1. Immunology and Inflammation
Download icon

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

  1. Guillem Angulo
  2. Jelena Zeleznjak
  3. Pablo Martínez-Vicente
  4. Joan Puñet-Ortiz
  5. Hartmut Hengel
  6. Martin Messerle
  7. Annette Oxenius
  8. Stipan Jonjic
  9. Astrid Krmpotić
  10. Pablo Engel
  11. Ana Angulo  Is a corresponding author
  1. Immunology Unit, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Spain
  2. Center for Proteomics, Faculty of Medicine, University of Rijeka, Croatia
  3. Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Croatia
  4. Institut d'Investigacions Biomèdiques August Pi i Sunyer, Spain
  5. Institute of Virology, University Medical Center, Albert-Ludwigs-University Freiburg, Germany
  6. Faculty of Medicine, Albert-Ludwigs-University Freiburg, Germany
  7. Institute of Virology, Hannover Medical School, Germany
  8. Institute of Microbiology, Department of Biology, ETH Zürich, Switzerland
Research Article
Cite this article as: eLife 2021;10:e59350 doi: 10.7554/eLife.59350
12 figures, 1 table and 1 additional file

Figures

Downregulation of ICOSL upon MCMV infection of antigen-presenting cells (APCs).

(A) Peritoneal macrophages (MΦ) 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.

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-GFPΔm128-m138, or MCMV-GFPΔm1-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 MCMVΔm138 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 MCMV∆m138 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.

Figure 3 with 1 supplement
Localization of m138 in MCMV-infected cells.

(A) NIH3T3 cells were infected with MCMV-GFP at an moi of 5 for 24 hr, fixed with 4% formaldehyde (panels d-f) or fixed and permeabilized with 0.05% Triton (panels a-c and g), and stained with the anti-m138 mAb followed by an anti-mouse IgG-A555. Nuclei were stained with the DAPI reagent. The cells were examined under a fluorescence microscope. Magnification, ×20 (a-f); an enlarged individual cell from panel c is shown in panel g. (B) MCMVwt-infected NIH3T3 cells were examined by flow cytometry for surface expression of m138 with the anti-m138 mAb (red histogram). Dotted line represents the isotype control. (C) 300.19 cells untransfected or stably transfected with the HA-m138 construct were fixed with 4% formaldehyde and stained with the anti-m138 mAb and DAPI and examined under a fluorescence microscope as indicated in A. Magnification, ×20. Overlaid images are shown. Transfected cells from the same cultures were analyzed by flow cytometry (right panels) with the anti-m138 mAb (red histogram), an anti-HA mAb (gray histogram) or isotype controls (dotted lines). (D) NIH3T3 cells were infected with MCMV at an moi of 5 for 24 hr, treated with LysoTracker DND99, fixed with 4% formaldehyde and permeabilized with 0.02% Saponin, and stained with the anti-m138 mAb followed by an anti-mouse IgG-A488. Nuclei were stained with the DAPI reagent. The cells were examined under a confocal microscope. Representative fields are shown in panels a, b, and c, with an enlargement of an individual and more exposed cell from panel c, in panel d. Magnification, ×63.

Figure 3—figure supplement 1
Localization of m138 in MCMV-infected DC2.4 cells.

D2.4 cells were infected with MCMV-GFP at an moi of 5 for 24 hr, fixed with 4% formaldehyde, permeabilized with 0.05% Triton, and stained with the anti-m138 mAb followed by an anti-mouse IgG-A555. Nuclei were stained with the DAPI reagent. The cells were examined under a fluorescence microscope. Magnification, ×20 (a-c); an enlarged individual cell from panel c is shown in panel d.

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 MCMV∆m138 (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 MCMV∆m138 (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.

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 μM 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.

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), m138∆Ig1-GFP (brown), m138∆Ig2-GFP (blue), or m138∆Ig2/3-GFP (red). The isotype for each antibody was used as a negative control (dotted lines). A representative experiment out of two performed is shown.

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), MCMVΔm138 (pink), MCMVm138ΔIg2 (blue), or MCMVm138ΔIg2/3 (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), MCMVΔm138 (pink), MCMVm138ΔIg2 (blue), or MCMVm138ΔIg2/3 (red), or left uninfected (gray) were further co-cultured with naïve CD8+ T cells from Maxi mice. After 6 hr, IFN-γ production by CD8+ T cells was determined by flow cytometry. Results are representative of three independent experiments. Results are expressed as the mean +/-SEM of the percentage values obtained for samples in each group.

