative of at least two independent experiments.

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody anti-mouse ICOSL (clone HK5.3) (Rat monoclonal) Biolegend San Diego, CA, USA Cat#: 107403 RRID:AB_ 345259 (Biotin) Cat#: 107405 RRID:AB_ 2248797 (PE) FACS (1:100) IF (1:50) Antibody anti-human ICOSL (Rabbit polyclonal) Elabscience Wuhan, Hubei, China Cat#: E-AB15519 WB (1:1000) Antibody anti-mouse ICOSL (clone 599841) (Rat monoclonal) R and D Systems, Minneapolis, MN, USA Cat#: MAB158 RRID:AB_ 10719415 IF (1:500) Antibody anti-m123/IE1 (MCMV) (clone IE1.01) (Mouse monoclonal) Center for Proteomics, Faculty of Medicine, University of Rijeka, Croatia Cat#: HRMCMV-12 Stock 1 mg/ mL IF (1:100) FC (1:100) WB (1:1000) Antibody anti-HA Agarose conjugate (Mouse monoclonal) Sigma-Aldrich, St. Louis, MO, USA Cat#: A2095 RRID:AB_ 257974 Cell line (M. musculus) NIH3T3 ATCC, Manassas, VA, USA Cat#: CRL1658 RRID: CVCL_ 0594 Cell line (M. musculus) DC2.4 Merck Millipore, Burlington, MA, USA Cat#: SCC142 RRID: CVCL_ J409 Cell line (M. musculus) MEFs p53 Dr. Jay Nelson, Health Sciences University, Oregon, USA Cell line (M. musculus) SVEC4-10 ATCC, Manassas, VA, USA Cat#: CRL2181 RRID: CVCL_ 4393 Cell line (M. musculus) IC-21 ATCC, Manassas, VA, USA Cat#: TIB186 RRID: CVCL_ 3726 Cell line (M. musculus) NS1 ECACC, Public Health England, Salisbury, UK Cat#: 85011427 RRID: CVCL_ 2155 Cell line (M. musculus) 300.19 Dr. Michel Streuli, Dana Farber Cancer Institute, MA, USA

Techniques: Infection, Flow Cytometry, Expressing, Derivative Assay, Cell Culture, Negative Control

Journal: eLife

Article Title: Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion

doi: 10.7554/elife.59350

Figure Lengend Snippet: Figure 2. Identification of the MCMV gene involved in the decreased cell surface expression of ICOSL. (A) IC-21 cells were mock-infected or infected for 72 hr with MCMV-GFP, MCMV-GFPDm128-m138, or MCMV-GFPDm1-m17 at an moi of 10 and examined by flow cytometry for surface expression of ICOSL. Gray histograms represent the expression of mock-infected cells, green histograms represent the expression on MCMV-infected (GFP+) cells, and blue histograms represent the expression on uninfected (GFP-) cells from the same culture. (B) IC-21 cells were mock-infected or infected with MCMVwt or MCMVDm138 for 72 hr at an moi of 20 and analyzed by flow cytometry for surface expression of ICOSL and CD84. Gray histograms as indicated in A, and brown histograms represent the expression on MCMVwt and MCMVDm138 IC-21-infected cells. (C) IC-21 cells were mock- transfected or transfected with the m138-GFP construct or with the control GFP empty vector and stained with the anti-ICOSL mAb or an isotype control (dotted lines). Dark green and red histograms represent cells from transfected cultures expressing or not expressing GFP, respectively, and gray histograms represent surface expression of ICOSL on untransfected cells. In A, B, and C, the isotype for each antibody was used as a negative control (dotted lines). Data are representative of at least two independent experiments.

