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Quantitative temporal viromics: an approach to investigate host-pathogen interaction.

Weekes MP, Tomasec P, Huttlin EL, Fielding CA, Nusinow D, Stanton RJ, Wang EC, Aicheler R, Murrell I, Wilkinson GW, Lehner PJ, Gygi SP - Cell (2014)

Bottom Line: QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets.Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined.QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK. Electronic address: mpw1001@cam.ac.uk.

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Validation of Temporal Profiles by Flow Cytometry and Immunoblot and Investigation of Novel Immune Ligands, Related to Figure 4(A) PM and WCL profiles of MHC-I (HLA-A11) and CD99, validated by flow cytometry and immunoblot. ≥ 95% of HFFF were routinely infected by HCMV, assessed by flow cytometry for cell surface MHC-I.(B) Representative intracellular flow cytometry of 24h-infected HFFF with anti-IE1 confirms > 95% infection efficiency.(C) Flow cytometry of HFFF infected with HCMV confirms proteomic profiles for five additional cell surface proteins.(D and E) NK degranulation assays suggest that CLEC1A and FAT1 are novel activating NK ligands. Top panels – validation of temporal PM and WCL profiles by flow cytometry and immunoblot. CLEC1A was not quantified in any WCL QTV experiments but accumulated by immunoblot of whole-cell lysates, while depleting from the PM. Bottom panels – target cells underwent siRNA knockdown of CLEC1A (D) or FAT1 (E) and were then incubated with stimulated polyclonal NK cells from each of three donors. Degranulation of NK cells in response to both CLEC1A and FAT1 knockdown targets was significantly reduced compared to control. Error bars: +/− SEM (donors A, B), +/− range (donor C). ∗ two-tailed p-value < 0.05, ∗∗ two-tailed p-value < 0.005. Cell surface MHC-I was unaffected by siRNA (right bottom panels, staining with W6-32 antibody or control Ig).(F) CD8+ T cell degranulation assay suggests that CEACAM1 is a novel inhibitory ligand for CMV-specific cytotoxic T cells. Top panel – validation of temporal PM profile by flow cytometry. Bottom panels (left) – flow cytometry of a CD8+ T cell line specific to the HCMV HLA-A2 restricted IE1 peptide VLEETSVML confirmed CEACAM1 surface expression. (right) HCMV peptide-specific CD8+ effector cells were incubated with autologous fibroblasts that had been infected with HCMV for 72h then pulsed with peptide or left unpulsed. Effectors and targets were treated with control Ig or anti-CEACAM1. HCMV-specific T cell degranulation was significantly increased with CEACAM1 block. Error bars ± SEM. ∗ two-tailed p-value < 0.0001.y-axes of QTV plots represent relative abundance, and y-axes of flow cytometry plots represent % of max.
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figs4: Validation of Temporal Profiles by Flow Cytometry and Immunoblot and Investigation of Novel Immune Ligands, Related to Figure 4(A) PM and WCL profiles of MHC-I (HLA-A11) and CD99, validated by flow cytometry and immunoblot. ≥ 95% of HFFF were routinely infected by HCMV, assessed by flow cytometry for cell surface MHC-I.(B) Representative intracellular flow cytometry of 24h-infected HFFF with anti-IE1 confirms > 95% infection efficiency.(C) Flow cytometry of HFFF infected with HCMV confirms proteomic profiles for five additional cell surface proteins.(D and E) NK degranulation assays suggest that CLEC1A and FAT1 are novel activating NK ligands. Top panels – validation of temporal PM and WCL profiles by flow cytometry and immunoblot. CLEC1A was not quantified in any WCL QTV experiments but accumulated by immunoblot of whole-cell lysates, while depleting from the PM. Bottom panels – target cells underwent siRNA knockdown of CLEC1A (D) or FAT1 (E) and were then incubated with stimulated polyclonal NK cells from each of three donors. Degranulation of NK cells in response to both CLEC1A and FAT1 knockdown targets was significantly reduced compared to control. Error bars: +/− SEM (donors A, B), +/− range (donor C). ∗ two-tailed p-value < 0.05, ∗∗ two-tailed p-value < 0.005. Cell surface MHC-I was unaffected by siRNA (right bottom panels, staining with W6-32 antibody or control Ig).(F) CD8+ T cell degranulation assay suggests that CEACAM1 is a novel inhibitory ligand for CMV-specific cytotoxic T cells. Top panel – validation of temporal PM profile by flow cytometry. Bottom panels (left) – flow cytometry of a CD8+ T cell line specific to the HCMV HLA-A2 restricted IE1 peptide VLEETSVML confirmed CEACAM1 surface expression. (right) HCMV peptide-specific CD8+ effector cells were incubated with autologous fibroblasts that had been infected with HCMV for 72h then pulsed with peptide or left unpulsed. Effectors and targets were treated with control Ig or anti-CEACAM1. HCMV-specific T cell degranulation was significantly increased with CEACAM1 block. Error bars ± SEM. ∗ two-tailed p-value < 0.0001.y-axes of QTV plots represent relative abundance, and y-axes of flow cytometry plots represent % of max.