Figure 8 with 1 supplement
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 of uninfected (GFP-) cells and green histograms represent the expression of MCMV-infected (GFP+) cells from the same mouse. The isotype for each antibody was used as a negative control (dotted lines). The results obtained from a representative infected mouse out of two are shown. (B) BALB/c mice (n = 4/group) were mock-infected or i.p. infected with 1 × 106 PFU of MCMVwt or MCMVΔm138. At 6 hpi, peritoneal exudate cells were extracted and surface expression of ICOSL and CD80 assessed by flow cytometry on the surface of DCs (CD11+ MHC II+ CD3-CD19- NKp46- cells), gating on m04-positive cells when derived from MCMVwt- or MCMVΔm138-infected mice. Results are expressed as the mean +/-SD of the MFI values obtained for samples from two independent experiments. (C) Table displaying the immunomodulatory effects of the different MCMVs on the cellular targets of m138. (D) On the top, a schematic representation of the MCMVΔm138 and MCMVm138ΔIg2 in vivo infection assay is shown. C57BL/6 mice (n = 5–7/group) with or without NK, or NK, CD4+ and CD8+ T-cell depletion, as indicated, were intravenously (i.v.) inoculated with 2 × 105 PFU/mouse of MCMVwt (yellow circles), MCMVΔm138 (pink triangles) or MCMVm138ΔIg2 (blue triangles). At day 14 post-infection mice were sacrificed and viral titers in salivary glands and lungs of individual mice were determined by standard plaque assays. Horizontal bars indicate the median values. The Kruskal-Wallis test was used to assess statistical differences between experimental groups. *p<0.05, **p<0.01. A representative experiment out of two performed is shown.

Figure 8—figure supplement 1
m138 contributes to immune evasion of CD4+ T cells in spleens and lungs of MCMV-infected mice.

On the top, a schematic representation of the adoptive transfer in vivo assay is shown. 1 × 105 M25-specific transgenic CD4+ T cells were transferred (i.v.) into C57BL/6 SCID mice (n = 4–5/group), which were depleted of NK cells as indicated, and either mock-infected or i.p. infected with 2 × 105 PFU/mouse of MCMVwt or MCMVΔm138. Seven dpi, spleens, lungs, and salivary glands were harvested, and analyzed by flow cytometry using anti-CD45.1 and anti-CD4-specific mAbs. The frequency of CD45.1+ CD4+ cells for each organ is indicated. Results are expressed as the mean +/-SEM of the percentage values obtained for samples in each group.

Figure 9 with 1 supplement
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 MCMVΔm138 and treated with (α-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 flow cytometry using anti-mouse CD3, CD4, CD8, CD44, CD62L, CXCR5, and PD1 for T-cell phenotype. For B cell phenotype B220, CD21, CD23, CD95, and GL7 mAbs were used. Percentages of Tfh cells (CD3+CD4+CXCR5hiPD1hi), germinal center (GC) B cells (CD95hiGL7hiB220+), follicular (FO) B cells (CD21+CD23+B220+), and CD3+CD4+ or CD3+CD8+ naive (CD44lowCD62Lhi) and memory (CD44hiCD62Llow) T cells from these two organs are shown. (B) Representative immunofluorescence of spleens from mice infected and treated with (panels b and d) or without (panels a and c) the anti-ICOSL mAb, as indicated in A. Two color colocalization with B220 in green and peanut agglutinin (PNA) in red are shown at ×4 and ×10 magnifications. Panels on the right show a graphic representation of follicle and GC relative areas from tissue sections of a representative mouse of each experimental group. In graphs shown in A and B, results are expressed as mean +/-SD. Mice treated with anti-ICOSL mAb are represented as red squares and mice not receiving 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, ****p<0.0001. Data are pooled from two independent experiments.

Figure 9—figure supplement 1
Effect of ICOSL blockade on the frequency of several lymphocyte subsets in the spleen and lymph nodes of MCMV-infected mice.

Cell suspensions obtained from spleens and lymph nodes of BALB/c mice infected and treated as indicated in Figure 8A were analyzed by flow cytometry using anti-mouse CD3, CD4, CD8, CD5, B220, CD23, CD21, CD138, and CD19 mAbs, and anti-mouse IgM polyclonal Ab. Percentages of total B cells (B220+), marginal zone (MZ) B cells (CD21+CD23-B220+), B1 B cells (CD19hiB220int), B1a (CD19hiB220intCD5+), and B1b (CD19hiB220intCD5-) subsets, CD3+CD4+ or CD3+CD8+ T cells, and plasma cells (CD138hiB220lowCD19-IgM-) are shown. Results are expressed as mean +/- SD. Mice treated with anti-ICOSL mAb are represented as red squares and mice untreated are represented as open circles. Two-tailed unpaired t-test was used to assess statistical differences between experimental groups.

