rstrem 1  (R&D Systems)

Journal: Science advances

Article Title: TREM-2 is a sensor and activator of T cell response in SARS-CoV-2 infection.

doi: 10.1126/sciadv.abi6802

Figure Lengend Snippet: Fig. 1. TREM-2 expression on CD4+ T cells was increased in patients with COVID-19. (A) The expression levels of TREM-1, TREM-2, TLT1, TLT2, and TLT4 in PBMCs from healthy donors or patients with COVID-19 were analyzed by real-time polymerase chain reaction (PCR). (B and C) The mRNA level of TREM-2 was determined in patients with nonsevere versus severe COVID-19 and patients with severe COVID-19 at the stage of pre-ICU, ICU, and post-ICU. (D) Flow cytometric analysis of TREM-2 expression in CD4+ T cells from healthy donors (n = 50) or patients with nonsevere (n = 83) and severe COVID-19 (n = 20). (E) Flow cytometric analysis of TREM-2 expression in CD4+ T cells from patients with severe COVID-19 (n = 20) at the stage of pre-ICU, ICU, and post-ICU. (F) PBMCs from healthy donors or patients with COVID-19 were double- stained with anti-CD4 (green) and anti–TREM-2 (red) Ab and then observed by fluorescent confocal microscopy. Scale bars, 10 m. (G) The number of TREM-2+CD4+ cells (yellow) was quantified in CD4+ cells (green) from patients with COVID-19 (n = 5). (H) The MFI of TREM-2 in PBMCs was quantified with ImageJ software (n = 5). (I) Immuno- fluorescence analysis of normal lung tissue (defined as healthy) versus pathological lung sections from one critical patient with COVID-19 who died from COVID-19. [4′,6-diamidino-2-phenylindole (DAPI), blue; CD4, green; TREM-2, red]. Scale bars, 50 m. (J) The MFI of TREM-2 was quantified with ImageJ software in five fields of the lung. (K) The sTREM-2 was determined by ELISA in the supernatant of lung tissues from normal lung tissues (defined as healthy) versus lung tissues from a critical patient with COVID-19 (n = 5). ns, not significant. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: To study the effect of TREM-2–Fc fusion protein, PP2, or SH-4-54 on T cell response, human CD4+ T cells and CD8+ T cells were pretreated with TREM-2–Fc fusion protein (300 ng/ml; 1828-T2, R&D Systems), TREM-1–Fc fusion protein (300 ng/ml; 1278-TR, R&D Systems), TREM-2 Ab (1 g/ml; clone 237920, R&D Systems), or isotype IgG (300 ng/ml; 110-HG, R&D Systems), PP2 (250 nM), or SH-4-54 (100 nM) versus dimethyl sulfoxide (DMSO) for 1 hour.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Confocal Microscopy, Software, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: TREM-2 is a sensor and activator of T cell response in SARS-CoV-2 infection.

doi: 10.1126/sciadv.abi6802

Figure Lengend Snippet: Fig. 2. TREM-2 bound SARS-CoV-2 M protein. (A and B) HEK293T cells were transfected with plasmids containing HA-tagged TREM-2, FLAG-tagged SARS-CoV-2 M protein, and FLAG-tagged SARS-CoV-2 N protein. The interaction of TREM-2 with SARS-CoV-2 M or N protein was analyzed by co-IP assay (A) and confocal scanning microscopy (B) (DAPI, blue; SARS-CoV-2 M, green; TREM-2, red). Scale bars, 10 m. (C) Plates coated with control IgG with same Fc, TREM-1–Fc, or TREM-2–Fc were incubated with increasing concen- trations of recombinant M protein, and bound M protein was detected using M protein Ab. (D) Plates coated with TREM-2–Fc were incubated with recombinant M protein in the presence of increasing concentrations of TREM-2–Fc or TREM-1–Fc, and bound M protein was detected using M protein Ab. The amount of M protein bound to TREM-2 was then quantified by normalizing the signal intensities of the TREM-2–M protein in which anti–M protein Ab was coated onto the plate and detected by another anti–M protein Ab. (E and F) HEK293T cells were transfected with plasmids encoding TREM-2 full-length (FL), Ig domain, or truncated forms of TREM-2 respectively deleting transmembrane (ΔTM), cytosolic domain (ΔCyto), or Ig domain (ΔIg). The interaction of TREM-2 and M protein was immunoprecipitated with anti-HA or FLAG Ab and determined with their Ab.

