Long-term T cell fitness and proliferation is driven by AMPK-dependent regulation of reactive oxygen species

  1. Anouk Lepez
  2. Tiphène Pirnay
  3. Sébastien Denanglaire
  4. David Perez-Morga
  5. Marjorie Vermeersch
  6. Oberdan Leo
  7. Fabienne Andris

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Abstract

The AMP-activated kinase (AMPK) is a major energy sensor metabolic enzyme that is activated early during T cell immune responses but its role in the generation of effector T cells is still controversial. Using both in vitro and in vivo models of T cell proliferation, we show herein that AMPK is dispensable for early TCR signaling and short-term proliferation but required for sustained long-term T cell proliferation and effector/memory T cell survival. In particular, AMPK promoted accumulation of effector/memory T cells in competitive homeostatic proliferation settings. Transplantation of AMPK-deficient hematopoïetic cells into allogeneic host recipients led to a reduced graft-versus-host disease, further bolstering a role for AMPK in the expansion and pathogenicity of effector T cells. Mechanistically, AMPK expression enhances the mitochondrial membrane potential of T cells, limits reactive oxygen species (ROS) production, and resolves ROS-mediated toxicity. Moreover, dampening ROS production alleviates the proliferative defect of AMPK-deficient T cells, therefore indicating a role for an AMPK-mediated ROS control of T cell fitness.

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  1. Version published to 10.1038/s41598-020-78715-2
  2. eLife

    ###Reviewer #3:

    The manuscript reexamines AMPK-deficiency in the T cell compartment using mixed bone marrow chimeras, to show that T cell cell expansion (and effector functioning) both in vitro and in vivo is compromised by AMPK deficiency, that this is despite any effect of this deficiency on early events during TCR signalling, and that ROS scavenging ameliorates these defects to some extent. While the data are interesting, they remain incremental at this point, since a role for AMPK in the functioning of the T cell lineage has been shown previously (including by the authors), as the authors cite. The potentially novel nuanced observations the authors report in the present manuscript are not accompanied by novel mechanistic insights as yet.

    The competitive bone marrow chimeras show the relative reduction of the AMPK-deficient genotype in the effector-memory T cell compartment, as would be predicted by previous literature. The more robust lack of AMPK-deficient T cells in the CXCR3-expressing subset and in gut lymphoid tissues is interesting, but no further mechanistic insights are offered into how AMPK specifically affects commitment to and or/survival in this compartment.

    Similarly, the authors show that, interestingly, AMPK-deficient T cells show much poorer homeostatic proliferation, in a number of models of such proliferation. The authors connect this deficit to increased mitochondrial turnover and to the generation of ROS in the absence of AMPK. Once again, these are potentially interesting data. However, the causal connectivity claimed between the mitochondrial phenotype and the homeostatic proliferation defect is not well supported by the data, which consists only of a partial pharmacological rescue by a ROS scavenger in vitro. Further, there are no data indicating any explanation for this apparent distinction between initial cognate activation-induced proliferation and homeostatic proliferation.

    Therefore, while this is a sound incremental manuscript of utility to the field, it does not as yet provide sufficient breadth of interest for a cross-disciplinary readership.

  3. eLife

    ###Reviewer #2:

    The manuscript by Anouk Lepez and colleagues examines the importance of AMPK in long term T cell fitness and proliferation and concludes that although AMPK is dispensable for early TCR signaling and short term proliferation it is required for sustained long-term T cell proliferation and effector/memory T cell survival. The authors demonstrate that AMPK aggravated the severity of graft vs host disease and mechanistically proposed that AMPK enhanced the mitochondrial membrane potential of T cells to limit ROS production and associated toxicity. As the authors acknowledge, previous work on AMPK has shown that its absence does not affect T cell proliferation, however earlier work has also established that absence of AMPK affects GVHD (Beezhold, K et al, Blood (2016) 128 (22): 806) and that AMPK maintains homeostasis through regulation of Mitochondrial ROS (Rabinovitch et al, Cell Rep 2017 Oct 3;21(1):1-9. Current work does not add any additional mechanistic insights to the already known functions of AMPK. Authors, however, have an interesting finding in the reduced population of gut lamina propria and intra-epithelial compartment but did not examine the outcomes of such defects.

    Major Concerns:

    1. AMPK was previously found to be dispensable for the generation of effector T cells (cited papers 15,16). Please expand on the reasons for differing results of this paper. Similarly, in vivo experiments have found AMPK-/- T cells to be largely immunocompetent (cited paper 17). The authors' focus seems to be on homeostatic expansion but it is not clear what the importance of the requirement of AMPK for homeostatic proliferation is. Additionally, if Lamina Propria and IEL compartments are most affected when AMPK is absent in T cells, what is its outcome on gut immunity? Authors fail to examine this.

    2. Much of the data presented in many of the figures is derived data presented as proportions or ratios of AMPK-KO to WT T cells.

