Peripheral natural killer cells in chronic hepatitis B patients display multiple molecular features of T cell exhaustion
- CIRI, Centre International de Recherche en Infectiologie, Team Innate Immunity in Infectious and Autoimmune Diseases, Univ Lyon, Inserm, Université Claude Bernard Lyon 1, CNRS, France
- Service d’Immunologie biologique, Hôpital Lyon Sud, Hospices Civils de Lyon, France
- CHU Limoges, Service d’Hépatogastroentérologie, U1248 INSERM, Université Limoges, France
- Département de Microbiologie, CHU de Limoges, Faculté de médecine-Université de Limoges, France
- Centre de Recherche en Cancérologie de Lyon (CRCL), INSERM, U1052, CNRS, Université de Lyon, France
Figures
Figure 1 with 3 supplements
NK cell functionality is impaired in CHB patients.
(A) PBMCs from HD (n = 30) or CHB patients (n = 32) were co-cultured with K562 during 4 hr, and the proportion of NK cells expressing CD107a was determined by immunostaining. Representative …
Figure 1—figure supplement 1
HCMV status.
(A) The concentrations of anti-CMV IgG antibodies were measured in serum from patients and HD to determine HCMV seropositivity. (B) The percentage of NKG2C+-adaptive NK cells in patients and HD was …
Figure 1—figure supplement 2
Gating strategy.
Gating strategy used for flow cytometry analysis. First, lymphocytes through SSC and FSC analyses were selected. Then doublets were eliminated as well as dead cells. We then removed CD4+, CD14+, and …
Figure 1—figure supplement 3
Functional properties.
(A) Dot plot showing the correlation between the percentage of IFN-γ and TNF-α-positive NK cells following K562 stimulation of HD and CHB patients PBMCs. Each dot represents an individual. …
Figure 2 with 1 supplement
NK cell from CHB patients display an altered phenotype.
(A) PBMCs from 30 HD and 32 CHB samples were stained with live/dead, CD4, CD14, CD19, CD3, CD7, and CD56 antibodies. The percentage of NK cells in each sample (± SD), and the proportion of CD56bright…
Figure 2—figure supplement 1
Phenotype of CHB patients NK cells along differentiation.
Mean fluorescence intensity (MFI) of indicated NK activating (A) and inhibitory (B) receptors was determined by flow cytometry on CD56Bright and CD56Dim NK cells among PBMCs from HD or CHB patients. …
Figure 3 with 1 supplement
mTOR activation is impaired in NK cells from patients.
(A) PBMCs from HD or CHB patients were stimulated or not with IL-15 at 100 ng/mL for 30 min prior to phospho-epitope staining (pS6 Ser235/236, pAKT S473, and pSTAT5 Y694). Overlays of representative …
Figure 3—figure supplement 1
Gating strategy, phospho-epitopes, and metabolic analysis.
(A) Gating strategy used for phospho-flow cytometry analysis: First, lymphocytes through SSC and FSC analysis were selected. Then doublets were eliminated. We then selected CD56+, CD3− cells. CD7 …
Figure 4 with 1 supplement
RNAseq analysis identifies an exhaustion-like signature in patient NK cells.
(A) Principal component analysis of the RNAseq data is shown. (B) Heatmap of the DEG genes between HD and CHB. (C) Gene Ontology analysis of DEG up-regulated in CHB patients using Metascape. …
Figure 4—figure supplement 1
Metascape analysis of CHB patients up-regulated genes.
Full results for the Gene Ontology analysis of DEG up-regulated in CHB patients performed with Metascape and presented in Figure 4C.
Figure 5 with 1 supplement
Validation of the exhausted phenotype at the protein level.
(A) Intracellular staining for TOX was performed on PBMCs from 9 HD and 10 CHB samples and the MFI measured. A representative FACS histogram overlay (left panel) as well as the median MFI and …
Figure 5—figure supplement 1
TOX expression in CD56Bright and CD56Dim NK cells.
(A) Intracellular staining for TOX was performed on PBMCs from 10 HD and 10 CHB samples and the MFI measured in CD56Bright and CD56Dim NK cells. The median MFI (± SD) and individual values for each …
Figure 6
RNAseq and in vitro modelling suggest that NK cell dysfunction is due to unbalanced Ca2+ signalling.
(A) A GSEA plot comparing HD and CHB patients is shown for the gene set regulated by partnerless NFAT. The normalised enrichment score (NES) and FDR q-values are indicated. (B) PBMCs from HD were …
Author response image 1
Correlation between percentages of IFN-γ+ following K562 stimulation and pS6 level following IL-15 stimulation.
PBMCs from HD (n=30) or CHB patients (n=32) were co-cultured with K562 during 4 hours and the proportion of NK cells expressing IFN-γ was determined by immunostaining. The same samples were …
Author response image 2
Representative histogram plots showing the expression level of the indicated markers as well as FMO and isotype control stainings on NK cells of HD or CHB patients.
Author response image 3
Correlation between pS6 level in CD56Dim and CD56Bright NK cells following IL-15 stimulation.
PBMCs from HD (n=30) or CHB patients (n=32) were stimulated with IL-15 at 100 ng/mL for 30 min, prior to phospho-epitope staining (pS6 Ser235/236). MFI values for each individual are represented.
Author response image 4
Representative FACS-plot showing TOX and T-BET coexpression in total NK cells of a CHB patient.
Author response image 5
Mean fluorescence intensity (MFI) of the indicated markers was determined by flow cytometry on CD56Bright, CD56Dim NKG2A+ and CD56Dim NKG2A- NK cells among PBMCs from HD or CHB patients.
Box-plots as well as values for each individual are represented.
Author response image 6
(A) Histogram overlays showing the level of the indicated phosphoepitope following 1h IL-15 stimulation in the presence or absence of the mTOR inhibitors Rapamycine (left) or Torin2 (right).
(B) Kinetic of Kynurenine uptake with or without excess Leucine or Lysine, inhibitors of Slc7a6 or Slc7a5 respectively. (C) Histogram overlays showing the level of mitochondrial ROS with or without …
Tables
Table 1
Characteristics of patients and HD enrolled in the study.
| Parameter | CHB Patients 1st cohort | Healthy Donors 1st cohort | CHB Patients 2nd cohort | Healthy Donors 2nd cohort |
|---|---|---|---|---|
| Number | 32 | 30 | 12 | 10 |
| Age (y) Mean Median (range) | 39 ± 13 38 (19–77) | 38 ± 13 36.5 (20–65) | 33 ± 13 30.5 (20–57) | 37 ± 8 38 (26–50) |
| Sex, n (%) Male Female | 20 (62.5) 12 (37.5) | 22 (73) 8 (27) | 8 (66.6) 4 (33.3) | 5 (50) 5 (50) |
| HBV load (IU/mL) Median | 2844 (10–62,225) | / | 1919 (18–32,785) | / |
| HBsAg (IU/mL) Median | 5770 (0.05–34,971) | / | 17,306(4,189–29,890) | / |
| ALT (IU/mL) Median | 20 (6–56) | NA | 21 (12–35) | NA |
| AST (IU/mL) Median | 21 (11–34) | NA | NA | NA |
| HCMV seropositive, % | 86 | 38 | NA | NA |
-
ALT, serum alanine aminotransferase levels; AST, aspartate transaminase levels; NA, not available.
Additional files
-
Supplementary file 1
Antibodies used.
- https://cdn.elifesciences.org/articles/60095/elife-60095-supp1-v2.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/60095/elife-60095-transrepform-v2.docx