Figure 10 with 1 supplement
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.

Figure 10—figure supplement 1
Effect of ICOSL blockade on MCMV neutralization in the presence of complement.

Same as in Figure 9B, except that the MCMV neutralization assay was performed in the presence of rabbit complement. 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. Results are expressed as mean +/-SD. Mice treated with anti-ICOSL mAb are represented as red squares and mice untreated are represented as empty circles. Two-tailed unpaired t-test was used to assess statistical differences between experimental groups. *p<0.05, **p<0.01.

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 μM 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.

Author response image 1
(A) Percentage (%) and (B) number (#) of total peritoneal DCs (live Lin- CD11c+ MHC II+) and MCMV-infected DCs (m04+ cells from total DCs).

BALB/c mice were i.p. infected with 106 PFU/mice of MCMVwt and MCMVΔm138. DCs were isolated from the peritoneal cavity 6 hr post-infection and analyzed by flow cytometry. Results represent two merged individual experiments (mock n=8, MCMVwt n=6, MCMVΔ138 n=7). One-way ANOVA test was used for statistical analysis. Graphs show mean with SEM as error bars. ****, p ≤0.0001; ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Antibodyanti-mouse
CD8 (clone
YTS 169.4)
(Rat monoclonal)
Bio X cell
West Lebanon, NH, USA
Cat#: BE0117
RRID:AB_10950145
in vivo depletion
(150 µg/injection)
Antibodyanti-mouse
CD4 (clone
GK1.5)
(Rat monoclonal)
Bio X cell
West Lebanon, NH, USA
Cat#: BE0003-3
RRID:AB_1107642
in vivo depletion
(150 µg/injection)
Antibodyanti-mouse
NK.1.1 (clone
PK136)
(Rat monoclonal)
Bio X cell
West Lebanon, NH, USA
Cat#: BE0036
RRID:AB_1107737
in vivo depletion
(250 µg/injection)
Antibodyanti-mouse ICOSL
(clone HK5.3)
(Rat monoclonal)
Bio X cell
West Lebanon, NH, USA
Cat#: BE0028
RRID:AB_1107566
in vivo
(100 µg/injection)
Antibodyanti-m04 (clone m04.17)
(Mouse monoclonal)
Center for Proteomics, Faculty of
Medicine, University of Rijeka, Croatia
PMID:31142589
Stock 1 mg/mL
FACS (1:100)
Antibodyanti-m04 (clone m04.16)
(Mouse monoclonal)
Center for Proteomics, Faculty of
Medicine, University of Rijeka, Croatia
PMID:31142589
Stock 1 mg/mL
FACS (1:100)
Antibodyanti-HA
(clone C2974)
(Mouse monoclonal)
Sigma-Aldrich, St. Louis, MO, USACat#: H9658
RRID:AB_260092
WB (1:2000)
Antibodyanti-human IE1
(clone 8B1.2)
(Mouse monoclonal)
Merck Millipore, Burlington, MA, USACat#: MAB810R
RRID:AB_11212266
WB (1:2000)
Antibodyanti-human Fc IgG (clone 29.5)
(Mouse monoclonal)
In house
Department of Biomedical Sciences, University of Barcelona, Spain
Stock 1 mg/mL
FACS (1:400)
Antibodyanti-m138
(clone m138.1.120)
(Mouse monoclonal)
In house
Department of Biomedical Sciences, University of Barcelona, Spain
This manuscriptStock 1 mg/mL
FACS (1:100)
IF (1:50)
Antibodyanti-mouse CD80 (clone 16-10A1) (Armenian hamster monoclonal)Biolegend
San Diego,
CA, USA
Cat#: 104714
RRID:AB_313134
FACS (1:200)
Antibodyanti-human ICOSL (clone MIH12)
(Mouse monoclonal)
Thermo Fischer Scientific, Waltham, MA, USACat#:
16-5889-82
RRID:AB_469129
