Fig 1: Pharmacological modulation of the inflammasome/dysfunctional T cell axis triggered by SARS-CoV-2 in vitro.(A) Flow cytometry study of TMEM176B-dependent Na+ transport in CHO cells loaded with the Na+-sensitive probe Asante NaTRIUM Green-2 (ANG-2). Cells were treated with 5 µM of the flavonoid isoquercetin (ISQ) or dimethyl sulfoxide (DMSO; vehicle control). MFI, mean fluorescence intensity. One experiment representative of four is shown. *P < 0.05; **P < 0.01, two-way ANOVA test. (B) Study of IL-1ß secretion by human primary monocytes infected with SARS-CoV-2 ± ISQ. Mock, not infected. One experiment representative of three is shown. ***P < 0.001; ****P < 0.0001, one-way ANOVA test. (C) Study of viral load in human primary monocytes infected with SARS-CoV-2 ± 5 µM ISQ. One experiment representative of three is shown. ****P < 0.0001, one-way ANOVA test. (D) Study of FLICA-1 MFI (active caspase-1) from a pool of three independent experiments, where human primary monocytes were not infected (Mock) or infected with SARS-CoV-2 and treated with DMSO (vehicle control) or with 5 µM ISQ. *P < 0.05; **P < 0.01, one-way ANOVA test. (E) Representative flow cytometry histograms of the experiments shown in (D). One experiment representative of three is shown. (F) Study of PD-1 expression by CD8+ TCR-ß+ cells cocultured with allogeneic monocytes ± cell culture medium (CCM) from SARS-CoV-2–infected monocytes ± 5 µM ISQ. One experiment representative of three is shown. (G and H) Flow cytometry study of IFN-? expression by CD8+ T cells cocultured with allogeneic monocytes ± CCM from uninfected monocytes (Mock) or SARS-CoV-2–infected cells ± anti–PD-1 or control antibody (20 µg/ml). The percentage of IFN-?+ cells is shown in (G), and the MFI of positive cells is shown in (H). The graphics show one experiment representative of three. *P < 0.05; **P < 0.01, one-way ANOVA test.
Fig 2: Inflammasome-dependent T cell exhaustion in Tmem176b-/- mice infected with MHV-A59.(A) Survival of MHV-A59–infected WT and Tmem176b-/- mice left untreated. In another group, infected WT mice were adoptively transferred with splenic DCs from infected Tmem176b-/- animals, and Tmem176b-/- mice were adoptively transferred with CD8+ T cells from infected WT animals. *P < 0.05; ***P < 0.001, log-rank (Mantel-Cox) test. (B) Percentage of MHV-A59–specific CD8-dependent in vivo lysis was calculated with the formula described in Materials and Methods. Twelve WT, 16 Tmem176b-/-, and 5 Tmem176b-/-Casp1-/- mice were studied at 5 dpi. ***P < 0.001; ****P < 0.0001, one-way ANOVA test. (C) Representative histograms of the experiments shown in (B). (D) Percentage of liver-infiltrating TOX+ TCF-1+ within PD-1+ CD44+ cells, gated in CD8+ MHV-A59–specific T cells. Animals from two independent experiments were analyzed. *P < 0.05, one-way ANOVA test. (E) Representative dot plots of the animals analyzed in (D). (F) Survival of Tmem176b-/- mice infected with MHV-A59 and treated with control IgG or anti–PD-1 antibody. ****P < 0.0001, log-rank (Mantel-Cox) test. (G) Viral load in the liver at 5 dpi with MHV-A59 in Tmem176b-/- mice treated with control IgG or anti–PD-1 antibodies. *P < 0.05, Mann-Whitney test. (H) Survival of WT mice infected with MHV-A59 and treated with control IgG or anti–PD-1 antibody. ns, nonsignificant. Log-rank (Mantel-Cox) test. (I) Viral load in the liver at 5 dpi with MHV-A59 in WT mice treated with control IgG or anti–PD-1 antibodies. Mann-Whitney test.
Fig 3: Exhausted T cells correlate with plasmatic active caspase-1 in critical COVID-19 and can be modulated by anti–PD-L1 antibodies.(A) Representative dot plots of a flow cytometry study of PD-1 and TOX in CD8+ TCR-ß+ CD38+ HLA-DR+ cells in non-ICU and ICU patients. (B) The graphic shows the individual percentages of PD-1+ TOX+ cells within CD38+ HLA-DR+ CD8+ TCR-ß+ for each studied patient. *P < 0.05, Student’s t test. (C) Network plot representing Pearson correlations of the indicated genes. Transcriptomic data from peripheral blood were analyzed from McClain et al. (46). (D) Correlation study between the MFI determined by flow cytometry of PD-1 and TOX within CD8+ TCR-ß+ CD38+ HLA-DR+ cells in non-ICU and ICU patients. (E) Network plot representing Pearson correlations of the indicated genes. Transcriptomic data from peripheral blood were analyzed from McClain et al. (46). (F) Correlation study between the percentage of PD-1+ TOX+ cells in CD8+ TCR-ß+ CD38+ HLA-DR+ cells and plasmatic active caspase-1 in non-ICU and ICU patients. (G) Flow cytometry study of the percentage of PD-1+ TOX+ cells within CD38+ HLA-DR+ CD8+ T cells from four lymphopenic ICU patients. Cells were incubated for 24 hours with control IgG (20 µg/ml; human IgG1) or anti–PD-L1 antibody. Then, 6 nmol of SARS-CoV-2 peptides was added or not to the culture for 6 hours. Representative dot plots are shown in the left panel. Data for the four studied patients are shown in the right panel. *P < 0.05; **P < 0.01; ****P < 0.0001, one-way ANOVA test. (H) Flow cytometry study of IFN-? production by CD8+ TCR-ß+ CD38+ HLA-DR+ PD-1+ TOX+ cells from the same ICU patients studied in (G) under the same in vitro treatments. Representative dot plots are shown in the left panel. Data for the four studied patients are shown in the right panel. *P < 0.05, one-way ANOVA test.
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