Article Snippet: To study the effect of TREM-2–Fc fusion protein, PP2, or SH-4-54 on T cell response, human CD4+ T cells and CD8+ T cells were pretreated with TREM-2–Fc fusion protein (300 ng/ml; 1828-T2, R&D Systems), TREM-1–Fc fusion protein (300 ng/ml; 1278-TR, R&D Systems), TREM-2 Ab (1 g/ml; clone 237920, R&D Systems), or isotype IgG (300 ng/ml; 110-HG, R&D Systems), PP2 (250 nM), or SH-4-54 (100 nM) versus dimethyl sulfoxide (DMSO) for 1 hour.

Techniques: Transfection, Co-Immunoprecipitation Assay, Microscopy, Control, Incubation, Recombinant, Immunoprecipitation

Journal: Science advances

Article Title: TREM-2 is a sensor and activator of T cell response in SARS-CoV-2 infection.

doi: 10.1126/sciadv.abi6802

Figure Lengend Snippet: Fig. 3. TREM-2 bound SARS-CoV-2 M protein and interacted with CD3/ZAP70. (A and B) HEK293T cells were transfected with plasmids containing HA-tagged TREM-2, FLAG-tagged SARS-CoV-2 M protein, and Myc-tagged CD3. The interaction of TREM-2, SARS-CoV-2 M protein, and CD3 was performed by co-IP assay. Blots of cell lysates (input) or anti-FLAG. (C and D) Co-IP assay was performed to analyze the interaction among TREM-2, CD3, and ZAP70 in CD4+ T cells isolated from healthy donors versus patients with COVID-19, as well as patients with nonsevere versus severe COVID-19. The intensity of anti–TREM-2 IP bands was quantified and normalized to input bands. (E) Sorted CD4+ T cells from patients with COVID-19 were incubated with pseudovirus reconstituted with SARS-CoV-2 M protein (VSV-M) or S protein (VSV-S). The interaction of TREM-2, ZAP70, CD3, S protein, or M protein was immunoprecipitated with anti–TREM-2 Ab and was determined with their Ab. (F) Lung tissues from healthy donor versus patient with COVID-19 were dissociated and immunoprecipitated with anti–TREM-2 Ab. The interaction of TREM-2, ZAP70, CD3, and M protein was determined with their Ab. The intensity of anti–TREM-2 IP bands was quantified and normalized to input -actin. *P < 0.05 and **P < 0.01.

Article Snippet: To study the effect of TREM-2–Fc fusion protein, PP2, or SH-4-54 on T cell response, human CD4+ T cells and CD8+ T cells were pretreated with TREM-2–Fc fusion protein (300 ng/ml; 1828-T2, R&D Systems), TREM-1–Fc fusion protein (300 ng/ml; 1278-TR, R&D Systems), TREM-2 Ab (1 g/ml; clone 237920, R&D Systems), or isotype IgG (300 ng/ml; 110-HG, R&D Systems), PP2 (250 nM), or SH-4-54 (100 nM) versus dimethyl sulfoxide (DMSO) for 1 hour.

