Peripheral natural killer cells in chronic hepatitis B patients display multiple molecular features of T cell exhaustion

  1. Marie Marotel
  2. Marine Villard
  3. Annabelle Drouillard
  4. Issam Tout
  5. Laurie Besson
  6. Omran Allatif
  7. Marine Pujol
  8. Yamila Rocca
  9. Michelle Ainouze
  10. Guillaume Roblot
  11. Sébastien Viel
  12. Melissa Gomez
  13. Veronique Loustaud
  14. Sophie Alain
  15. David Durantel
  16. Thierry Walzer
  17. Uzma Hasan
  18. Antoine Marçais

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Abstract

Antiviral effectors such as natural killer (NK) cells have impaired functions in chronic hepatitis B (CHB) patients. The molecular mechanism responsible for this dysfunction remains poorly characterised. We show that decreased cytokine production capacity of peripheral NK cells from CHB patients was associated with reduced expression of NKp30 and CD16, and defective mTOR pathway activity. Transcriptome analysis of patients NK cells revealed an enrichment for transcripts expressed in exhausted T cells suggesting that NK cell dysfunction and T cell exhaustion employ common mechanisms. In particular, the transcription factor TOX and several of its targets were over-expressed in NK cells of CHB patients. This signature was predicted to be dependent on the calcium-associated transcription factor NFAT. Stimulation of the calcium-dependent pathway recapitulated features of NK cells from CHB patients. Thus, deregulated calcium signalling could be a central event in both T cell exhaustion and NK cell dysfunction occurring during chronic infections.

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  1. Version published to 10.7554/elife.60095 on eLife
  2. eLife

    ###Reviewer #3:

    General comment: Marotel et al present a detailed characterization of the peripheral NK cells phenotype and function in patients with chronic hepatitis B. The cohorts are well designed and used in an appropriate way that makes the conclusions interesting. The manuscript is well written and the figures easy to navigate. Supplementary information is relevant. Interesting parallels with T cell exhaustion mechanisms are made. Weakness might relate to relative lack of selective/precise analysis of subsets (bright vs dim, and maturation stratification) for example in RNAseq, calcium experiments, phosflow and mitochondria analysis.

    Major comment 1: Figure 2 - As it seems, results display total NK cells which makes sometimes differences difficult to interpret, if possible, please provide in supplement at least phenotype of Bright vs DIM NKG2A+ vs DIM NKG2A-

    Major comment 2: Figure 3 - Phosflow as well as mitochondrial analysis are always difficult to perform due to technical specificities, efficient detection of epitopes, atypical fluorescence leakages or analysis of small shift differences. For both techniques, in order to highlight the quality of the datasets, please provide representative histograms as well as positive and negative controls, and gating strategy to further convince the readers.

    Major comment 3: Figure 6 - Regarding calcium related mecanisms - Mechanistic investigations might be completed to support the current statements such as highlighted in the abstract "when stimulating Ca2+-dependent pathway in isolation, we recapitulated the dysfunctional phenotype" (based on n=3, total NK cells from Healthy individuals). Cells from patients might be investigated. Also, beside the ionomycine treatment performed, calcium flux experiment in sorted cells based on the phenotypes described would have been elegant.

    Major comment 4: A large part of the manuscript relates to TOX and its involvement in exhaustion. However, a recent article (Sekine et al, Science immunology 2020) demonstrated that TOX is expressed by most circulating effector memory CD8+ T cell subsets and not exclusively linked to exhaustion.

    This is an important piece of work where such data might be integrated and invite reinterpretation of results and conclusions.

  3. eLife

    ###Reviewer #2:

    In this manuscript, Marcais laboratory defines the molecular basis of NK cell dysfunction in patients with Hepatitis B. They use NK cells derived from the peripheral blood of Hep-B patients and healthy cohorts. The key finding is that the NK cells derived from the Hep-B patients were able to mediate cytotoxicity while they were significantly impaired to producing inflammatory cytokines, including IFN-g. Employing phenotypic, functional, and transcriptomic analyses, authors conclude that NFAT-mediated Ca2+-dependent cellular exhaustion as the potential mechanism results in dysfunctional peripheral NK cells. This study provides newer insights into the molecular mechanisms associated with NK cell dysfunction. However, addressing the following concerns can vastly improve the contribution of this work.

    1. Given significant differences between the published characteristics of T cell exhaustion and authors' findings in this current work, it is not fair to call them similar. This applies to both phenotypic and functional changes. For example, in multiple viral infection models, the decrease in IFN-g production occurs in a step-wise manner during the progress of T cell exhaustion. In the current work, the authors show a significant and complete reduction of IFN-g production in all the patients analyzed. Importantly, the number of T cells that produce multiple cytokines such as IFN-g and TNF-a are reduced. However, it does not appear that these two cytokines are concurrently reduced in Hep-B patients. Another difference is that the NK cells from Hep-B patients are able to mediate normal cytotoxicity against K562 cells while the exhausted T cells are impaired in mediating this effector function. While it may be true that the NK cells in the Hep-B patients undergoing exhaustion, it may not be fair to call this phenomenon as that of T cells.

