01.2018 | THE SCIENTIST 57G
iven the roles of CD4+ and CD8+ T cells
as searchers and destroyers, it is no
surprise that much of the chimeric antigen
receptor (CAR)-T cell development centers on
“conventional” T cells. In contrast, regulatory T cells
(Tregs) reside more in the background. However, as
stewards of immune homeostasis, their role is no
less important.
Interest in genetically engineered Tregs, including
CAR-Tregs, for therapeutic use is growing.
Leonardo Ferreira is a postdoctoral fellow at the
University of California, San Francisco, where
he studies the effects of cell therapies on the
immune system. We spoke to him about his
work with CAR-Tregs.
The functional differences between CAR-Tregs
and “conventional” CAR-T cells
While conventional CAR-T cell therapies are
designed to kill specific cells, CAR-Tregs are
designed to prevent cell killing. “Most T cells
are dedicated to defending our body from
pathogenic invaders and cancer. They recognize
peptides derived from viruses, bacteria, or
proteins mutated in cancer and target the cells
presenting those peptides for destruction. Tregs
are dedicated to suppressing immune responses
and maintaining homeostasis of the immune
system; they are not dedicated to searching and
destroying,” Ferreira said. As such, CAR-Tregs can
be designed to protect and maintain the integrity
of specific targets. For example, they can be
keyed to islet-specific antigens in order to protect
the pancreatic islets from autoimmune attacks.
Utilizing CRISPR to edit cell therapy products
The added precision from technologies like CRISPR
has been very important. “CRISPR allows one to
integrate the CAR gene in the native T cell receptor
(TCR) locus. This means that, unlike lentiviral and
retroviral transduction, which introduces multiple
copies of the CAR gene per cell at random places in
the genome driven by a non-physiological promoter,
a CAR knocked in the TCR locus is under the native
gene regulation of the TCR and is present at a
controlled copy number. This makes a big difference
in terms of the uniformity of CAR expression, T cell
expansion, and function,” Ferreira said.
Because CRISPR can produce off-target effects,
Ferreira uses a variety of approaches, both in vitro
and in vivo, to characterize CRISPR-edited cells.
“The first step is always to confirm successful
deletion or knock-in. I always start with functional
in vitro assays before moving on to in vivo studies,”
he said. “In vivo studies always require more cells
and have longer timelines, but they are the ultimate
gold-standard.”Using single cell analyses in CAR-Treg
development
After confirming successful gene editing, the next
step is to investigate cellular heterogeneity, which
can dramatically impact therapeutic efficacy
and consistency. “Single cell analyses are key to
developing and validating any kind of cell therapy,”
said Ferreira. “For CAR Tregs in particular, it is key to
perform single cell techniques to accurately trace
Treg stability, cytokine production, and receptor
expression, as Tregs are known to be heterogeneous.”“As an immunologist, I tend to favor protein
over RNA,” said Ferreira. “Our objects of interest
at the molecular level are often proteins, be it
surface receptors, cytokines, or lineage-defining
transcription factors. Single cell proteomics is thus
key to speeding up [our understanding of] both
the basic biology and the clinical translation of
CAR-Tregs.”In his research, Ferreira uses single cell proteomics
data to characterize the impact of CAR-mediated
signaling on CAR-Treg survival, stability, and function,
as well as to dissect differences in cellular signaling
between Tregs and conventional T cells. “Using
IsoPlexis, which provides single-cell cytokine data,
we found very clear patterns showing differences not
just between different CAR signaling architectures
in Tregs, but also between Tregs and conventional T
cells activated via the same CAR. This tells us that
all the large number of studies on the impact of
CAR signaling on T cell function do not necessarily
translate to CAR-Tregs.”Unlocking the potential of CAR-Tregs
When it comes to potential future uses for CAR-Tregs,
Ferreira is full of ideas. “For CAR-Tregs specifically,
I believe that the first application will be in graft-versus-host disease and organ transplantation. We
know [mismatches in human leukocyte antigens
(HLAs)] are responsible for transplant rejection. So,
one can engineer CAR-Tregs specifically targeting
an HLA allele present in the transplanted organ but
not in the recipient and create tolerance specifically
toward that foreign organ.”CAR-Tregs also hold more flexibility than conventional
cell therapies when it comes to delivery timing. “For
autoimmune disease, one will only deliver CAR-Tregs
after the disease has been detected.” Ferreira began,
“but in organ transplantation, one could, in theory,
deliver CAR-Tregs before the organ is transplanted.
This can re-educate the patient’s immune system,
creating immune tolerance before the organ goes in.”Finally, Tregs may go beyond protection to aid with
repair. “Tregs secrete tissue repair molecules,” said
Ferreira. “There are currently clinical trials using
Tregs against amyotrophic lateral sclerosis (ALS), a
neurodegenerative disease affecting motor neurons.
In the future, I predict we will be able to use CAR-
Tregs not only to suppress autoimmunity and prevent
organ rejection, but also to treat inflammation and
aid with wound healing and regeneration.”How single cell proteomics data can drive CAR-Treg-based
therapies—an interview with Leonardo Ferreira
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