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody anti-mouse ICOSL (clone HK5.3) (Rat monoclonal) Biolegend San Diego, CA, USA Cat#: 107403 RRID:AB_ 345259 (Biotin) Cat#: 107405 RRID:AB_ 2248797 (PE) FACS (1:100) IF (1:50) Antibody anti-human ICOSL (Rabbit polyclonal) Elabscience Wuhan, Hubei, China Cat#: E-AB15519 WB (1:1000) Antibody anti-mouse ICOSL (clone 599841) (Rat monoclonal) R and D Systems, Minneapolis, MN, USA Cat#: MAB158 RRID:AB_ 10719415 IF (1:500) Antibody anti-m123/IE1 (MCMV) (clone IE1.01) (Mouse monoclonal) Center for Proteomics, Faculty of Medicine, University of Rijeka, Croatia Cat#: HRMCMV-12 Stock 1 mg/ mL IF (1:100) FC (1:100) WB (1:1000) Antibody anti-HA Agarose conjugate (Mouse monoclonal) Sigma-Aldrich, St. Louis, MO, USA Cat#: A2095 RRID:AB_ 257974 Cell line (M. musculus) NIH3T3 ATCC, Manassas, VA, USA Cat#: CRL1658 RRID: CVCL_ 0594 Cell line (M. musculus) DC2.4 Merck Millipore, Burlington, MA, USA Cat#: SCC142 RRID: CVCL_ J409 Cell line (M. musculus) MEFs p53 Dr. Jay Nelson, Health Sciences University, Oregon, USA Cell line (M. musculus) SVEC4-10 ATCC, Manassas, VA, USA Cat#: CRL2181 RRID: CVCL_ 4393 Cell line (M. musculus) IC-21 ATCC, Manassas, VA, USA Cat#: TIB186 RRID: CVCL_ 3726 Cell line (M. musculus) NS1 ECACC, Public Health England, Salisbury, UK Cat#: 85011427 RRID: CVCL_ 2155 Cell line (M. musculus) 300.19 Dr. Michel Streuli, Dana Farber Cancer Institute, MA, USA

Techniques: Expressing, Infection, Flow Cytometry, Transfection, Construct, Control, Plasmid Preparation, Staining, Negative Control

Journal: eLife

Article Title: Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion

doi: 10.7554/elife.59350

Figure Lengend Snippet: Figure 4. m138 leads to a reduction of the expression of ICOSL on the cell surface of infected cells but not of its overall intracellular levels. (A) NIH3T3 cells stably transfected with HA-ICOSL were mock-infected (gray histograms) or infected with MCMVwt or MCMVDm138 (brown or pink histograms, respectively) and analyzed by flow cytometry for surface expression of ICOSL and MHC I. (B) NIH3T3 HA-ICOSL cells non-transfected (gray histograms) or transfected with the m138-GFP construct (green histograms) were analyzed by flow cytometry for surface expression of ICOSL and CD44. In A and B, the isotype for each antibody was used as a negative control (dotted lines). (C) NIH3T3 HA-ICOSL cells were mock-infected (lane 2), treated with trypsin (lane 3), or infected with MCMVwt (lane 4) or MCMVDm138 (lane 5). Untransfected NIH3T3 cells were used as a control (lane 1). Samples were analyzed by western blot using an anti-HA mAb, followed by anti-rabbit IgG-HRP. An anti-IE1 and an anti-actin mAbs followed by anti-mouse IgG-HRP, were used as controls of MCMV infection and loading, respectively. (D) Flow cytometry analysis of NIH3T3 HA-ICOSL treated with trypsin (open histogram) or mock-treated (gray histogram) and stained with an anti-HA mAb or an isotype control (dotted line), followed by anti-mouse IgG PE. (E) NIH3T3 cells (lane 1) or NIH3T3 HA-ICOSL (lane 2) were surface labeled with biotin, immunoprecipitated with an anti-HA mAb, and analyzed by western blot analysis using streptavidin-HRP conjugate. (F) NIH3T3 HA-ICOSL cells were mock-infected (lane 2), or infected with MCMVwt (lane 3) or MCMVwt UV-inactivated (lane 4). Samples were lysed and processed as indicated in C. A lysate of untransfected NIH3T3 cells (lane 1) was also subjected to western blot and employed as a control. All infections were performed at an moi of 5 for 24 hr. Data are representative of at least two independent experiments. In C, E, and F, the size of the bands corresponding to ICOSL are indicated on the right margin in kilodaltons (kDa). In E, the sizes of the molecular weight markers are shown on the left side.

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody anti-mouse ICOSL (clone HK5.3) (Rat monoclonal) Biolegend San Diego, CA, USA Cat#: 107403 RRID:AB_ 345259 (Biotin) Cat#: 107405 RRID:AB_ 2248797 (PE) FACS (1:100) IF (1:50) Antibody anti-human ICOSL (Rabbit polyclonal) Elabscience Wuhan, Hubei, China Cat#: E-AB15519 WB (1:1000) Antibody anti-mouse ICOSL (clone 599841) (Rat monoclonal) R and D Systems, Minneapolis, MN, USA Cat#: MAB158 RRID:AB_ 10719415 IF (1:500) Antibody anti-m123/IE1 (MCMV) (clone IE1.01) (Mouse monoclonal) Center for Proteomics, Faculty of Medicine, University of Rijeka, Croatia Cat#: HRMCMV-12 Stock 1 mg/ mL IF (1:100) FC (1:100) WB (1:1000) Antibody anti-HA Agarose conjugate (Mouse monoclonal) Sigma-Aldrich, St. Louis, MO, USA Cat#: A2095 RRID:AB_ 257974 Cell line (M. musculus) NIH3T3 ATCC, Manassas, VA, USA Cat#: CRL1658 RRID: CVCL_ 0594 Cell line (M. musculus) DC2.4 Merck Millipore, Burlington, MA, USA Cat#: SCC142 RRID: CVCL_ J409 Cell line (M. musculus) MEFs p53 Dr. Jay Nelson, Health Sciences University, Oregon, USA Cell line (M. musculus) SVEC4-10 ATCC, Manassas, VA, USA Cat#: CRL2181 RRID: CVCL_ 4393 Cell line (M. musculus) IC-21 ATCC, Manassas, VA, USA Cat#: TIB186 RRID: CVCL_ 3726 Cell line (M. musculus) NS1 ECACC, Public Health England, Salisbury, UK Cat#: 85011427 RRID: CVCL_ 2155 Cell line (M. musculus) 300.19 Dr. Michel Streuli, Dana Farber Cancer Institute, MA, USA