FACS (1:50)
Antibodyanti-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)
Antibodyanti-human ICOSL
(Rabbit polyclonal)
Elabscience Wuhan, Hubei, ChinaCat#:
E-AB-15519
WB (1:1000)
Antibodyanti-mouse ICOSL (clone 599841)
(Rat monoclonal)
R and D Systems, Minneapolis, MN, USACat#: MAB158
RRID:AB_10719415
IF (1:500)
Antibodyanti-m123/IE1 (MCMV) (clone IE1.01)
(Mouse monoclonal)
Center for Proteomics, Faculty of
Medicine, University of Rijeka, Croatia
Cat#: HR-MCMV-12Stock 1 mg/mL
IF (1:100)
FC (1:100)
WB (1:1000)
Antibodyanti-HA−Agarose conjugate
(Mouse monoclonal)
Sigma-Aldrich, St. Louis, MO, USACat#: A2095
RRID:AB_257974
Cell line (M. musculus)NIH3T3ATCC, Manassas, VA, USACat#: CRL-1658
RRID:CVCL_0594
Cell line (M. musculus)DC2.4Merck Millipore, Burlington, MA, USACat#: SCC142
RRID:CVCL_J409
Cell line (M. musculus)MEFs p53Dr. Jay Nelson, Health Sciences University, Oregon, USA
Cell line (M. musculus)SVEC4-10ATCC, Manassas, VA, USACat#: CRL-2181
RRID:CVCL_4393
Cell line (M. musculus)IC-21ATCC, Manassas, VA, USACat#: TIB-186
RRID:CVCL_3726
Cell line (M. musculus)NS1ECACC, Public Health England, Salisbury, UKCat#: 85011427
RRID:CVCL_2155
Cell line (M. musculus)300.19Dr. Michel Streuli, Dana Farber Cancer Institute, MA, USA
Cell line (Homo-sapiens)THP-1ATCC, Manassas, VA, USACat#: TIB-202
RRID:CVCL_0006
Cell line (Homo-sapiens)HFFATCC, Manassas, VA, USACat#: SCRC-1041
RRID:CVCL_3285
Cell line (Homo-sapiens)HEL299ATCC, Manassas, VA, USACat#: CCL-137
RRID:CVCL_2480
Cell line (Cercopithecus aethiops)VeroECACC, Public Health England, Salisbury, UKCat#: 84113001
RRID:CVCL_0059
Cell line (Cercopithecus aethiops)COS-7ATCC, Manassas, VA, USACat#: CRL-1651
RRID:CVCL_0224
Strain, strain background-
(Mouse cytomegalovirus)
wild type (BAC-derived strain pSM3fr)PMID:10400809
Strain, strain background-
(Mouse cytomegalovirus)
BAC-derived strain pSM3fr-GFPPMID:12660946
Strain, strain background-
(Mouse cytomegalovirus)
Mutant MCMV-GFPΔ15PMID:16831899Lacks ORFs m128-m138
Strain, strain background-
(Mouse cytomegalovirus)
Mutant MCMV-GFPΔm138PMID:16831899Lacks ORF m138
Strain, strain background-
(Mouse cytomegalovirus)
Mutant MCMV-GFPΔ1Brune et al., 2006Lacks ORFs m1-m17
Strain, strain background-
(Mouse cytomegalovirus)
Mutant MCMV-m138ΔIg2PMID:16831899Lacks Ig2 domain of m138
Strain, strain background-
(Mouse cytomegalovirus)
Mutant MCMV-m138ΔIg2/Ig3PMID:16831899Lacks Ig2 and Ig3 domains of m138
Strain, strain background-
(Human cytomegalovirus)
HCMV-GFP (strain TB40/E)PMID:18198366
Strain, strain background
(M. musculus)
MAXI miceCentral Animal Facility, Faculty of Medicine, University of Rijeka, Croatia
PMID:22046127
Strain, strain background
(M. musculus)
BALB/c miceCentral Animal Facility, Faculty of Medicine, University of Barcelona, Spain and
Central Animal Facility, Faculty of Medicine, University of Rijeka, Croatia
Strain, strain background
(M. musculus)
C57BL/6 miceCentral Animal Facility, Faculty of Medicine, University of Rijeka, Croatia
Strain, strain background
(M. musculus)
M25-II miceCentral Animal Facility, Faculty of Medicine, University of Rijeka, Croatia
PMID:22876184
Strain, strain background
(M. musculus)