Mentions: We confirmed temporal profiles of protocadherins PCDHγC3 and FAT1 in addition to eight other cell-surface proteins by flow cytometry and immunoblot (Figures 4D and S4A, S4C, and S4E). To provide proof-of-principle validation of our functional predictions, we performed siRNA-mediated knockdown of the C-type lectin CLEC1A (Table S5A), which is downregulated 7-fold by HCMV and is a potential NK ligand by homology to CLEC2D. Polyclonal NK-cells from three of three independent donors demonstrated reduced degranulation to autologous targets upon knockdown of CLEC1A suggesting that this molecule may be a novel activating NK ligand (Figure S4D). We observed similar results for the protocadherin FAT1, providing initial confirmatory evidence that members of this family may indeed be involved in immunoregulation (Figure S4E). CEACAM-1 (immunoglobulin family) was upregulated 20-fold by HCMV infection and may have roles in T cell regulation. Using a blocking antibody, we demonstrated increased degranulation of CD8+ T cells specific for an HCMV HLA-A2 restricted peptide epitope suggesting that upregulation of this molecule in infected cells may inhibit cytotoxic T cell-mediated lysis (Figure S4F).


Quantitative temporal viromics: an approach to investigate host-pathogen interaction.

Weekes MP, Tomasec P, Huttlin EL, Fielding CA, Nusinow D, Stanton RJ, Wang EC, Aicheler R, Murrell I, Wilkinson GW, Lehner PJ, Gygi SP - Cell (2014)

Validation of Temporal Profiles by Flow Cytometry and Immunoblot and Investigation of Novel Immune Ligands, Related to Figure 4(A) PM and WCL profiles of MHC-I (HLA-A11) and CD99, validated by flow cytometry and immunoblot. ≥ 95% of HFFF were routinely infected by HCMV, assessed by flow cytometry for cell surface MHC-I.(B) Representative intracellular flow cytometry of 24h-infected HFFF with anti-IE1 confirms > 95% infection efficiency.(C) Flow cytometry of HFFF infected with HCMV confirms proteomic profiles for five additional cell surface proteins.(D and E) NK degranulation assays suggest that CLEC1A and FAT1 are novel activating NK ligands. Top panels – validation of temporal PM and WCL profiles by flow cytometry and immunoblot. CLEC1A was not quantified in any WCL QTV experiments but accumulated by immunoblot of whole-cell lysates, while depleting from the PM. Bottom panels – target cells underwent siRNA knockdown of CLEC1A (D) or FAT1 (E) and were then incubated with stimulated polyclonal NK cells from each of three donors. Degranulation of NK cells in response to both CLEC1A and FAT1 knockdown targets was significantly reduced compared to control. Error bars: +/− SEM (donors A, B), +/− range (donor C). ∗ two-tailed p-value < 0.05, ∗∗ two-tailed p-value < 0.005. Cell surface MHC-I was unaffected by siRNA (right bottom panels, staining with W6-32 antibody or control Ig).(F) CD8+ T cell degranulation assay suggests that CEACAM1 is a novel inhibitory ligand for CMV-specific cytotoxic T cells. Top panel – validation of temporal PM profile by flow cytometry. Bottom panels (left) – flow cytometry of a CD8+ T cell line specific to the HCMV HLA-A2 restricted IE1 peptide VLEETSVML confirmed CEACAM1 surface expression. (right) HCMV peptide-specific CD8+ effector cells were incubated with autologous fibroblasts that had been infected with HCMV for 72h then pulsed with peptide or left unpulsed. Effectors and targets were treated with control Ig or anti-CEACAM1. HCMV-specific T cell degranulation was significantly increased with CEACAM1 block. Error bars ± SEM. ∗ two-tailed p-value < 0.0001.y-axes of QTV plots represent relative abundance, and y-axes of flow cytometry plots represent % of max.
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Related In: Results  -  Collection