    3. The GVHD data presented in figure 3 makes the point that absence of AMPK reduces the severity of GVHD. Is this due to defective cytokine production/defective division/defective survival of transferred cells? Moreover these findings were already published in Blood in 2016.

    4. The in vitro data do not substantially add to the author's point that homeostatic proliferation is defective in the absence of AMPK.

    5. With regards to mitochondrial fitness, this was demonstrated in fibroblasts in the paper published in Cell Reports in 2017. Although it is interesting that AMPK has conserved properties in fibroblasts and T cells, this is not a conceptual leap.

    6. The final figure in the paper has major caveats.(Figure 7H,I) Rescue of T cell proliferation in the presence of ROS scavenger. This experiment should be extended to show if the ROS scavenger rescues other defects like priming in the IL7+DC condition, IFNg production, Cxcr3 expression, GVHD pathogenicity.

  4. eLife

    ###Reviewer #1:

    The study by Lepez et al, investigates the requirement for the metabolic sensor AMPK in the T-cell lineage. The analysis builds on genetic ablation that results in functional deficit of AMPK in the lineage to assess cellular response in homeostatic conditions, in response to antigen and in an in vitro cell culture system. The experiments are well executed and generally carefully controlled. The cell culture system allows the interrogation of mechanistic underpinnings at the cellular level in vitro and can be coupled with the validation of predictions in vivo.

    AMPK regulation of cellular ROS homeostasis is one of the main outcomes reported in this work. However, the data supporting the latter are somewhat preliminary. Overall, in my view this work offers some advance on current knowledge but sufficient mechanistic insight is lacking at this juncture.

    Concerns:

    The experiments connecting AMPK signaling and ROS homeostasis are interesting but the evidence that ROS toxicity is inhibited by AMPK is largely correlative.

    Nutrient sensing modalities are undoubtedly affected in AMPK deficient cells and the implications of these for ROS homeostasis are not evident in the analysis or discussion. For instance, AMPK control of redox regulation by the maintenance of cellular NADPH (Chandel's group) has been described and is a potential target that could be assessed in T-cells.

    In Figure 7D, the WT cells show 70% mortality and the KO ~90% with differences maintained in the dose response analysis (S11). An important control would be the demonstration that (WT) cells are protected following treatment with an anti-oxidant/ scavenger. Further, does modulation of AMPK in WT cells - activation or inhibition - replicate the results seen with WT and KO cells?

    The inclusion of another ROS perturbation such as mitochondria-targeted MitoParaquat will strengthen the assessment of differential susceptibilities in the survival/ ROS toxicity assays.

    Given the rich literature on ROS regulation of T-cell function, the identity and characterisation of the ROS component[s] regulated by AMPK is necessary. This is relevant, as not only are there several sources of cellular ROS, their requirements are thought to be distinct in T-cell subsets.

    Finally, the data presented do not account for the differential requirement of AMPK in T-cell subsets, which appears to be a major objective of the study. The conclusions of the study would be strengthened with an effort that establishes the identity of the ROS component and its interaction or regulation by AMPK.

  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 3 of the manuscript.

    ###Summary:

    The manuscript examines the importance of AMPK in long term T cell fitness and proliferation and concludes that although AMPK is dispensable for early TCR signaling and short term proliferation, it is required for sustained long-term T cell proliferation and effector/memory T cell survival. The authors demonstrate that AMPK aggravates the severity of graft vs host disease and propose that AMPK enhances mitochondrial membrane potential in T cells to limit ROS production and associated toxicity and that ROS scavenging ameliorates these defects to an extent. However, causal connectivity claimed between the mitochondrial phenotype and the homeostatic proliferation defect is not established. The competitive bone marrow chimeras show the relative reduction of the AMPK-deficient genotype in the effector-memory T cell compartment, as predicted by previous literature. The more robust lack of AMPK-deficient T cells in the CXCR3-expressing subset and in gut lymphoid tissues is interesting, but no further mechanistic insights are offered into how AMPK specifically affects commitment to and or/survival in this compartment. Previous work on AMPK has shown that its absence does not affect T cell proliferation and also established that absence of AMPK affects GVHD (Beezhold, K et al, Blood (2016) 128 (22): 806) and that AMPK maintains homeostasis through regulation of Mitochondrial ROS (Rabinovitch et al, Cell Rep 2017 Oct 3;21(1):1-9. AMPK regulation of mitochondrial fitness, is previously demonstrated in fibroblasts (Cell Reports in 2017), and sufficient insight in constraining T-cell function is not provided. While the experiments are well executed and carefully controlled with several potentially interesting new observations, the study does not provide a sufficient advance to current knowledge or offer novel mechanistic insights into AMPK signalling in the mature T-cell compartment.

  6. Version published to 10.1101/2020.03.06.978791 on bioRxiv