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Page 14 Graner. Extracell Vesicles Circ Nucleic Acids 2020;1:3-19 I http://dx.doi.org/10.20517/evcna.2020.08

The final Virus session speaker was Daniel Pinto (Walter Reed Army Institute of Research, US) who told us
of the role of EV in transmission and spread of the HTLV-1 virus. This virus can cause adult T cell leukemia/
lymphoma, or a neurocognitive disorder known as HAM/TSP. A prevalent infectious motif is cell-cell
contact. During EV preparations using density gradient centrifugation, they noted that viral components
could end up in EV fractions that were separated from the virus itself, and from EVs containing virions.
This begged the question, which of these are infectious? Curiously, none of them were separately infectious,
nor were they infectious if all were combined. However, the treated cells displayed clustering/aggregation
phenotypes similar to that seen in bona fide HTLV-1 infection. This perhaps enables the cell-cell contact
that is necessary for viral spread. EVs from infected cells enhanced viral infection (measured by viral RNA
load in recipient cells), and this could be blocked by antibodies against cell adhesion molecules CD45 and
ICAM1 or by treating cells with siRNAs. In an animal model, injecting EVs prior to an infection with HTLV-
1 increased the viral load in the blood and numerous target tissues of the mice. Differential centrifugation
yielding 2K × g, 10K × g, and 100K × g fractions showed that most of the detrimental effects (and most of
the viral components) were in the 2K and 10K fractions, along with differential cytokines (and surface vs.
internal localizations). Thus, different EV subtypes may have unique effects on cells that ultimately lead to
more efficient viral infection and spread.

ASEMV2020 Day 4 started with the beneficial uses and therapeutic effects of EVs, hosted by Saumya Das
(Massachusetts General Hospital, US) and Kendall van Keuren-Jensen (TGEN, US). Robert Blelloch (UCSF,
US) opened up the session with his group’s studies showing how exosomes suppress anti-tumor immune
responses. Unsurprisingly, this focused on the immune checkpoint inhibitor PDL1, part of the PD1/PDL1
checkpoint axis. T cell activation can result in expression of PD1 as a means of immune control; cells
expressing its ligand PDL1 (often other immune cells, but also cancer cells) can suppress T cell activity as
a control against autoimmunity. For instance, as activated T cells infiltrate tumors, tumor PDL1 could bind
T cell PD1 to deactivate the T cell effector function, and this is the standard paradigm for use of immune
checkpoint inhibitors. Robert’s lab believes there may be an important role of PDL1 far earlier in the priming
phase of T cell activation. PDL1 is found on tumor-derived exosomes, but not all tumors may localize
it there, suggesting that this may be an active process. Their additional data indicate that the vesicles are
specifically exosomes and not other EV types. They employed a mouse prostate cancer model (TRAMP-C2)
that is resistant to anti-PDL1 antibody (checkpoint inhibitor) therapy. However, the loss of PDL1 or reduced
exosome formation (RAB27A or SMPD3 knockout) dramatically reduced tumorigenicity, implying PDL1 as
critical to tumor progression; this also involved the immune system via T cell activation. Adding back PDL1-
containing exosomes reversed the effects. This was particularly notable in draining lymph nodes, suggesting
that T cell priming may be key. PDL1 on plasma exosomes is correlated with a lack of response to anti-PD1
therapy in patients across several cancer types. Their model invokes a tumor cell surface pool of PDL1 (more
sensitive to checkpoint inhibition) and an exosome pool of PDL1, which impacts T cell priming in lymph
nodes that remains resistant to such therapies.

Ryan Reshke (University of Ottawa, Canada) introduced a method for loading and delivery of siRNA in
EVs. While siRNAs are quite effective, the delivery systems beyond liver targets (liver serves as a depot
for lipoparticle delivery) can lead to systemic (and liver) toxicity, so better delivery means are needed.
The number of small RNAs in EVs remains controversial, but appears to be limited, suggesting a need for
efficient loading to improve therapeutic capacity. One novel concept involves the use of pre-miR-451, which
is a uniquely short pre-miR hairpin that is too short for processing by DICER and is sliced by AGO2, and is
highly enriched in EVs. The short hairpin is responsible for this lack of DICER processing and allows pre-
miR-451 preferential EV loading. Other RNAs, such as siRNAs, can also be efficiently loaded, provided
they have the miR-451 hairpin, allowing for their enrichment and packaging. Ryan’s group demonstrated
this in a GFP silencing model, showing that in comparison to liposomal delivery, they could use 10-100-
fold less siRNA to achieve silencing. In a translational setting of a murine model, they could knock down