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Research Article
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Interferon lambda 4 can directly activate human CD19+ B cells and CD8+ T cells

Mairene Coto-Llerena, Marco Lepore, View ORCID ProfileJulian Spagnuolo, Daniela Di Blasi, View ORCID ProfileDiego Calabrese, Aleksei Suslov, Glenn Bantug, Francois HT Duong, Luigi M Terracciano, View ORCID ProfileGennaro De Libero  Correspondence email, View ORCID ProfileMarkus H Heim  Correspondence email
Mairene Coto-Llerena
1Department of Biomedicine, Hepatology, University Hospital and University of Basel, Basel, Switzerland
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Marco Lepore
2Department of Biomedicine, Experimental Immunology, University Hospital and University of Basel, Basel, Switzerland
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Julian Spagnuolo
2Department of Biomedicine, Experimental Immunology, University Hospital and University of Basel, Basel, Switzerland
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Daniela Di Blasi
1Department of Biomedicine, Hepatology, University Hospital and University of Basel, Basel, Switzerland
2Department of Biomedicine, Experimental Immunology, University Hospital and University of Basel, Basel, Switzerland
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Diego Calabrese
1Department of Biomedicine, Hepatology, University Hospital and University of Basel, Basel, Switzerland
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Aleksei Suslov
1Department of Biomedicine, Hepatology, University Hospital and University of Basel, Basel, Switzerland
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Glenn Bantug
3Department of Biomedicine, Immunobiology, University Hospital and University of Basel, Basel, Switzerland
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Francois HT Duong
1Department of Biomedicine, Hepatology, University Hospital and University of Basel, Basel, Switzerland
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Luigi M Terracciano
4Molecular Pathology Division, Institute of Pathology, University Hospital Basel, Basel, Switzerland
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Gennaro De Libero
2Department of Biomedicine, Experimental Immunology, University Hospital and University of Basel, Basel, Switzerland
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  • For correspondence: gennaro.delibero@unibas.ch
Markus H Heim
1Department of Biomedicine, Hepatology, University Hospital and University of Basel, Basel, Switzerland
5Division of Gastroenterology and Hepatology, Clarunis, University Center for Gastrointestinal and Liver Diseases, Basel, Switzerland
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  • ORCID record for Markus H Heim
  • For correspondence: markus.heim@unibas.ch
Published 6 November 2020. DOI: 10.26508/lsa.201900612
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  • Figure 1.
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    Figure 1. Identification of IFNλ-responsive cell populations in PBMCs.

    (A) PBMCs were stimulated for 15 min with 1,000 IU/ml IFNα or 100 ng/ml of IFNλ1. Western blot detection of phosphorylated STAT1 (pY-STAT1), total STAT1, and actin protein in total cell lysates of PBMCs obtained from three different donors. (B) Purified human PBMCs were stimulated with 100 ng/ml of IFNλ1 for 15, 30, and 60 min or 1,000 IU/ml of IFNα for 15 min (left panel) or with 50, 100, and 200 ng/ml of IFNλ1 or 1,000 IU/ml of IFNα for 15 min. Shown are representative blots from two different donors. (A, C) Total PBMCs and purified CD19+ B cells, CD3+ cells, CD8+ T cells, CD4+ T cells, CD3−/CD16+ NK cells, and CD14+ monocytes were stimulated for 15 min with 1,000 IU/ml IFNα or 100 ng/ml IFNλ1 and analyzed as described in (A). A representative blot from two experiments is shown. (A, D) PBMC-derived CD19+ B cells and CD8+ T cells were stimulated for 15 min with 1,000 IU/ml IFNα, 100 ng/ml IFNλ1, or 100 ng/ml IFNλ4 and analyzed as described in (A). (E) qRT-PCR analysis of IFNLR1, IFNAR1, and IL10RB transcripts in total RNA isolated from the indicated PBMC subpopulations and primary human hepatocytes. Transcript levels are expressed as the ΔΔCT relative to GAPDH. Results are shown as mean ± SEM; n = 3.