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figs4: Validation of Temporal Profiles by Flow Cytometry and Immunoblot and Investigation of Novel Immune Ligands, Related to Figure 4(A) PM and WCL profiles of MHC-I (HLA-A11) and CD99, validated by flow cytometry and immunoblot. ≥ 95% of HFFF were routinely infected by HCMV, assessed by flow cytometry for cell surface MHC-I.(B) Representative intracellular flow cytometry of 24h-infected HFFF with anti-IE1 confirms > 95% infection efficiency.(C) Flow cytometry of HFFF infected with HCMV confirms proteomic profiles for five additional cell surface proteins.(D and E) NK degranulation assays suggest that CLEC1A and FAT1 are novel activating NK ligands. Top panels – validation of temporal PM and WCL profiles by flow cytometry and immunoblot. CLEC1A was not quantified in any WCL QTV experiments but accumulated by immunoblot of whole-cell lysates, while depleting from the PM. Bottom panels – target cells underwent siRNA knockdown of CLEC1A (D) or FAT1 (E) and were then incubated with stimulated polyclonal NK cells from each of three donors. Degranulation of NK cells in response to both CLEC1A and FAT1 knockdown targets was significantly reduced compared to control. Error bars: +/− SEM (donors A, B), +/− range (donor C). ∗ two-tailed p-value < 0.05, ∗∗ two-tailed p-value < 0.005. Cell surface MHC-I was unaffected by siRNA (right bottom panels, staining with W6-32 antibody or control Ig).(F) CD8+ T cell degranulation assay suggests that CEACAM1 is a novel inhibitory ligand for CMV-specific cytotoxic T cells. Top panel – validation of temporal PM profile by flow cytometry. Bottom panels (left) – flow cytometry of a CD8+ T cell line specific to the HCMV HLA-A2 restricted IE1 peptide VLEETSVML confirmed CEACAM1 surface expression. (right) HCMV peptide-specific CD8+ effector cells were incubated with autologous fibroblasts that had been infected with HCMV for 72h then pulsed with peptide or left unpulsed. Effectors and targets were treated with control Ig or anti-CEACAM1. HCMV-specific T cell degranulation was significantly increased with CEACAM1 block. Error bars ± SEM. ∗ two-tailed p-value < 0.0001.y-axes of QTV plots represent relative abundance, and y-axes of flow cytometry plots represent % of max.
Mentions: We confirmed temporal profiles of protocadherins PCDHγC3 and FAT1 in addition to eight other cell-surface proteins by flow cytometry and immunoblot (Figures 4D and S4A, S4C, and S4E). To provide proof-of-principle validation of our functional predictions, we performed siRNA-mediated knockdown of the C-type lectin CLEC1A (Table S5A), which is downregulated 7-fold by HCMV and is a potential NK ligand by homology to CLEC2D. Polyclonal NK-cells from three of three independent donors demonstrated reduced degranulation to autologous targets upon knockdown of CLEC1A suggesting that this molecule may be a novel activating NK ligand (Figure S4D). We observed similar results for the protocadherin FAT1, providing initial confirmatory evidence that members of this family may indeed be involved in immunoregulation (Figure S4E). CEACAM-1 (immunoglobulin family) was upregulated 20-fold by HCMV infection and may have roles in T cell regulation. Using a blocking antibody, we demonstrated increased degranulation of CD8+ T cells specific for an HCMV HLA-A2 restricted peptide epitope suggesting that upregulation of this molecule in infected cells may inhibit cytotoxic T cell-mediated lysis (Figure S4F).

Bottom Line: QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets.Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined.QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK. Electronic address: mpw1001@cam.ac.uk.

Show MeSH
Related in: MedlinePlus