Isoplexis_July2021_AcceleratingDevelopmentCurativeMedicines - 11

ACCELERATING THE DEVELOPMENT OF CURATIVE MEDICINES WITH FUNCTIONAL IMMUNE PROFILING
CD8+, but not CD4+ TEM
expansion. Additionally,
when ECs were treated with IL-1Ra before co-culture
with T cells, the stimulatory effects of PRA on CD4+
and CD8+ TEM
proliferation but had less effect than
anti-IL-15 on T cell polyfunctionality. IsoPlexis'
identification of these treatments enables the
suppression of a potentially significant driver of
mixed allograft rejection.
IsoPlexis' highly multiplexed functional single-cell
cells identified
proteomics analysis of allogeneic TEM
polyfunctional responder subsets that were
upregulated by complement and alloantibodyactivated
ECs, driving alloimmune rejection. The
significance of polyfunctional T cell subsets is
supported by research not only in alloimmunity, but
also in inflammation, cancer, infectious disease, and
beyond. These cells which are capable of co-producing
multiple cytokines can be key drivers of
disease pathogenesis as well as anti-tumor response
and are uniquely revealed by IsoPlexis' functional
proteomics. While methods such as single-cell
RNA-seq provide insight into gene expression, the
connection between gene expression and function
is tenuous. Functional single-cell proteomics can
help to determine the " true protein secretory pattern
from live cells, " including identifying the highly
polyfunctional effector cell subsets that lend deep
insights into in vivo biology. The study's findings that
TEM
cell polyfunctionality is increased by interactions
with MAC-activated ECs elucidates the relationship
between alloantibody and T cell-mediated allograft
rejection.2
n
Key drivers of transplant
rejection identified with
functional single-cell proteomics
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A
llograft rejection is a complicated process through
which immune systems reject a transplanted organ or
tissue. Rejection can be mediated by both cells and
antibodies through a process known as mixed rejection, which
often results in poor outcomes for patients. Previous research
suggests that donor-specific antibodies can augment T cell
responses, contributing to mixed rejection, but studies looking
at bulk T cell populations have not been able to determine
whether some individual T cells are more intensely activated
than others. Functional heterogeneity of immune cells prevents
researchers from drawing conclusions about the effect on
individual T cells within a population. Functional single-cell
proteomics resolves this heterogeneity with highly multiplexed
analysis of each single cell, allowing researchers to uncover the
individual immune cell subpopulations driving mixed rejection.
In a paper published in the American Journal of Transplantation,
Catherine Xie et al. from the Yale University School of Medicine
used highly multiplexed single-cell secreted proteomic analysis
to uncover the unique drivers of T cell polyfunctionality, which
was found to be driving mixed allograft rejection. The
researchers used this same technology to identify antagonists
that suppressed the detrimental effect of interferon-γ (IFN-γ)primed
human endothelial cells treated with human alloantibodies
called panel-reactive antibodies (PRAs).
Complement and alloantibody-activated ECs trigger
altered functions and heterogeneous responders
Graft antibodies trigger CD4+
and CD8+ effector memory T
cells to proliferate as well as enhance total cytokine production.
The magnitude of the T cell activation response depends
on both the strength of the antigen and the costimulatory signal
released by the endothelial cell. Stronger signals produce
more potent and complex effector cells, which release complex
combinations of effector molecules. Researchers presume
that this is how donor-specific antibodies can increase T
1
Advanced visualizations from the IsoSpeak software enable
stratification of distinct functional cell subsets with unique cytokine
signatures, revealing increased polyfunctional TEM
heterogeneity promoted by alloimmune responses.
with cellular
cell responses and contribute to mixed allograft rejection.
Consistent with previous findings, Xie et al. found that
treatment with human alloantibodies in the form of PRA
enhances the ability of IFN-γ-primed human endothelial
cells to stimulate alloreactive CD4+
and CD8+ effector
memory T cell proliferation. To more deeply assess the
functionality of the activated effector memory T cells,
the scientists used a proteomically barcoded microchip
enabling automated quantification of over 32 secreted
cytokines across thousands of single cells. Compared to
control endothelial cells (treated with GVB), PRA-treated
endothelial cells triggered a significant upregulation in
the number of polyfunctional allogeneic CD4+
and CD8+
effector memory T cells, i.e., those that secreted more than
two cytokines simultaneously. A large number of these cells
secreted three to five cytokines simultaneously.
In response to PRA activation of endothelial cells, individual T
cells secreted higher overall levels of cytokines, predominantly
GENengnews.com | 11
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Isoplexis_July2021_AcceleratingDevelopmentCurativeMedicines

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