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    Figure S1. IFNλ4 induces STAT1 phosphorylation and weakly stimulates neutrophil migration.

    (A) Neutrophils were stimulated with IFNλ1 (100 ng/ml) for 15, 30 and 60 min or 1,000 IU/ml of IFNα for 15 min. (B) Neutrophils were placed in Transwell plate and stimulated with IFNλ1 (100 ng/ml) and IFNλ4 (100 ng/ml) alone and in the presence of fMLP (100 nM) for 4 h. Cell supernatants were collected and migrated neutrophils were counted using FACS. Results shown as mean ± SEM from two independent experiments.

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    Figure 2. IFNλ4 has no co-stimulatory activity on B-cell activation.

    B cells were isolated from PBMCs and stimulated with IFN and/or CpG ODN2006 for 48 h and analyzed for B-cell activation. (A) B cells isolated from seven donors were stimulated with IFNα (1,000 IU/ml), IFNλ1 (100 ng/ml) and IFNλ4 (100 ng/ml), fixed, and analyzed by CD69-specific mAbs by FACS. Percentages of CD69+ cells within the B-cell population (upper panel) or median fluorescence intensity (MFI) of CD69+ cells within the B-cell population (lower panel) are shown. Significant changes between IFN-treated and control samples are denoted by a thick line. (A, B) B cells isolated from three donors were stimulated with IFNα (1,000 IU/ml), IFNλ1 (100 ng/ml), and IFNλ4 (100 ng/ml) alone or in combination and analyzed as described in (A). Significant changes between IFN-treated and control samples as well as combination versus single IFN treatment are denoted by a thick line. (C) IFI27, MX1, OAS1, and IFIT1 transcript levels in B cells stimulated with individual or combinations of IFNα, IFNλ1, and IFNλ4 were analyzed by qRT-PCR of total RNA. Results are shown as ΔΔCT relative to GAPDH (mean ± SEM; n = 2). (D, E) B cells were stimulated with IFNα (1,000 IU/ml), IFNλ1 (100 ng/ml), and IFNλ4 (100 ng/ml) alone (D) or in combination (E) in the presence or absence of CpG (0.8 μg/ml). (A) Cells were analyzed as described in (A). (A, B, D, E) Colors depict individual donors and thin horizontal lines indicate the mean; n = 7 for (A) and (D); n = 3 for (B) and (E). *P < 0.05, **P < 0.01, and ***P < 0.001 (paired t test).

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    Figure 3. IFNλ4 induces IL-10 production in B cells.

    (A) B cells (n = 4) were stimulated with IFNα (1,000 IU/ml), IFNλ1 (100 ng/ml), and IFNλ4 (100 ng/ml) alone or in combination and in the presence or absence of CpG (2.5 μg/ml) for 48 h. Cell supernatants were collected and released IL-10 was determined by ELISA. Results shown as ± SEM from two independent experiments. Significant changes between IFN-treated and control samples as well as IFN combinations versus single IFN treatment are denoted by a thick line. (B) Memory (CD27+ CD19+) and naïve (CD27− CD19+) B cells were stimulated with IFNα (1,000 IU/ml), IFNλ1 (100 ng/ml), and IFNλ4 (100 ng/ml) alone or in combination for 48 h either in the absence or presence of CpG (2.5 μg/ml). IL-10 present in supernatants of TLR9-stimulated and unstimulated cells in the presence of IFN was analyzed by ELISA. Results show mean ± SEM concentration (n = 4). (C) Memory B cells were treated with IFNα (1,000 IU/ml), IFNλ1 (100 ng/ml) or IFNλ4 (100 ng/ml) in the presence of CpG (2.5 μg/ml) for 48 h. The presence of IgG in the supernatants of stimulated and unstimulated controls was analyzed by ELISA. Results show the mean ± SEM concentration (n = 4). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control condition (paired t test).

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    Figure 4. IFNλ4 provides a co-stimulatory role during CD8+ T-cell activation.

