Predicted structure of CD73 via AlphaFold.

Imagine the 2004 USA Soccer Team without Mia Hamm. Certainly, there would have been excellence, but a gold medal? Maybe not? Now imagine Hamm taking on eleven players of Team Brazil, completely solo. Absurd! In a similar way, bioscientists often like to ponder, hypothesize, and then test whether a key variable is “sufficient” and/or “necessary” for an impactful result.

Like a soccer team, the biological activity of extracellular vesicles (EVs) or exosomes from mesenchymal stem/stromal cells (MSCs) seems to be the combined effort of numerous molecular players. However, investigators today still search for the constituents that exert the most zing across the therapeutic exosome field. Which ones are lynchpins (~necessary) and which ones are good enough (~sufficient)? We’ve heard a lot about CD73 (NT5E; Ecto-5’-nucleotidase). This blog will evaluate the recent rationale to enrich CD73’s “star power” within Team Exosome.

Figure 1: CD73 is beginning to appear on more recent ISEV Annual Meeting Abstracts

CD73 is one of three canonical markers (along with CD90 and CD105) that positively define multipotent mesenchymal stromal cells according to the ISCT, [1] where it localizes to the plasma membrane via its GPI anchor and converts extracellular cAMP into adenosine (ADO; expertly reviewed by Aldcedo, et. al 2021). [2] As a metabolite of cAMP, ADO is downstream of extracellular ATP via CD39’s Ecto-ATPDase activity. ADO acts as a signaling messenger to engage heterotrimeric G protein coupled receptors like ADORA2 and trigger pleiotropic effects. In addition to cell surfaces, CD73 may be found highly expressed on MSCs’ secreted EVs and/or exosomes. MSCs’ therapeutic activity has been largely attributed to their secretomes, [3, 4] of which exosomes may be a key ingredient. [5, 6, 7, 8] The presence of enzymatically active CD73 has recently been proposed to serve as an activity-coupled biomarker to gauge the potency of exosome/EV preparations. [9, 10, 11, 12]

Figure 2: CD73 is part of a cascade to convert extracellular ATP into AMP and adenosine.

Why does the cell bother with CD73 in nature? The answer is of course complex. It’s known that sudden influx of extracellular ATP into a local milieu is read as a chemoattractant damage  signal [13, 14] that flags down cellular first responders of the innate immune system: macrophages [11, 15] and neutrophils. [16] However, excessive inflammation can also propagate injury beyond the initial benefit of anti-infection protection (e.g., cytokine storm and toxic shock). [17, 18] Degradation of ATP by CD39 in concert with CD73 to yield ADO may thus help dissipate the signal transduction of inflammasomes.

Transmission of active signaling of ADO via its adenosine receptors (ARs) may also matter. While A₂R (ADORA2A) and A₃R may decrease inflammation by repolarizing macrophages [19] and T cells, [20] ADO can also act through A₁R to decrease oxygen demand and A_2BR to promote angiogenesis. [2, 21] Downstream of these ARs, the ADO signal may be potentiated in still other distinct ways, depending on the recipient cell type and the equilibrium of compatible G_s, G_i, and beta subunits within it. With CD73 expression “downstream” from HIF-1a and hypoxia, its role may be rheostatic in nature such that it helps dial back from “DEFCON 1” and gradually rebalance the inflamed microenvironment into a normal, healed tissue. Given ADO’s stimulation of wortmannin-sensitive signaling “upstream” of AKT/PKB, [11, 22] a possible mechanism for its survival benefit vs. cellular insults may be a positive feedback loop in favor of increased HIF-1a and suppression of apoptotic effectors.

Figure 3: CD73 elicits downstream effects by (i), removal of extracellular ATP and (ii), contribution to a pleotropic signaling cascade involving cell-specific anti-inflammatory (↓reduced NF-kappaB) and anti-apoptotic (↑elevated AKT/PKB) activities. Schematic inspired by Figure 1 of de Leve, et al (2019) [36]

Adding to CD73’s complexity, we know that homozygous knockout mice deficient in CD73 are viable and fertile. These mice do, however, exhibit enhanced graft-versus-host disease, [23] exacerbated organ injury in response to ischemic hypoxia, [18] and other subtle defects that show up with some prodding. [24] Genetic disruptions of CD73’s upstream (CD39) and downstream (A_2BR) mediators show similar subtle problems. Surprisingly, mouse CD73 -/- phenotypes manifest much more mildly compared with pathologies reported in association with human CD73 polymorphisms. In humans, these may include aneurysm formation, atherosclerosis, joint calcification and arthritis, and heart failure. [24]

Exosomes enriched in CD73 are reported to ameliorate various injury insult models, and many efforts are made to show at least partial dependency of the effect on CD73/adenosine activity. [6, 11, 21, 22, 25, 26] Nevertheless, other studies compellingly argue that multiple cell types must be working concertedly for CD73+ exosomes to make a difference in vivo. (The right context is essential?) For example, Kerkela et al report (2016) [27] that there must be copresence of CD39-expressing T cells to contribute AMP such that MSC-EVs can convert it into a potent ADO immunosuppressive signal. Do, et al (2021) likewise observed [28] that the cooperative CD39/CD73 signaling between MSCs and Tregs is necessary for MSCs’ mitochondrial transfer to Tregs, thereby sustaining their immunosuppressive function. Perplexingly, it was recently noted that exosomes/EVs can induce immunomodulatory capacities irrespective of CD73 activity, [29] and that an mdMLR (multi-donor Mixed Lymphocyte Reaction) assay would thus be better suited as a batch-to-batch indicator of exosome potency in vivo.

Therapeutic effects of EVs/exosomes might be varied and can encompass bioactivities of anti-apoptosis (survival), immune modulation, neovascularization and angiogeneis, and cell motility. The molecular basis for this broad scope thus almost certainly involves players unrelated to adenosine signaling cascades. [6]  Other important molecular cargoes that travel either “on” or “inside” EVs may include HSP70 for blockade of cell death, [30] PD-L1 for immune down-modulation, [31] transcription factors for angiogenesis, [32] matrix metalloproteinases (MMPs) for migration, [33] and possibly miRNAs for miscellaneous cytoprotective results, [34, 35] to name a few.

Thus, like an Olympic gold-winning soccer club, there are surely many team members on the “EV squad” who can more than pull their weight when acting together. However, there will also be instances where only one key player can be decisive—and where her absence would be sorely missed. If CD73 is a minimally definitive constituent of the MSC plasma membrane, [1] perhaps this molecule or its bioactivity might also serve as an assayable exemplar quality metric for MSC-EVs…something that is “necessary but not sufficient?”

References

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