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ADD discussion to aim 2b

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Nathan Dwarshuis 2021-07-28 22:51:31 -04:00
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tex/thesis.tex
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@ -40,6 +40,7 @@
\renewcommand{\glossarysection}[2][]{} % remove glossary title
\makeglossaries
\newacronym{act}{ACT}{adoptive cell therapies}
\newacronym{qc}{QC}{quality control}
\newacronym{tcm}{T\textsubscript{cm}}{central memory T cell}
\newacronym{tscm}{T\textsubscript{scm}}{stem-memory T cell}
\newacronym{car}{CAR}{chimeric antigen receptor}
@ -1823,32 +1824,6 @@ exhaustion, and apoptosis23. Despite evidence for the importance of CD4 T cells,
more work is required to determine the precise ratios of CD4 and CD8 T cell
subsets to be included in CAR T cell therapy given a disease state.
% TODO this might be more appropriate for aim 2b where I actually talk about
% the signaling and why this might matter
There are several plausible explanations for the observed phenotypic differences
between beads and DMSs. First, the DMSs are composed of a collagen derivative
(gelatin); collagen has been shown to costimulate activated T cells via
\gls{a2b1} and \gls{a2b2}, leading to enhanced proliferation, increased
IFN$\upgamma$ production, and upregulated CD25 (IL2R$\upalpha$) surface
expression8,10,11,41,42. Second, there is evidence that providing a larger
contact area for T cell activation provides greater stimulation16,43; the DMSs
have a rougher interface than the 5 µm magnetic beads, and thus could facilitate
these larger contact areas. Third, the DMSs may allow the T cells to cluster
more densely compared to beads, as evidenced by the large clusters on the
outside of the DMSs (Figure 1f) as well as the significant fraction of DMSs
found within their interiors (Supplemental Figure 2a and b). This may alter the
local cytokine environment and trigger different signaling pathways.
Particularly, IL15 and IL21 are secreted by T cells and known to drive memory
phenotype44–46. We noted that the IL15 and IL21 concentration was higher in a
majority of samples when comparing beads and DMSs across multiple timepoints
(Supplemental Figure 18) in addition to many other cytokines. IL15 and IL21 are
added exogenously to T cell cultures to enhance memory frequency,45,47 and our
data here suggest that the DMSs are better at naturally producing these
cytokines and limiting this need. Furthermore, IL15 unique signals in a trans
manner in which IL15 is presented on IL15R to neighboring cells48. The higher
cell density in the DMS cultures would lead to more of these trans interactions,
and therefore upregulate the IL15 pathway and lead to more memory T cells.
% TODO this mentions the DOE which is in the next aim
When analyzing all our experiments comprehensively using causal inference, we
found that all three of our responses were significantly increased when
@ -2423,6 +2398,88 @@ gains its advantages via the \gls{il15} pathway.
\section{discussion}
This work provides insight for how the \gls{dms} operates and may be optimized
further. The data showing increased \pthp{} when \glspl{dms} are added and the
reverse when removed is consistent with other data we produced via \gls{doe}
showing that higher \gls{dms} concentrations lead to higher \pthp{}
(\cref{fig:doe_responses_cd4,fig:add_rem_cd4}). The difference in this case is
that we showed that altering activation signal analogously affects the \pthp{}
in the dimension of time as well as space. A similar trend was observed with
memory T cells in this aim. Our previous \gls{doe} data showed that, to a point,
lower \gls{dms} concentration leads to higher \ptmemp{}
(\cref{fig:doe_responses_mem}). In this aim, we showed that decreasing
activation signal temporally by removing \glspl{dms} leads to the same effect in
the \gls{tcm}, \gls{tscm} and `transitory' \gls{tscm} populations, (all of which
are included in the \ptmem{} phenotype). Taken together, these imply that
temporally or spatially altering the \gls{dms} concentration, and thus the
activation signal, has similar effects.