    2. The link that authors are providing between mTOR-S6-NK cell exhaustion is not clear. The reduction in the phosphorylation of AKT is significant; but, moderate. Is this physiologically relevant? Does the alternate pathway mediated by PIM kinases is the one primarily affected in the NK cells from the Heo-B patients?

    3. Apart from NFAT, T-bet, BATF, EOMES, FOXO1, BLIMP1, and IRF4 have been implicated in playing a significant role in causing T cell exhaustion. What are the reasons that the gene signatures representing these transcription factors did not come through from the RNA sequencing analyses?

    4. It is not clear how treating with a higher concentration of ionomycin can mimic NK cell exhaustion that occurs over a period of months or years. Theoretically, it cannot be a transient over-flux of calcium that initiates the expression of TOX and leading to NK cell exhaustion. NFAT/Calcineurin could play a role in the formation of NK cell exhaustion. However, the over-activation of NK cells from healthy control does not prove that this mechanism is the cause of the pathological outcome.

  4. eLife

    ###Reviewer #1:

    Marotel et al. study the mechanisms of NK cell exhaustion in patients with chronic hepatitis B infection (CHB). They first confirm several previous findings, such as reduction of IFNg production by NK cells accompanied by a change in phenotype in CHB patients. Furthermore, they show that mTOR activation is impaired in CD56bright NK cells upon IL-15 stimulation, and at the same time total NK cells do not show differences in selected metabolic parameters. They also performed RNAseq analysis which indicated transcriptional similarities of CHB NK cells and exhausted CD8+ T cells. In line with RNAseq, CHB NK cells showed increased expression of TOX transcription factor and inhibitory receptor LAG3 in CHB NK cells. The authors suggest that this is due to NFAT signaling, and show that NK cells have reduced ability to produce IFNg following incubation with target cells if they were previously stimulated with ionomycin overnight to support their hypothesis of NFAT involvement.

    In conclusion, while presented observations are interesting and relevant, they are still preliminary and largely descriptive. In addition, conclusions are not fully supported by the data.

    1. Figure 3. The authors focus on CD56bright NK cells when measuring mTOR activation, as CD56bright NK cells are more responsive to IL-15. They show that in HBV patients CD56bright NK cells have impaired response to mTOR activation. They correlate this finding with several metabolic parameters in total NK cells. Since CD56bright NK cells represent only a small fraction of NK cells it is not clear why the metabolic parameters were not analyzed only on CD56bright population as well, or vice versa, why the total NK cells were not compared in both cases (mTOR activation and metabolic characteristics). At the current state, no conclusion can be reached by comparing these two sets of data. Also, it is not clear if cells that have reduced ability to activate mTOR upon IL-15 stimulation contribute to other observations presented, e.g. if this finding would explain reduced NK cell ability to produce IFNg, changes in NK phenotype or transcriptome.

    2. Several metabolic parameters are studied, however, it is not clear how they were selected as there are many other metabolic processes involved in NK cell response which could be important and deregulated in CHB. In addition, only basal metabolic state was analyzed, but it remains unclear if CHB NK cells show the same metabolic characteristics upon activation.

    3. Figure 5 - isotype controls are missing in all histograms. The authors state in the text 'Increased TOX expression was seen mainly in the CD56dim subset in CHB patients.', however, they do not provide data for this statement. As mentioned previously, the effects of CHB on NK mTOR signaling are the highest in the CD56bright population, so it is not clear how these data do relate one to each other.

    4. The authors provide evidence that expression of transcription factor TOX is increased and T-bet expression is reduced to support the transcriptome data on the similarity of CHB NK cells and exhausted CD8+ T cells. However, they do not provide the evidence on the co-expression of these transcription factors, and if their changed expression directly correlates with reduced functional properties of NK cells, e.g. if NK cells having high TOX and low T-bet will produce less IFNg.

    5. To address their hypothesis on NFAT involvement in NK cell exhaustion and TOX expression the authors stimulate NK cells in vitro with ionomycin and show that pretreatment with ionomycin renders NK cells hyporesponsive. They titrate the effect of ionomycin and find an ionomycin concentration which is inducing a reduction of IFNg response without affecting degranulation. While the reduction of IFNg response in this experiment is observed as in chronic HBV infection, this model should be validated before making any claims. For example, the phenotype and transcription profile of the ionomycin treated cells should be analyzed, as well as the expression of transcription factors. A similar experiment has been published previously, so the novelty is minor without additional experiments addressing above mentioned issues.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

  6. Version published to 10.1101/2020.06.16.154419 on bioRxiv