    (A, B) CD8+ T cells were plated for 48 h either (A) on a 96-well plate pre-coated with three different concentrations of anti-CD3 (0.1; 0.3 and 1 μg/ml) in the presence of IFNα (1,000 IU/ml), IFNλ1 (100 ng/ml), or IFNλ4 (100 ng/ml) or (B) on a 96-well plate pre-coated with 0.1 μg/ml of anti-CD3 in the presence of increasing doses of IFNλ1 and IFNλ4. Released IFNγ was determined by ELISA. Data are representative of three independent experiments. (C, D, E, F, G, H) CD8+ T cells were plated for 48 h either on a 96-well plate pre-coated with three different concentrations of anti-CD3 (0.1 and 0.3 μg/ml) in the presence of IFNα (1,000 IU/ml) or IFNλ1 (100 ng/ml). (C, D, E, F, G, H) Released IFNγ (C), IL-10 (D), IL-22 (E), GM-CSF (F), TNFα (G), and IL-1β (H) were determined using Meso Scale Discovery assay. Data are representative of three independent experiments. (I) Total CD8+ and purified TEMRA, naïve, EM, and CM CD8+ T-cell populations were stimulated with IFNα (1,000 IU/ml) or IFNλ1 (100 ng/ml) for 15 min. Phosphorylated STAT1 (pY-STAT1), total STAT1, and actin protein were analyzed by Western blotting using total cellular extracts. A representative blot from two independent experiments is shown. (B, J) CD8+ T cells were treated with individual or combinations of IFNα (1,000 IU/ml), IFNλ1 (100 ng/ml), and IFNλ4 (100 ng/ml) for 48 h in the presence or absence of anti-CD3 mAbs stimulation and analyzed as described in (B). (A, D) Mean ± SEM from three independent experiments (n = 3 for A, D) are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 (paired t test).

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    Figure 5. Detection of CD3+/PD1+ cells in the liver of chronic hepatitis C patients.

    (A) Paraffin-embedded liver biopsies were stained using mAbs specific for CD3 and PD1 (A) Frequency of CD3+/PD1+ cells in the liver of chronic hepatitis C patients with ΔG and TT IFNλ4 genotype. (B, C) Representative bright-field images of CD3 and PD1 staining of liver sections of a patient of TT/ΔG genotype and (C) TT/TT genotype. Red, CD3 signal; brown, PD1 signal.

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    Figure 6. Phenotypic characterization and multidimensional analysis of patient liver biopsies.

    Intrahepatic lymphocytes (IHLs) were isolated from fresh liver biopsy tissue obtained from chronically hepatitis C virus–infected patients either carrying the IFNλ4 ΔG or TT allele. IHLs were subjected to multicolor FACS analysis. (A) t tests identified no significant differences (P > 0.05) in the percent of cells with a positive phenotype for any cell surface marker between patients with ΔG (n = 9) or TT (n = 4) IFNλ4 alleles. (B, C) tSNE dimensional reduction and clustering enabled identification of clusters enriched for either IFNλ4 allele (C) using a Poisson test (false discovery rate < 0.001; colored dots) while maintaining within-cluster representation of patients with each IFNλ4 allele (B). Cells not selected for further analysis are gray. (B, C, D) Hierarchical clustering of the median marker expression of selected clusters from (B) and (C), revealing three groups (1, 2, 3) of CD8+ clusters with opposing expression of exhaustion and senescence markers CD57, CD127, KLRG1, PD1, and 2B4.

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    Figure 7. Pseudotime ordering of CD8+, CD4+, and double-negative cell populations indicates differentiation state-dependent changes in exhaustion and senescence markers.

    IFNλ4 allele enriched clusters were embedded by diffusion mapping and temporally ordered based on the co-expression of CD57, KLRG1, PD1, and TIM3. Curves fitted to the temporally ordered cells indicate smoothed average of marker expression.

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    Figure 8. Distribution of cells across pseudotime.