Techniques: Expressing, Infection, Stable Transfection, Transfection, Flow Cytometry, Construct, Negative Control, Control, Western Blot, Staining, Labeling, Immunoprecipitation, Molecular Weight

Journal: eLife

Article Title: Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion

doi: 10.7554/elife.59350

Figure Lengend Snippet: Figure 5. m138 impedes ICOSL maturation redirecting it to lysosomal degradation. (A) Western blot analysis of NIH3T3 cells untransfected (lane 1) or transiently co-transfected with either the HA-ICOSL and the control-GFP (CTL-GFP) constructs (lanes 2 and 3), or the HA-ICOSL and the m138-GFP constructs (lanes 4 and 5) and, when indicated, treated with 250 mM of leupeptin and 20 nM of bafilomycin A1 (lanes 3 and 5). The expression of ICOSL and actin was assessed as described in Figure 4C, and for the expression of the m138-GFP and CTL-GFP proteins a polyclonal antibody anti-GFP followed by anti-rabbit IgG-HRP were used. (B) NIH3T3 HA-ICOSL were infected with MCMVwt at an moi of 5 for 24 hr in the absence (-; panels a, b, c and d) or presence of leupeptin and bafilomycin (panels e, f, g and h). Cells were fixed, permeabilized, and stained with anti-m138 and anti-ICOSL mAbs followed by an anti-mouse IgG-A555 and anti-rat IgG-A488, respectively. Nuclei were stained with the DAPI reagent and samples were examined under a confocal fluorescence microscope. Shown are representative cells from cultures stained for m138 (panels a, e), ICOSL (b, f), and overlaid images (c, d, g, and h). Enlarged and more exposed individual cells from panels c and g are presented on panels d and h, respectively. Magnification, 63. (C) NIH3T3 HA-ICOSL infected and treated with the lysosomal inhibitors as in B were fixed, permeabilized with the Foxp3 staining buffer set, and analyzed by flow cytometry with anti-m138 and anti-ICOSL mAbs followed by an anti-mouse IgG-A555 and anti-rat IgG-A488, respectively. Conditions were analyzed in triplicates and median fluorescence intensity (MFI) values +/- SD were extracted from pre-gated m138 positive cells. (D) NIH3T3 cells were co-transfected with the m138-GFP and HA-ICOSL constructs. HA-ICOSL was immunoprecipitated from 0.5% Triton-treated lysates using an anti-HA mAb or an anti-hFc as a control. Recovered immunoprecipitates were subjected to SDS-PAGE and western blot with antibodies against GFP and HA, as described in A and Figure 4C. Data are representative of at least two independent experiments. In A and D, the size of the two bands corresponding to ICOSL, and in D, the band corresponding to m138-GFP, are indicated on the right margin in kilodaltons (kDa). In D, the sizes of the molecular weight markers are shown on the left side.

