Journal: Molecular Therapy. Nucleic Acids

Article Title: Novel Fas-TNFR chimeras that prevent Fas ligand-mediated kill and signal synergistically to enhance CAR T cell efficacy

doi: 10.1016/j.omtn.2023.04.017

Figure Lengend Snippet: Fas-CD40 activates NF-κB and induces strong proliferation upon FasL binding (A) Left: upon binding FasL, Fas trimerization recruits FADD, initiating apoptosis. Middle: FasΔDD acts as a decoy receptor to FasL by being unable to recruit FADD. Right: schematic of Fas-TNFR structure; the ectodomain and transmembrane domain of Fas are fused to the endodomains of TNFRs. Upon FasL binding the Fas-TNFR chimera converts the death signal into a survival/growth signal. (B) Members of the TNFR superfamily. Those highlighted in gold were included in the Fas-TNFR screen. (C) Schematic of polycistronic transgene transduced into human T cells. 19-ζ: Fmc63 binder fused to the endodomain of CD3ζ via a CD8 stalk/transmembrane domain. (D) NF-κB reporter Jurkat cells transduced to express either 19-ζ alone or co-express FasΔDD or the Fas-TNFRs were cultured with or without immobilized recombinant FasL (20 μg/mL) overnight and NF-κB activity was measured. Experiment performed with technical triplicates, error bars are SEM. (E) Human T cells (5 × 10 4 ) expressing 19-ζ and FasΔDD or the Fas-TNFRs were cultured with or without immobilized recombinant FasL (20 μg/mL) for 5 days, at which point cell counts were analyzed by flow cytometry. Due to the large list of Fas-TNFR chimeras, they were tested over two separate experiments (screens 1 and 2) with the data being compiled onto one graph. The conditions were identical between screens having the same 19-ζ and FasΔDD controls. Five independent donors were tested in screen 1 and four independent donors were tested in screen 2, error bars are SEM. (F) Human T cells from five independent donors were transduced to express 19-ζ or co-express FasΔDD or the stated Fas-TNFRs and then cultured with or without immobilized recombinant FasL (20 μg/mL) for 3 days, at which point RNA was extracted and analyzed using the nCounter NanoString platform with the CAR-T Characterization Panel. 19-ζ cells co-expressing FasΔDD or the Fas-TNFRs were normalized to 19-ζ alone, and the number of significantly (p < 0.05) upregulated differentially expressed genes (DEGs) were categorized by pathway involvement. (G) Significant DEGs relative to 19-ζ with greatest Log 2 fold change (FC) from the experiment described in (F). (H) Volcano plot from the experiment described in (F) of Fas-CD40-19-ζ cells compared with 19-ζ alone after incubation with immobilized FasL. (I) Significant DEGs relative to FasΔDD-19-ζ with greatest Log 2 (FC) from the experiment described in (F). (J and K) 19-ζ cells were cultured in the presence or absence of immobilized FasL (20 μg/mL) for 5 days and then stained for CCR8, ICOSL, and ICOS expression by flow cytometry (I), or the cell culture supernatant analyzed for CCL1, CXCL10, and CXCL13 secretion (J). Six independent donors tested, error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two-way ANOVA. (L) Top: TCF7 expression from the experiment described in (F). Bottom left: 19-ζ cells were stained for TCF-1, representative flow cytometry plots from one donor. Bottom right: TCF-1 expression from three independent donors, error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, two-way ANOVA.

Article Snippet: Cytokine concentrations in cell culture supernatants were measured by ELISA using kits to detect IFN-γ (BioLegend, 430104), IL-2 (BioLegend, 431804), CCL1 (R&D Systems, DY272), CXCL10 (R&D Systems, DY266), and CXCL13 (R&D Systems, DY801) according to the manufacturer’s instructions using a Multiskan FC microplate photometer (Thermo Scientific).

Techniques: Binding Assay, Cell Culture, Recombinant, Activity Assay, Expressing, Flow Cytometry, Incubation, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Novel Fas-TNFR chimeras that prevent Fas ligand-mediated kill and signal synergistically to enhance CAR T cell efficacy

doi: 10.1016/j.omtn.2023.04.017

Figure Lengend Snippet: Upregulated DEGs specific to Fas-CD40, Fas-Fn14, and Fas-BCMA expression

Article Snippet: Cytokine concentrations in cell culture supernatants were measured by ELISA using kits to detect IFN-γ (BioLegend, 430104), IL-2 (BioLegend, 431804), CCL1 (R&D Systems, DY272), CXCL10 (R&D Systems, DY266), and CXCL13 (R&D Systems, DY801) according to the manufacturer’s instructions using a Multiskan FC microplate photometer (Thermo Scientific).

