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: Cell Communication and Signaling : CCS

Article Title: CD69 + CD103 + CD8 + tissue-resident memory T cells possess stronger anti-tumor activity and predict better prognosis in colorectal cancer

doi: 10.1186/s12964-024-01990-3

Figure Lengend Snippet: Relationship of different CD8 + TRM subset with B cells and TLS in CRC tumor tissues. ( A ) Volcano plot showing differentially expressed genes between DP CD8 + TRM subsets and SP CD8 + TRM subsets of the GSE108989 dataset. ( B ) The FFPE CRC tumor tissue slides from patients with CRC were stained with DAPI (blue), CD20 (green), CD8 (yellow), CD103 (orange), and CD69 (red). ( C ) Relationship between the proportion of different CD8 + TRM cells and the proportion of B cells in CRC tumor tissues detected by flow cytometry ( n = 40) and relationship between the proportion of different CD8 + TRM cells and the count of B cells and the count and area of TLS in CRC tumor tissues detected by mIHC ( n = 64). Statistical analysis was performed by Pearson correlation analysis. ( D ) Schematic overview of the transwell migration assay design. ( E ) B cell migration rate when cultured either alone or with different CD8 + TRM subsets ( n = 10) and B cell migration rate when cultured with DP CD8 + TRM cells with or without anti-CXCL13 neutralizing antibody ( n = 7). Statistical analysis was performed by non-parametric Wilcoxon’s matched-pairs tests and Friedman test with Dunn’s multiple comparisons test. (* p < 0.05; ** p < 0.01)

Article Snippet: To disrupt CXCL13/CXCR5 interactions during chemotaxis, neutralizing antibody against CXCL13 (2 µg/mL, MAB801, R&D Systems) was added to the culture medium.

Techniques: Staining, Flow Cytometry, Transwell Migration Assay, Migration, Cell Culture

Journal: eBioMedicine

Article Title: Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer

doi: 10.1016/j.ebiom.2024.105003

Figure Lengend Snippet: The depletion of hMENA 11a down-regulates LTβR and up-regulates FN1. In node-negative patients with NSCLC, hMENA 11a expression associates with intratumoral TLS . a. Volcano plots showing differentially expressed genes (q-value <0.05, n = 1096, GSE217451) in Si-hMENA 11a versus control H1650 NSCLC cells (n = 3). Reported are the negative log10-transformed adjusted P values plotted against the log2 fold changes. Dots represent individual genes. b . qRT-PCR analysis of LTβR mRNA expression in the indicated cell lines transfected with control (Si-CNTR), and hMENA 11a pool SiRNAs (Si-hMENA 11a ) or with plasmid control (CNTR) and hMENA 11a expressing vector (hMENA 11a ). The control of hMENA 11a expression in the transfected cells by WB is reported in Supplementary Figure S3a . Data are reported as the mean ± SD of technical triplicates which are representative of two independent experiments. P value was calculated by 2-tailed Student's t test. c. qRT-PCR analysis showed that LTβR and hMENA 11a mRNA expression level correlate in NSCLC cell lines. The value of Spearman correlation is reported. d. Consecutive sections of a representative NSCLC primary tumor hMENA 11a positive, showing low stromal FN1, TLS IT localization and high CXCL13. Magnification 8×. Scale bar 300 μm. Right, histograms relative to the IHC analysis of 94 node-negative NSCLC tissues showing that hMENA 11a positive cases more frequently show TLS within the tumor area (TLS IT). e. Consecutive sections of a representative case of lung adenocarcinoma hMENA 11a negative, showed high stromal FN1, TLS PT and low CXCL13. Right, histograms are relative to the IHC analysis of 94 node-negative NSCLC tissues showing that hMENA 11a negative (hMENA 11a low /hMENA (t) high ) cases more frequently show peritumoral TLS (TLS PT). P value was estimated with Fisher Exact test.

Article Snippet: CXCL13 detection was obtained by the use of human CXCL13 Antibody (AF801 R&D Systems).

Techniques: Expressing, Control, Transformation Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: eBioMedicine

Article Title: Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer

doi: 10.1016/j.ebiom.2024.105003

Figure Lengend Snippet: hMENA/hMENAΔv6 influences the expression and signaling of LTβR and the secretion of CXCL13 in CAFs. The ‘epithelial’ hMENA 11a isoform in tumor cells affects CXCL13 production by T RM cells . a. qRT-PCR analysis of LTβR mRNA expression in the CAFs obtained from four different patients with NSCLC (#359, #358, #391, #405), transfected with control (Si-CNTR), and hMENA(t) pool siRNAs (Si-hMENA(t)). Data reported are the mean of technical triplicates. P value of paired 2-tailed Student's t test is reported. b. Representative WB analysis with the indicated Abs of protein extracts from CAF #302 transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA, untreated or treated with 50 ng/mL of LIGHT for 24 h. Fold change of P-p65/p65 and of p52/p100 staining intensity (right). Data reported are the mean of 4 different experiments ± SD. Adjusted P values of One-way ANOVA followed by Tukey's multiple comparisons procedures are reported when significant. c. CXCL13 production evaluated by ELISA assay of CAFs derived from three different patients (#302; #358; #571) transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA. Data reported are the mean of technical triplicates of pg/mL normalized for total protein content in three biological replicates. P value of paired two tailed t test, is reported d. Percentage of CXCL13 chemokine, evaluated by multiparametric flow cytometry, in ex-vivo TILs isolated from eight patients with NSCLC, after culture (24 h) with CM from H1650 tumor cells silenced for hMENA 11a (Si-hMENA 11a ), or control (Si-CNTR). Results within CD8 + T RM (CD103 + CD69 + , left panel) or CD4 + T RM (CD103 + CD69 + , right panel) are shown. Statistical significance was determined using non-parametric Wilcoxon rank test.

Article Snippet: CXCL13 detection was obtained by the use of human CXCL13 Antibody (AF801 R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Staining, Enzyme-linked Immunosorbent Assay, Derivative Assay, Two Tailed Test, Flow Cytometry, Ex Vivo, Isolation

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