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody anti-mouse ICOSL (clone HK5.3) (Rat monoclonal) Biolegend San Diego, CA, USA Cat#: 107403 RRID:AB_ 345259 (Biotin) Cat#: 107405 RRID:AB_ 2248797 (PE) FACS (1:100) IF (1:50) Antibody anti-human ICOSL (Rabbit polyclonal) Elabscience Wuhan, Hubei, China Cat#: E-AB15519 WB (1:1000) Antibody anti-mouse ICOSL (clone 599841) (Rat monoclonal) R and D Systems, Minneapolis, MN, USA Cat#: MAB158 RRID:AB_ 10719415 IF (1:500) Antibody anti-m123/IE1 (MCMV) (clone IE1.01) (Mouse monoclonal) Center for Proteomics, Faculty of Medicine, University of Rijeka, Croatia Cat#: HRMCMV-12 Stock 1 mg/ mL IF (1:100) FC (1:100) WB (1:1000) Antibody anti-HA Agarose conjugate (Mouse monoclonal) Sigma-Aldrich, St. Louis, MO, USA Cat#: A2095 RRID:AB_ 257974 Cell line (M. musculus) NIH3T3 ATCC, Manassas, VA, USA Cat#: CRL1658 RRID: CVCL_ 0594 Cell line (M. musculus) DC2.4 Merck Millipore, Burlington, MA, USA Cat#: SCC142 RRID: CVCL_ J409 Cell line (M. musculus) MEFs p53 Dr. Jay Nelson, Health Sciences University, Oregon, USA Cell line (M. musculus) SVEC4-10 ATCC, Manassas, VA, USA Cat#: CRL2181 RRID: CVCL_ 4393 Cell line (M. musculus) IC-21 ATCC, Manassas, VA, USA Cat#: TIB186 RRID: CVCL_ 3726 Cell line (M. musculus) NS1 ECACC, Public Health England, Salisbury, UK Cat#: 85011427 RRID: CVCL_ 2155 Cell line (M. musculus) 300.19 Dr. Michel Streuli, Dana Farber Cancer Institute, MA, USA

Techniques: Western Blot, Transfection, Control, Construct, Expressing, Infection, Staining, Fluorescence, Microscopy, Flow Cytometry, Immunoprecipitation, SDS Page, Molecular Weight

Journal: eLife

Article Title: Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion

doi: 10.7554/elife.59350

Figure Lengend Snippet: Figure 6. Identification of the m138 domains involved in the downmodulation of ICOSL and CD80. (A) Schematic representation (not drawn to scale) of m138-GFP protein mutants. (B) NIH3T3 ICOSL-HA cells were untransfected or transfected with m138-GFP, or m138-GFP mutant constructs and analyzed by flow cytometry for ICOSL and CD80 surface levels. Panels on the right represent the MFI of each sample minus the MFI of the control isotypes. Gray histograms represent the expression of untransfected cells and colored histograms represent the expression of cells transfected with m138-GFP (green), m138DIg1-GFP (brown), m138DIg2-GFP (blue), or m138DIg2/3-GFP (red). The isotype for each antibody was used as a negative control (dotted lines). A representative experiment out of two performed is shown.

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody anti-mouse ICOSL (clone HK5.3) (Rat monoclonal) Biolegend San Diego, CA, USA Cat#: 107403 RRID:AB_ 345259 (Biotin) Cat#: 107405 RRID:AB_ 2248797 (PE) FACS (1:100) IF (1:50) Antibody anti-human ICOSL (Rabbit polyclonal) Elabscience Wuhan, Hubei, China Cat#: E-AB15519 WB (1:1000) Antibody anti-mouse ICOSL (clone 599841) (Rat monoclonal) R and D Systems, Minneapolis, MN, USA Cat#: MAB158 RRID:AB_ 10719415 IF (1:500) Antibody anti-m123/IE1 (MCMV) (clone IE1.01) (Mouse monoclonal) Center for Proteomics, Faculty of Medicine, University of Rijeka, Croatia Cat#: HRMCMV-12 Stock 1 mg/ mL IF (1:100) FC (1:100) WB (1:1000) Antibody anti-HA Agarose conjugate (Mouse monoclonal) Sigma-Aldrich, St. Louis, MO, USA Cat#: A2095 RRID:AB_ 257974 Cell line (M. musculus) NIH3T3 ATCC, Manassas, VA, USA Cat#: CRL1658 RRID: CVCL_ 0594 Cell line (M. musculus) DC2.4 Merck Millipore, Burlington, MA, USA Cat#: SCC142 RRID: CVCL_ J409 Cell line (M. musculus) MEFs p53 Dr. Jay Nelson, Health Sciences University, Oregon, USA Cell line (M. musculus) SVEC4-10 ATCC, Manassas, VA, USA Cat#: CRL2181 RRID: CVCL_ 4393 Cell line (M. musculus) IC-21 ATCC, Manassas, VA, USA Cat#: TIB186 RRID: CVCL_ 3726 Cell line (M. musculus) NS1 ECACC, Public Health England, Salisbury, UK Cat#: 85011427 RRID: CVCL_ 2155 Cell line (M. musculus) 300.19 Dr. Michel Streuli, Dana Farber Cancer Institute, MA, USA

Techniques: Transfection, Mutagenesis, Construct, Flow Cytometry, Control, Expressing, Negative Control