Techniques: Transfection, Co-Immunoprecipitation Assay, Isolation, Incubation, Immunoprecipitation

Journal: Science advances

Article Title: TREM-2 is a sensor and activator of T cell response in SARS-CoV-2 infection.

doi: 10.1126/sciadv.abi6802

Figure Lengend Snippet: Fig. 4. TREM-2 enhanced CD3/ZAP70/STAT1 signal pathway in SARS-CoV-2 M protein–stimulated CD4+ T cell. (A to F) Sorted CD4+ T cells from patients with COVID-19 (n = 10) were cultured and stimulated with pseudovirus of SARS-CoV-2 M protein for 12 hours, and no stimulation (Mock) and empty pseudovirus vector (Vector) stimulation were used as controls (A, B, and E). Sorted CD4+ T cells from patients with COVID-19 (n = 10) were cultured and stimulated with pseudovirus of SARS-CoV-2 M protein in the presence of TREM-2–Fc or TREM-1–Fc fusion protein or isotype IgG for 12 hours, and no stimulation (Mock) and only pseudovirus of SARS-CoV-2 M protein stimulation (Control) were used as control (C, D, and F). MFI of phosphorylated CD3 (p-CD3) (A and C), ZAP70 (p-ZAP70) (B and D), and STAT1 (p-STAT1) (E and F) in TREM-2− versus TREM-2+CD4+ T cells were analyzed by flow cytometry. (G) Purified CD4+ T cells were treated with lentivirus containing TREM-2 and then stimulated with pseudovirus of SARS-CoV-2 M protein. Flow cytometric analysis of surface TREM-2 in CD4+ T cells overexpressed TREM-2. p-CD3, p-ZAP70, and p-STAT1 were analyzed by Western blot. (H) The intensity of phosphorylated bands in (G) was quantified and normalized to total bands. (I to K) Sorted CD4+ T cells from patients with COVID-19 (n = 10) were cultured and stimulated with recombinant M protein or anti-CD3 Ab in the presence of TREM-2–Fc fusion protein or isotype IgG for 12 hours. MFI of p-CD3 (I), p-ZAP70 (J), and p-STAT1 (K) in CD4+ T cells were analyzed by flow cytometry. (L) Sorted CD4+ T cells from patients with COVID-19 were stimulated with pseudovirus of SARS-CoV-2 M protein in the presence of ZAP-70 inhibitor PP2 or dimethyl sulfoxide (DMSO). MFI of p-STAT1 in TREM-2− versus TREM-2+CD4+ T cells were analyzed by flow cytometry. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: To study the effect of TREM-2–Fc fusion protein, PP2, or SH-4-54 on T cell response, human CD4+ T cells and CD8+ T cells were pretreated with TREM-2–Fc fusion protein (300 ng/ml; 1828-T2, R&D Systems), TREM-1–Fc fusion protein (300 ng/ml; 1278-TR, R&D Systems), TREM-2 Ab (1 g/ml; clone 237920, R&D Systems), or isotype IgG (300 ng/ml; 110-HG, R&D Systems), PP2 (250 nM), or SH-4-54 (100 nM) versus dimethyl sulfoxide (DMSO) for 1 hour.

Techniques: Cell Culture, Plasmid Preparation, Control, Flow Cytometry, Purification, Western Blot, Recombinant

Journal: Science advances

Article Title: TREM-2 is a sensor and activator of T cell response in SARS-CoV-2 infection.