    The proportion of cells is differentially distributed across pseudotime in a lineage-dependent manner. Total CD8+ TT cells (n = 14,081) are distributed late in the temporal ordering, whereas total CD8+ ΔG cells (n = 6,230) are distributed earlier. No difference was observed in the temporal distribution of CD8+ CD161+ TT or ΔG cells (n = 4,226 and 513, respectively). In contrast, an IFNλ4-dependent differential distribution of cells was observed in CD8+ CD161− TT and ΔG cells (n = 9,855 and 5,717, respectively). CD4+ TT and ΔG cells (n = 1,591 and 6,959, respectively) were temporally distributed similarly to CD8+ TT cells. Double-negative (DN) TT and ΔG cells (n = 6,722 and 437, respectively) were distributed similarly to CD8+ CD161+ cells.

Tables

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    Table 1.

    Characteristic of patients included in the study.

    NrGenderAge at biopsyrs815 (IFNL4)HCV genotypeLog10 (viral load)MetavirFACSImmunohistochemistry
    1f64ΔG/ΔG1b5.44A2/F1XX
    2m58ΔG/ΔG3a5.47A3/F3XX
    3m24ΔG/ΔG14.81A1/F0XX
    4m49ΔG/ΔG46.50A1/F1X
    5m61ΔG/ΔG1bn.aA2/F2X
    6m51ΔG/ΔG1b5.75A2/F4X
    7m40ΔG/ΔG3a5.66A1/F1X
    8f53ΔG/ΔG4a/c/d5.90A3/F3X
    9m48ΔG/ΔG4a/c/d6.03A2/F2X
    10m30ΔG/ΔG1a5.03A1/F1X
    11m24ΔG/ΔG1a5.26A1/F0X
    12m52TT/ΔG3a6.69A2/F2XX
    13m56TT/ΔG3a5.75A3/F4XX
    14m50TT/ΔG1a6.92A3/F2XX
    15f37TT/ΔG1an.aA1/F2XX
    16m46TT/ΔG4a/c/d5.06A3/F4XX
    17m45TT/ΔG1b5.25A1/F1XX
    18M15TT/ΔG1a5.44A1/F1X
    19f47TT/ΔG1a6.78A2/F4X
    20m45TT/ΔG1a6.21A2/F2X
    21m42TT/TT3a6.98A2/F1XX
    22f52TT/TT4n.aA2/F2XX
    23m77TT/TT1b5.92A2/F3XX
    24m61TT/TT1an.aA2/F1XX
    25m43TT/TT15.97A1/F1X
    26f45TT/TT3a5.06A1/F1X
    27f45TT/TT1b6.38A2/F1X
    28m53TT/TTn.a6.47A2/F2X
    29f52TT/TT1a6.09A2/F1X
    30m36TT/TT3a5.90A1/F1X
    31m43TT/TT1a3.60A1/F1X
    32m39TT/TT3a6.41A1/F1X
    33f41TT/TT1b6.44A2/F3X
    34m50TT/TT3a6.52A2/F1X
    35m53TT/TT3a6.52A3/F4X
    36m56TT/TT1a6.80A3/F3X
    37m45TT/TT3a5.87A2/F4X
    38f37TT/TT1a6.19A2/F3X
    39m53TT/TT46.12A1/F1X

Supplementary Materials

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  • Table S1 Reagents and antibodies.

  • Table S2 Primer sequences for quantitative RT-PCR.

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IFNl4 stimulates human CD8+ T cells
Mairene Coto-Llerena, Marco Lepore, Julian Spagnuolo, Daniela Di Blasi, Diego Calabrese, Aleksei Suslov, Glenn Bantug, Francois HT Duong, Luigi M Terracciano, Gennaro De Libero, Markus H Heim
Life Science Alliance Nov 2020, 4 (1) e201900612; DOI: 10.26508/lsa.201900612

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IFNl4 stimulates human CD8+ T cells
Mairene Coto-Llerena, Marco Lepore, Julian Spagnuolo, Daniela Di Blasi, Diego Calabrese, Aleksei Suslov, Glenn Bantug, Francois HT Duong, Luigi M Terracciano, Gennaro De Libero, Markus H Heim
Life Science Alliance Nov 2020, 4 (1) e201900612; DOI: 10.26508/lsa.201900612
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