Journal: eLife

Article Title: Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion

doi: 10.7554/elife.59350

Figure Lengend Snippet: Figure 7. m138 is capable to restrict CD8+ T-cell activation by downregulating ICOSL on the surface of antigen-presenting cells (APCs). (A) C57BL/6- derived BMDCs were mock-infected or infected for 24 hr with MCMVwt or MCMVm138 mutants at an moi of 3 and analyzed by flow cytometry for surface expression of ICOSL and CD80 using specific mAbs against each of these molecules. Gray histograms represent the expression of mock- infected cells and colored histograms represent the expression of cells infected (positive for the MCMV m04 protein) with MCMVwt (yellow), MCMVDm138 (pink), MCMVm138DIg2 (blue), or MCMVm138DIg2/3 (red). The isotype for each antibody was used as a negative control (black line histograms). Panels on the right represent the MFI of each sample minus the MFI of the control isotypes. Results are representative of two independent experiments (B) On the left, schematic representation of the in vitro antigen-presentation assay. BMDCs infected as indicated in A with MCMVwt (yellow), MCMVDm138 (pink), MCMVm138DIg2 (blue), or MCMVm138DIg2/3 (red), or left uninfected (gray) were further co-cultured with naı¨ve CD8+ T Figure 7 continued on next page

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody anti-mouse ICOSL (clone HK5.3) (Rat monoclonal) Biolegend San Diego, CA, USA Cat#: 107403 RRID:AB_ 345259 (Biotin) Cat#: 107405 RRID:AB_ 2248797 (PE) FACS (1:100) IF (1:50) Antibody anti-human ICOSL (Rabbit polyclonal) Elabscience Wuhan, Hubei, China Cat#: E-AB15519 WB (1:1000) Antibody anti-mouse ICOSL (clone 599841) (Rat monoclonal) R and D Systems, Minneapolis, MN, USA Cat#: MAB158 RRID:AB_ 10719415 IF (1:500) Antibody anti-m123/IE1 (MCMV) (clone IE1.01) (Mouse monoclonal) Center for Proteomics, Faculty of Medicine, University of Rijeka, Croatia Cat#: HRMCMV-12 Stock 1 mg/ mL IF (1:100) FC (1:100) WB (1:1000) Antibody anti-HA Agarose conjugate (Mouse monoclonal) Sigma-Aldrich, St. Louis, MO, USA Cat#: A2095 RRID:AB_ 257974 Cell line (M. musculus) NIH3T3 ATCC, Manassas, VA, USA Cat#: CRL1658 RRID: CVCL_ 0594 Cell line (M. musculus) DC2.4 Merck Millipore, Burlington, MA, USA Cat#: SCC142 RRID: CVCL_ J409 Cell line (M. musculus) MEFs p53 Dr. Jay Nelson, Health Sciences University, Oregon, USA Cell line (M. musculus) SVEC4-10 ATCC, Manassas, VA, USA Cat#: CRL2181 RRID: CVCL_ 4393 Cell line (M. musculus) IC-21 ATCC, Manassas, VA, USA Cat#: TIB186 RRID: CVCL_ 3726 Cell line (M. musculus) NS1 ECACC, Public Health England, Salisbury, UK Cat#: 85011427 RRID: CVCL_ 2155 Cell line (M. musculus) 300.19 Dr. Michel Streuli, Dana Farber Cancer Institute, MA, USA

Techniques: Activation Assay, Derivative Assay, Infection, Flow Cytometry, Expressing, Negative Control, Control, In Vitro, Immunopeptidomics, Cell Culture

Journal: eLife

Article Title: Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion

doi: 10.7554/elife.59350

Figure Lengend Snippet: Figure 8. Via targeting ICOSL, m138 reduces T-cell mediated control of MCMV infection in vivo. (A) BALB/c mice were intraperitoneally (i.p.) inoculated with 2 106 PFU MCMV-GFP. Two days post-infection mice were sacrificed and peritoneal exudate cells were extracted and analyzed by flow cytometry for surface expression of ICOSL and MHCI, using specific mAbs against each of these molecules. Blue histograms represent the expression Figure 8 continued on next page

