Preprint Club
A cross-institutional Journal Club Initiative

Keywords

●  Pannexin-1

●  Antigen-specific CD8 T cells responses

●  Metabolite sensing

Main Findings

After antigen priming, CD8+ T cells activate signalling pathways, leading to effector function and clonal expansion, further differentiating into memory precursors (MP) or terminal effectors (TE). This process is regulated by a network of supporting cells and extracellular signals, including interactions with innate immune cells and cytokine sensing. In addition, metabolites released from dead cells or via transmembrane channels could also play a role. Nevertheless, the exact origins of these metabolites and their impact on intracellular pathways of CD8+ T cells remain unknown. In this preprint, Vardam-Kaur et. al, focused on Pannexin-1 (Panx1), a metabolite transporter, that promotes ATP export and is expressed in T cells and other immune cells. The authors sought to investigate the in vivo function of Panx1 in regulating the generation of antigen-specific CD8+ T cells using virus infection murine models.

The preprint reports the following main findings:

1. Through Panx1, CD8 T cells release multiple metabolites for their own sensing. Ablation of Panx1, leads to defective proliferation of effector CD8+ T upon antigen stimulation and apoptosis of long-lived memory CD8+ T cells. Therefore, authors describe Panx1 is a” master transporter” of metabolites essential for antigen-specific CD8+ T cell.

2. Panx1 promotes antigen-specific CD8+ T cell activation, proliferation and survival by exerting an opposing control of memory and effector CD8+ T cell metabolisms’.

3. During effector phase, Panx1 induces effector CD8+ T cells expansion and function by release and gathering of lactate in the extracellular space, which originates from enhanced glycolytic pathways. Extracellular lactate is further recycled into the TCA cycle leading to the biosynthesis of phosphatidyl inositol (PI), essential for lipid raft, immunological synapse generation and MAPK-pathway sustained activation.

4. In contrast, the authors showed that, in the course of memory phase, memory CD8+ T cells survival is mainly driven by Panx1-released extracellular ATP (eATP). This mechanism is dependent on P2RX7 sensing of eATP, which will further induce mitochondrial function and upregulation of pro-survival genes.

3.  Finally, the authors show the importance of Panx1 CD8+ T cell expression for melanoma tumor control.

In conclusion, the authors reported the importance of Panx1 in the establishment, function and survival of antigen specific effector and memory CD8 T cells, including Panx1 opposite control of effector (Lactate) and memory (eATP) CD8+ T cell metabolism.

Limitations

1.  The use of CD4-Cre Panx1fl/fl will lead to KO of Panx1 in CD8+ T cells as well as in CD4+ T cells. It would be interesting to understand if the differentiation and function of CD4 T cells are influenced by Panx1-mediated secretion of ATP and lactate. Also, the authors claim that CD8 T cells sense their own secreted metabolites via Panx1. It is unclear what experiments were performed to support this statement. It would be interesting to understand if it is indeed an autocrine mechanism or if also bystander CD8 and/or CD4 T cells influence the differentiation and function of CD8 T cells through the secretion of ATP and lactate via Panx1.

2.  The authors employed an in vitro model of CD8+ T cell activation and differentiation by using IL15 or IL2 to recapitulate the memory-like or effector-like CD8 T cells and performed RNA-seq, flow cytometry, efflux analysis and GC-MS metabolomics for extracellular and intracellular metabolites. While these models provide important mechanistic insights, they may not fully recapitulate the complexity and dynamics of the immune response in vivo. Although the authors confirm this data with in vivo experiments, it is advised to sort effector and memory CD8+ T cells from their CD4-Cre Panx1fl/fl LCMV infected mice to perform downstream analyses in order to better recapitulate the in vivo context, instead of employing an in vitrostimulation using IL-2 or IL-15.

3.  In their melanoma tumor model, it is importance to study how Panx1 deficiency in CD8+ T cells shapes the tumor microenvironment. Moreover, for translational applicability, it is important to understand if Panx1 expression can be downregulated in CD8+ T cells in the tumor microenvironment. In addition, does the tumor microenvironment reduce Lactate and eATP levels so CD8+ effector and memory T cells are not able to persist? Moreover, to strengthen their tumor data, authors could employ in vitro co-cultures of T cell vs Tumor and explore the killing and degranulation capacities of Panx1 KO CD8+ T cells vs. WT.

4.  Although the study includes animal models to investigate the role of Panx1 in CD8+ T cell responses, the specific findings may not directly translate to human CD8+ T cell biology. Further studies are necessary to validate the relevance of these findings in human immune responses.

Significance/Novelty

1.  The authors have made significant discoveries regarding the role of Panx1 in CD8+ T cell function. While previous research has explored the involvement of Panx1 as an ATP transporter in T cells and other immune cells, this study reveals a novel aspect of Panx1's function; Panx1 is not only an ATP transporter but also a transporter of other metabolites, transforming it into a "master transporter." Nevertheless, although the study mainly focuses on the role of extracellular ATP and lactate as metabolites regulated by Panx1, there may be other metabolites that are influenced by Panx1-mediated export and have potential implications for CD8+ T cell function. Therefore, this study opens new possibilities of exploring the export of additional metabolites which could provide a more comprehensive understanding of the metabolic regulation in CD8+ T cells. For instance, as the authors mention, future studies could explore whether there are specific metabolites that are selectively exported in different dynamics between effector and memory CD8+ T cells.

2.  Secondly, the authors have addressed an essential question regarding how memory effector CD8 T cells can become long-lived and persist even in the absence of a pathogen. In this case, this preprint shows how memory CD8+ T cells release ATP that sustains their survival through Panx1

Finally, by unraveling the role of Panx1 in CD8+ T cell function, this study provides important insights into the fundamental mechanisms of CD8+ T cell metabolism and function. This can aid to improve immunity in various diseases or to develop potential therapeutic applications. For instance, by using B16.GP33 melanoma tumor bearing mice and adoptive transfer of WT or Panx-1 KO P14 GP33-antigen specific CD8+ T cells, the authors show the relevance of Panx1 expression in CD8+ T cells for melanoma tumor control. Their findings lay the foundation to exploit clinical interventions and how to apply them into generating more effective immunotherapies.

Credit

Reviewed by Mireia Cruz De los Santos as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interests in relation to their involvement in the review.