doi: 10.1126/sciadv.abi6802

Figure Lengend Snippet: Fig. 5. TREM-2 facilitated TH1 cytokine production in CD4+ T cells in COVID-19. (A and B) Sorted CD4+ T cells from patients with COVID-19 (n = 10) were cultured and stimulated with pseudovirus of SARS-CoV-2 M protein for 12 hours, and no stimulation (Mock) and pseudovirus vector (Vector) stimulation were used as controls. (C and D) Sorted CD4+ T cells from patients with COVID-19 (n = 10) were cultured and stimulated with pseudovirus of SARS-CoV-2 M protein in the presence of TREM-2–Fc fusion protein or isotype IgG for 12 hours, and no stimulation (Mock) and only pseudovirus of SARS-CoV-2 M protein stimulation (control) were used as controls. (E and F) Purified CD4+ T cells were treated with lentivirus containing TREM-2 (F) or vector (E) and then stimulated with pseudovirus of SARS-CoV-2 M protein. (G and H) Sorted CD4+ T cells from a patient with COVID-19 were stimulated with pseudovirus of SARS-CoV-2 M protein in the presence of ZAP-70 inhibitor PP2, pan-STATs inhibitor SH-4-54 or DMSO. (I and J). Sorted CD4+ T cells from a patient with COVID-19 patient were stimulated with recombinant M protein or anti-CD3 Ab in the presence of TREM-2–Fc fusion protein or isotype IgG for 12 hours. Cells were then stimulated with phorbol 12-myristate 13-acetate (50 nM), ionomycin (1 g/ml), and brefeldin A (1 g/ml) for 6 hours. Percentages of IFN-– and TNF-–producing cells were analyzed by flow cytometry. ns, not significant; **P < 0.01; ***P < 0.001. mAb, monoclonal Ab.

Article Snippet: To study the effect of TREM-2–Fc fusion protein, PP2, or SH-4-54 on T cell response, human CD4+ T cells and CD8+ T cells were pretreated with TREM-2–Fc fusion protein (300 ng/ml; 1828-T2, R&D Systems), TREM-1–Fc fusion protein (300 ng/ml; 1278-TR, R&D Systems), TREM-2 Ab (1 g/ml; clone 237920, R&D Systems), or isotype IgG (300 ng/ml; 110-HG, R&D Systems), PP2 (250 nM), or SH-4-54 (100 nM) versus dimethyl sulfoxide (DMSO) for 1 hour.

Techniques: Cell Culture, Plasmid Preparation, Control, Purification, Recombinant, Flow Cytometry

Journal: Science advances

Article Title: TREM-2 is a sensor and activator of T cell response in SARS-CoV-2 infection.

doi: 10.1126/sciadv.abi6802

Figure Lengend Snippet: Fig. 6. TREM-2 promoted TH1-mediated host defense against coronavirus infection. (A) Flow cytometric analysis of TREM-2 expression on CD4+ T cells from the lungs and spleen of MHV-A59–infected WT mice versus MHV-A59–infected CD4–TREM-2 KO mice. (B) Survival rate of WT mice versus CD4–TREM-2 KO mice infected with MHV-A59. (C and D) The expression levels of MHV N gene (C) and viral load (D) in lungs of infected mice were analyzed by real-time PCR and plaque assay. (E) CT imaging of uninfected mice and WT mice versus CD4–TREM-2 KO mice. (F) Hematoxylin and eosin staining of lung sections was examined in uninfected mice and WT mice versus CD4–TREM-2 KO mice. (G and H) The expression of CD69 (G) and CD134 (H) was determined in lung-infiltrating CD4+ T cells by flow cytometry. (I and J) The mRNA expression in the lung (I) and protein level in the plasma (J) for IFN- and TNF- were analyzed in MHV-A59–infected WT mice versus CD4–TREM-2 KO mice by real-time PCR and ELISA. (K to N) MFI of p-CD3 (K), p-ZAP70 (L), and p-STAT1 (M) as well as T-bet expression (N) in WT versus CD4–TREM-2 KO CD4+ T cells was analyzed by flow cytometry. The uninfected mice were as control (Mock). Scale bars, 50 m. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: To study the effect of TREM-2–Fc fusion protein, PP2, or SH-4-54 on T cell response, human CD4+ T cells and CD8+ T cells were pretreated with TREM-2–Fc fusion protein (300 ng/ml; 1828-T2, R&D Systems), TREM-1–Fc fusion protein (300 ng/ml; 1278-TR, R&D Systems), TREM-2 Ab (1 g/ml; clone 237920, R&D Systems), or isotype IgG (300 ng/ml; 110-HG, R&D Systems), PP2 (250 nM), or SH-4-54 (100 nM) versus dimethyl sulfoxide (DMSO) for 1 hour.

Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, Plaque Assay, Imaging, Staining, Flow Cytometry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Control