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody anti-mouse ICOSL (clone HK5.3) (Rat monoclonal) Biolegend San Diego, CA, USA Cat#: 107403 RRID:AB_ 345259 (Biotin) Cat#: 107405 RRID:AB_ 2248797 (PE) FACS (1:100) IF (1:50) Antibody anti-human ICOSL (Rabbit polyclonal) Elabscience Wuhan, Hubei, China Cat#: E-AB15519 WB (1:1000) Antibody anti-mouse ICOSL (clone 599841) (Rat monoclonal) R and D Systems, Minneapolis, MN, USA Cat#: MAB158 RRID:AB_ 10719415 IF (1:500) Antibody anti-m123/IE1 (MCMV) (clone IE1.01) (Mouse monoclonal) Center for Proteomics, Faculty of Medicine, University of Rijeka, Croatia Cat#: HRMCMV-12 Stock 1 mg/ mL IF (1:100) FC (1:100) WB (1:1000) Antibody anti-HA Agarose conjugate (Mouse monoclonal) Sigma-Aldrich, St. Louis, MO, USA Cat#: A2095 RRID:AB_ 257974 Cell line (M. musculus) NIH3T3 ATCC, Manassas, VA, USA Cat#: CRL1658 RRID: CVCL_ 0594 Cell line (M. musculus) DC2.4 Merck Millipore, Burlington, MA, USA Cat#: SCC142 RRID: CVCL_ J409 Cell line (M. musculus) MEFs p53 Dr. Jay Nelson, Health Sciences University, Oregon, USA Cell line (M. musculus) SVEC4-10 ATCC, Manassas, VA, USA Cat#: CRL2181 RRID: CVCL_ 4393 Cell line (M. musculus) IC-21 ATCC, Manassas, VA, USA Cat#: TIB186 RRID: CVCL_ 3726 Cell line (M. musculus) NS1 ECACC, Public Health England, Salisbury, UK Cat#: 85011427 RRID: CVCL_ 2155 Cell line (M. musculus) 300.19 Dr. Michel Streuli, Dana Farber Cancer Institute, MA, USA

Techniques: Control, Infection, In Vivo, Flow Cytometry, Expressing

Journal: eLife

Article Title: Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion

doi: 10.7554/elife.59350

Figure Lengend Snippet: Figure 9. Blockade of ICOSL reduces the frequency of particular lymphocyte subsets in the spleen and lymph nodes of MCMV-infected mice. (A) BALB/ c mice (n = 11–12/group) were i.p. inoculated with 2 106 PFU of MCMVDm138 and treated with (a-ICOSL) or without (CTL) 100 mg of anti-ICOSL mAb. At day 14 post-infection mice were sacrificed, and spleens and lymph nodes were isolated and disaggregated. Cell suspensions were analyzed by Figure 9 continued on next page

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody anti-mouse ICOSL (clone HK5.3) (Rat monoclonal) Biolegend San Diego, CA, USA Cat#: 107403 RRID:AB_ 345259 (Biotin) Cat#: 107405 RRID:AB_ 2248797 (PE) FACS (1:100) IF (1:50) Antibody anti-human ICOSL (Rabbit polyclonal) Elabscience Wuhan, Hubei, China Cat#: E-AB15519 WB (1:1000) Antibody anti-mouse ICOSL (clone 599841) (Rat monoclonal) R and D Systems, Minneapolis, MN, USA Cat#: MAB158 RRID:AB_ 10719415 IF (1:500) Antibody anti-m123/IE1 (MCMV) (clone IE1.01) (Mouse monoclonal) Center for Proteomics, Faculty of Medicine, University of Rijeka, Croatia Cat#: HRMCMV-12 Stock 1 mg/ mL IF (1:100) FC (1:100) WB (1:1000) Antibody anti-HA Agarose conjugate (Mouse monoclonal) Sigma-Aldrich, St. Louis, MO, USA Cat#: A2095 RRID:AB_ 257974 Cell line (M. musculus) NIH3T3 ATCC, Manassas, VA, USA Cat#: CRL1658 RRID: CVCL_ 0594 Cell line (M. musculus) DC2.4 Merck Millipore, Burlington, MA, USA Cat#: SCC142 RRID: CVCL_ J409 Cell line (M. musculus) MEFs p53 Dr. Jay Nelson, Health Sciences University, Oregon, USA Cell line (M. musculus) SVEC4-10 ATCC, Manassas, VA, USA Cat#: CRL2181 RRID: CVCL_ 4393 Cell line (M. musculus) IC-21 ATCC, Manassas, VA, USA Cat#: TIB186 RRID: CVCL_ 3726 Cell line (M. musculus) NS1 ECACC, Public Health England, Salisbury, UK Cat#: 85011427 RRID: CVCL_ 2155 Cell line (M. musculus) 300.19 Dr. Michel Streuli, Dana Farber Cancer Institute, MA, USA