C57BL/6 SCIDCentral Animal Facility, Faculty of Medicine, University of Rijeka, Croatia
Chemical compound, drugAmaxa Cell Line Nucleofactor Kits R and VLonza, Basel,
Switzerland
Cat#: VCA-1001 and VCA-1003
Chemical compound, drugG418Invivogen, Toulouse, FranceCat#: ant-gn-11.2 mg/mL
Chemical compound, drugleupeptinSigma-Aldrich, St. Louis, MO, USACat#: L2884250 μM
Chemical compound, drugbafilomycin A1Sigma-Aldrich, St. Louis, MO, USACat#: B179320 nM
Chemical compound, drugBiotinSigma-Aldrich, St. Louis, MO, USACat#: B51612 mg/million of cells
Chemical compound, drugProtein G Sepharose 4 Fast FlowGE Healthcare, Chicago, IL, USACat#: 17-0618-01
Chemical compound, drugTissue-Tek O.C.T. CompoundSakura, Torrance, CA, USACat#: 4583
Chemical compound, drugPNA biotinSigma-Aldrich, St. Louis, MO, USACat#: L6135
Chemical compound, drugBrefeldin AeBioscience, Thermo Fischer Scientific, Waltham, MA, USACat#: 00-4506-511 µg/ml
Chemical compound, drugLysoTracker Red DND-99Invitrogen, Thermo Fischer Scientific, Waltham, MA, USACat#: L7528
Chemical compound, drugIntracellular Fixation and Permeabilization Buffer SeteBioscience, Thermo Fischer Scientific, Waltham, MA, USACat#: 88-8824-00
Chemical compound, drugFoxp3/
Transcription Factor Fixation/Permeabilization
eBioscience, Thermo Fischer Scientific, Waltham, MA, USACat#: 00-5521-00
Cell Isolation kitCD4+ T Cell Isolation Kit, mouseMiltenyi Biotec, Bergisch Gladbach, GermanyCat#: 130-104-454
Recombinant DNA reagentpGEM-T (plasmid)Promega, Madison, WI, USACat#: A3600
Recombinant DNA reagentpDisplay (plasmid)Invitrogen, Thermo Fischer Scientific, Waltham, MA, USACat#: V660-20
Recombinant DNA reagentpEGFP-N3 (plasmid)Clontech, Takara, Tokio, JapanCat#: 6080–1
Recombinant DNA reagentpCMV6-mouse ICOSL (plasmid)Origene, Rockville, MD, USACat#: MR204667
Sequence-based reagentm138PstIFor: 5’-CTGCAGGCATCAATTACCTGCGTGCCAG-3’Sigma-Aldrich, St. Louis, MO, USA
Sequence-based reagentm138PstIRev: 5’-CTGCAGTTACGTGTGACGTACGCAACC-3’Sigma-Aldrich, St. Louis, MO, USA
Sequence-based reagentm138BamFor: 5’-GGATCCATGGCGCCTTCGACGCTGATC-3’Sigma-Aldrich, St. Louis, MO, USA
Sequence-based reagentm138BamRev: 5’-GGATCCCGTGTGACGTACGCAACCCGG-3’Sigma-Aldrich, St. Louis, MO, USA
Sequence-based reagentSOEm138∆Ig1Rev: 5’-AGTCCCGGTGGAGTCGGTGATCAGTTGCGT-3’Sigma-Aldrich, St. Louis, MO, USA
Sequence-based reagentSOEm138∆Ig1For: 5’-ACGCAACTG ATCACCGACTCCACCGGGACT-3’Sigma-Aldrich, St. Louis, MO, USA
Sequence-based reagentSOEm138∆Ig2Rev: 5’-CATCAGCG AC CGGCCAGT CCCGGTGGAGTC-3’Sigma-Aldrich, St. Louis, MO, USA
Sequence-based reagentSOEm138∆Ig2For: 5’-GACTCCACC GGGACTGGCCGGTCGCTGATG-3’Sigma-Aldrich, St. Louis, MO, USA
Sequence-based reagentSOEm138∆Ig2/3Rev: 5’-CTGAG GGGACGTGACAGTCCCGGTGGAGTC-3’Sigma-Aldrich, St. Louis, MO, USA
Sequence-based reagentSOEm138∆Ig2/3For: 5’-GACTC CACCGGGACTGTCACGTCCCCTCAG-3’Sigma-Aldrich, St. Louis, MO, USA
Sequence-based reagentICOSLmurineSalIFor: 5’-GTCGACAGAGACTGAAGTCGGTGCAATG-3’Sigma-Aldrich, St. Louis, MO, USA
Sequence-based reagentICOSLmurineNotIRev: 5’-GCGGCC GCTTAGGCGTGGTCTGTAAGTTCA-3’Sigma-Aldrich, St. Louis, MO, USA

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)