% TODO this sounds like background?
% There are several plausible explanations for the observed phenotypic differences
% between beads and DMSs. First, the DMSs are composed of a collagen derivative
% (gelatin); collagen has been shown to costimulate activated T cells via
% \gls{a2b1} and \gls{a2b2}, leading to enhanced proliferation, increased
% \gls{ifng} production, and upregulated CD25 (IL2R$\upalpha$) surface
% expression8,10,11,41,42.
While we did not find support for our hypothesis that the \gls{dms} signal
through the \gls{a2b1} and/or \gls{a2b2} receptors, we can speculate as to why
either this experiment failed and may be done better in the future, or why these
receptors may simply be irrelevant for our system.
On the first point, we did not verify that these \glspl{mab} indeed blocked the
receptor we were targeting. There has been evidence from other groups that these
particular clones work at the concentrations we used {\#}. This does not
necessarily mean that the \glspl{mab} we obtained were functional in blocking
their intended targets (although they were from a reputable manufacturer, \bl).
Furthermore, we can safely rule out the possibility that the \glspl{mab} never
reached their targets, as they were added immediately after the T cells were
resuspended as required for cell counting, hence their resting clustered state
was disrupted.
% TODO define Bite
On the second point, the collagen domains may not even be relevant to our system
depending on the nature of the \gls{stp} coating. We intended by design for the
system to be fully coated or nearly fully-coated with \gls{stp}
(\cref{fig:stp_coating}). Thus the domains that \gls{a2b1} and \gls{a2b2} may be
targeting could be sterically hindered by a layer of \gls{stp}, and if not that,
also a layer of CD3/CD28 \glspl{mab}. The other possibility is that these
domains are simply denatured to beyond recognition due to the fabrication
process for the microcarriers we used (which involves a proprietary
cross-linking step to make the material autoclave-safe). Either of these could
be tested and verified by staining the \glspl{dms} with a fluorescently-tagged
\gls{mab} (or something smaller such as a BiTE to reduce the possibility of
steric hindrance) and verifying binding via confocal microscopy or indirect
protein quantification as we do for the \gls{qc} of the \gls{dms}. If this test
came back negative, we would be fairly confident that the \gls{a2b1} and
\gls{a2b1} domains are either unreachable or unrecognizable.
% DISCUSSION not sure exactly how to explain this
We also failed to uphold our hypothesis that the \gls{dms} system gains its
advantage via \gls{il15} signaling.
% TODO not sure if this belongs here, although it might make sense to offer
% alternative explanations of why the DMSs "work" given this negative data
% Second, there is evidence that providing a larger
% contact area for T cell activation provides greater stimulation16,43; the DMSs
% have a rougher interface than the 5 µm magnetic beads, and thus could facilitate
% these larger contact areas. Third, the DMSs may allow the T cells to cluster
% more densely compared to beads, as evidenced by the large clusters on the
% outside of the DMSs (Figure 1f) as well as the significant fraction of DMSs
% found within their interiors (Supplemental Figure 2a and b). This may alter the
% local cytokine environment and trigger different signaling pathways.
% Particularly, IL15 and IL21 are secreted by T cells and known to drive memory
% phenotype44–46. We noted that the IL15 and IL21 concentration was higher in a
% majority of samples when comparing beads and DMSs across multiple timepoints
% (Supplemental Figure 18) in addition to many other cytokines. IL15 and IL21 are
% added exogenously to T cell cultures to enhance memory frequency,45,47 and our
% data here suggest that the DMSs are better at naturally producing these
% cytokines and limiting this need. Furthermore, IL15 unique signals in a trans
% manner in which IL15 is presented on IL15R to neighboring cells48. The higher
% cell density in the DMS cultures would lead to more of these trans interactions,
% and therefore upregulate the IL15 pathway and lead to more memory T cells.
\chapter{aim 3}\label{aim3}
\section{introduction}