Techniques: Infection, Isolation

Journal: eLife

Article Title: Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion

doi: 10.7554/elife.59350

Figure Lengend Snippet: Figure 10. ICOSL blockade limits the generation of MCMV specific antibodies. (A) Total levels of IgG, IgG1, IgG2a, IgG2b, IgG3 (at dilutions 1:200 and 1:400), and IgM (at dilutions 1:25 and 1:50) were determined by ELISA from sera collected at day 14 of the same infected BALB/c mice indicated in Figure 8A. (B) Sera (at dilutions 1:10 and 1:100) as in A were tested in a viral neutralization assay. Results are presented as the percentage of plaque reduction determined by the ratio of the number of plaques counted in the sample wells relative to the ratio of plaques in wells containing the serum of an uninfected mouse used as a negative control. In A and B, results are expressed as mean +/- SD. Mice treated with anti-ICOSL mAb are represented as red squares and mice that did not receive the mAb are represented as open circles. Two-tailed unpaired t-tests were used to assess statistical differences between experimental groups. *p<0.05, **p<0.01, ***p<0.001. Data are pooled from two independent experiments. The online version of this article includes the following figure supplement(s) for figure 10:

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody anti-mouse ICOSL (clone HK5.3) (Rat monoclonal) Biolegend San Diego, CA, USA Cat#: 107403 RRID:AB_ 345259 (Biotin) Cat#: 107405 RRID:AB_ 2248797 (PE) FACS (1:100) IF (1:50) Antibody anti-human ICOSL (Rabbit polyclonal) Elabscience Wuhan, Hubei, China Cat#: E-AB15519 WB (1:1000) Antibody anti-mouse ICOSL (clone 599841) (Rat monoclonal) R and D Systems, Minneapolis, MN, USA Cat#: MAB158 RRID:AB_ 10719415 IF (1:500) Antibody anti-m123/IE1 (MCMV) (clone IE1.01) (Mouse monoclonal) Center for Proteomics, Faculty of Medicine, University of Rijeka, Croatia Cat#: HRMCMV-12 Stock 1 mg/ mL IF (1:100) FC (1:100) WB (1:1000) Antibody anti-HA Agarose conjugate (Mouse monoclonal) Sigma-Aldrich, St. Louis, MO, USA Cat#: A2095 RRID:AB_ 257974 Cell line (M. musculus) NIH3T3 ATCC, Manassas, VA, USA Cat#: CRL1658 RRID: CVCL_ 0594 Cell line (M. musculus) DC2.4 Merck Millipore, Burlington, MA, USA Cat#: SCC142 RRID: CVCL_ J409 Cell line (M. musculus) MEFs p53 Dr. Jay Nelson, Health Sciences University, Oregon, USA Cell line (M. musculus) SVEC4-10 ATCC, Manassas, VA, USA Cat#: CRL2181 RRID: CVCL_ 4393 Cell line (M. musculus) IC-21 ATCC, Manassas, VA, USA Cat#: TIB186 RRID: CVCL_ 3726 Cell line (M. musculus) NS1 ECACC, Public Health England, Salisbury, UK Cat#: 85011427 RRID: CVCL_ 2155 Cell line (M. musculus) 300.19 Dr. Michel Streuli, Dana Farber Cancer Institute, MA, USA

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Neutralization, Negative Control, Two Tailed Test

Journal: eLife

Article Title: Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion

doi: 10.7554/elife.59350

Figure Lengend Snippet: Figure 11. HCMV, HSV-1, and HSV-2 also limit cell surface expression of ICOSL on antigen-presenting cells (APCs). (A) Primary monocyte-derived macrophages (upper panels) and PMA-treated THP-1 cells (bottom panels) were mock-infected or infected for 72 hr with HCMV-GFP at an moi of 10 and analyzed by flow cytometry for cell-surface expression of human ICOSL or CD70 using specific mAbs against each of these receptors. Gray histograms represent the expression of mock-infected cells, green histograms represent the expression on HCMV-infected (GFP+) cells, and blue histograms represent the expression on uninfected (GFP-) cells from the same culture. (B) PMA-treated THP-1 cells were mock-infected (time 0) or infected with HCMV-GFP as in A and analyzed by flow cytometry for surface expression of ICOSL at the different time points after infection indicated. (C) Same as in A, except that an moi of 20 was used, and THP-1 cells were also exposed for 72 hr to the same amount of HCMV-GFP UV-inactivated (red histogram). (D) Equal amounts of lysates from PMA-treated THP-1 cells mock-infected (lane 1) or infected for 72 hr at an moi of 20 with HCMV-GFP (lanes 2 and 3), and when indicated, treated with 250 mM leupeptin and 20 nM of bafilomycin A1 (lane 3), were lysed and analyzed by western blot with antibodies against ICOSL and actin, followed by anti-rabbit IgG-HRP (ICOSL) or anti-mouse IgG-HRP (actin). (E) PMA-treated THP-1 cells were mock- infected or infected with HSV-1-GFP, HSV-1-GFP UV-inactivated, HSV-2-GFP or HSV-2-GFP UV-inactivated at an moi of 100 (HSV-1) or 200 (HSV-2). Twenty-four hr later, the expression of human ICOSL and CD70 were analyzed as in A. Green histograms represent the expression of ICOSL on infected cells, red histograms represent the expression on UV-inactivated HSV infected cells, and gray histograms represent the expression on mock-infected cells. The isotype for the ICOSL antibody was used as a negative control (dotted lines). A representative experiment out of three performed is shown. (F) PMA-treated THP-1 cells were mock-infected (lane 1) or infected with HSV-1-GFP (lane 2) and HSV-2-GFP (lane 3) as indicated in E. Cell lysates were prepared, and subjected to western blot analysis using anti-human ICOSL or anti-actin mAbs as in D. The molecular weight of the band corresponding to ICOSL is indicated in D and E in kilodaltons on the right margin.