Techniques: Phospho-proteomics

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: Untreated hyperacute HIV, but not ART early-treated hyperacute HIV, is associated with elevation of plasma cytokines that have distinct kinetics. a Interferon gamma-induced protein 10 (IP-10/CXCL-10). b Monokine induced by gamma interferon (MIG/CXCL-9). c Monocyte chemoattractant protein 1 (MCP-1). d Interleukin 12 (IL-12). e Soluble IL-2 receptor (IL-2R). f Interleukin 8 (IL-8). g Interferon gamma (IFN-gamma). h Interleukin-1 receptor antagonist (IL-1RA). i B cell-activating factor (BAFF/BLYS/TNFSF13B). j Chemokine (C-X-C motif) ligand 13 (CXCL13). k Soluble CD14. l Interferon alpha (IFN-alpha). N = 12 for untreated hyperacute HIV-infected participants (except CXCL13 and BAFF with N = 10). N = 8 for ART early-treated hyperacute HIV-infected individuals (except CXCL13 and BAFF with N = 6 and IFN-alpha with N = 7). Cytokine levels for one of the untreated participants were measured 434 days instead of 238–263 days after the detection of viremia. Each symbol represents an individual participant. Except for IFN-alpha, red symbols show the plasma levels in untreated participants and blue symbols show the plasma levels in ART early-treated participants. Horizontal lines and error bars in the scatter plots represent the median and interquartile range. In l (IFN-alpha), every colored line represents a participant. Statistical test used: Wilcoxon matched-pairs signed-rank test. P values < 0.05 were considered significant. * P < 0.05, ** P < 0.01, *** P < 0.001. “Pre” refers to the pre-infection time point

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics, Infection

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: CXCL13 is positively associated with delayed suppression of viremia in early-treated individuals. a Duration to viral suppression in days among early-treated participants. b Correlation between duration to viral suppression in days and viral load at the time of initiating ART in early-treated individuals. c Correlation between duration to viral suppression and plasma CXCL13 levels at 3 months. d Correlation between viral load at the time of initiating ART and plasma CXCL13 levels at 3 months. Each symbol represents an individual participant ( N = 6). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: The magnitude of plasma cytokines predicts CD4 + T cell and viral load dynamics in untreated hyperacute HIV infection. a Correlation between peak IFN-alpha and peak viremia. b Correlation between hyperacute soluble IL-2 receptor and peak viremia. c Correlation between hyperacute IL-1RA and viral load set point. d Correlation between hyperacute CXCL13 and nadir CD4 + T cell counts. e Correlation between hyperacute soluble IL-2 receptor and nadir CD4 + T cell counts. f Correlation between hyperacute IL-1RA and set point CD4 + T cell counts. Each symbol represents an individual participant ( N = 12 except CXCL13 with N = 10). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics, Infection

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: Plasma cytokines/chemokines are associated with reduced blood counts of lymphocytes, eosinophils, and basophils in untreated acutely HIV-infected patients. a Correlation between CXCL13 and total lymphocytes. b Correlation between CXCL13 and eosinophils. c Correlation between CXCL13 and basophils. d Correlation between MIG/CXCL9 and total lymphocytes. e Correlation between MIG/CXCL9 and eosinophils. f Correlation between MIG/CXCL9 and basophils. g Correlation between soluble IL-2 receptor and total lymphocytes. h Correlation between soluble IL-2 receptor and eosinophils. i Correlation between soluble IL-2 receptor and basophils. The measurements of cytokines and blood cell counts were in the hyperacute phase of HIV infection. Each symbol represents an individual participant ( N = 12 except CXCL13 ( a – c ) with N = 10). Statistical test: Spearman’s rank-order correlation. P values < 0.05 were considered significant

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Clinical Proteomics, Infection

Journal: BMC Medicine

Article Title: Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

doi: 10.1186/s12916-020-01529-6

Figure Lengend Snippet: Correlation network showing a summary of the relationships between cytokines, CD4 + T cell dynamics, viral load dynamics, Gag-driven viral replication capacity, and hematological parameters in untreated hyperacute HIV infection. Statistical test used: Spearman’s rank-order correlation. Red lines show significant positive correlations. Blue lines show significant inverse correlations. The width of the line indicates the strength of Spearman’s correlation coefficient (rho). Only correlations that have P < 0.05 are shown. Gag RC, Gag-driven viral replication capacity ( N = 12 except CXCL13 and BAFF with N = 10)

Article Snippet: The plasma levels of BAFF had been measured in a previous study pre-infection, during the hyperacute infection phase (4–11 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using Human BAFF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) while CXCL13 had been measured pre-infection, during the hyperacute infection phase (1–4 days after the detection of viremia), at two time points after peak viremia (13–18 days and 24–32 days after the detection of viremia), and during the early chronic phase (77–95 days after the detection of viremia) using human CXCL13 Quantikine ELISA kit (R&D Systems) [ ].

Techniques: Infection