Article Snippet: resource Designation Source or reference Identifiers Additional information Antibody anti-mouse ICOSL (clone HK5.3) (Rat monoclonal) Biolegend San Diego, CA, USA Cat#: 107403 RRID:AB_ 345259 (Biotin) Cat#: 107405 RRID:AB_ 2248797 (PE) FACS (1:100) IF (1:50) Antibody anti-human ICOSL (Rabbit polyclonal) Elabscience Wuhan, Hubei, China Cat#: E-AB15519 WB (1:1000) Antibody anti-mouse ICOSL (clone 599841) (Rat monoclonal) R and D Systems, Minneapolis, MN, USA Cat#: MAB158 RRID:AB_ 10719415 IF (1:500) Antibody anti-m123/IE1 (MCMV) (clone IE1.01) (Mouse monoclonal) Center for Proteomics, Faculty of Medicine, University of Rijeka, Croatia Cat#: HRMCMV-12 Stock 1 mg/ mL IF (1:100) FC (1:100) WB (1:1000) Antibody anti-HA Agarose conjugate (Mouse monoclonal) Sigma-Aldrich, St. Louis, MO, USA Cat#: A2095 RRID:AB_ 257974 Cell line (M. musculus) NIH3T3 ATCC, Manassas, VA, USA Cat#: CRL1658 RRID: CVCL_ 0594 Cell line (M. musculus) DC2.4 Merck Millipore, Burlington, MA, USA Cat#: SCC142 RRID: CVCL_ J409 Cell line (M. musculus) MEFs p53 Dr. Jay Nelson, Health Sciences University, Oregon, USA Cell line (M. musculus) SVEC4-10 ATCC, Manassas, VA, USA Cat#: CRL2181 RRID: CVCL_ 4393 Cell line (M. musculus) IC-21 ATCC, Manassas, VA, USA Cat#: TIB186 RRID: CVCL_ 3726 Cell line (M. musculus) NS1 ECACC, Public Health England, Salisbury, UK Cat#: 85011427 RRID: CVCL_ 2155 Cell line (M. musculus) 300.19 Dr. Michel Streuli, Dana Farber Cancer Institute, MA, USA

Techniques: Expressing, Derivative Assay, Infection, Flow Cytometry, Western Blot, Negative Control, Molecular Weight

Journal: Cancer cell

Article Title: Lymphoma accelerates T cell and tissue aging

doi: 10.1016/j.ccell.2025.07.023

Figure Lengend Snippet: (A) Gating strategy and quantification of naive (N), effector (E), central memory (CM), and CD62L − CD44 lo CD4 + T cell populations ( n = 5). (B–E) Cell surface expression and representative histograms on CD4 + T cells ( n = 5): (B) CD39, (C) CD69, (D) KLRG1, and (E) PD-1. Line in flow plots represents where positivity was determined. (F–H) Intracellular expression and representative histograms of transcription factors in the indicated CD4 + T cells ( n = 3–4) (F) Tbet, (G) Tox, and (H) Foxp3. (I) Gating strategy for CD11b/CD27 NK cell maturation subtypes ( n = 3). (J) Cell surface expression and representative histogram of CXCR3 in NK cells ( n = 3/group). YC, YL, AC, AL are defined in . Data are mean ± SEM and are analyzed by a two-way ANOVA and Sidak’s test (A and I) or Student’s t test (B–H and J). Data are compiled from at least two independent experiments, and each dot represents an individual mouse. * p < 0.05 ** p < 0.01, and *** p < 0.001, **** p < 0.0001, n.s. = not significant. See also and .

Article Snippet: KLRG1 – APC , Tonbo , Cat # 20-5893; RRID:AB_2621607.

Techniques: Expressing