ADD experiments for finding the mechanism

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Nathan Dwarshuis 2021-09-02 16:22:39 -04:00
parent 9c23941ef0
commit 7c427b39d8
2 changed files with 115 additions and 11 deletions

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@ -2742,6 +2742,32 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec
publisher = {Elsevier {BV}},
}
@Article{Wang1984,
author = {James C. Wang},
journal = {Journal of Materials Science},
title = {Young{\textquotesingle}s modulus of porous materials},
year = {1984},
month = {mar},
number = {3},
pages = {801--808},
volume = {19},
doi = {10.1007/bf00540451},
publisher = {Springer Science and Business Media {LLC}},
}
@Article{Ju2017,
author = {Lining Ju and Cheng Zhu},
journal = {Biophysical Journal},
title = {Benchmarks of Biomembrane Force Probe Spring Constant Models},
year = {2017},
month = {dec},
number = {12},
pages = {2842--2845},
volume = {113},
doi = {10.1016/j.bpj.2017.10.013},
publisher = {Elsevier {BV}},
}
@Comment{jabref-meta: databaseType:bibtex;}
@Comment{jabref-meta: grouping:

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@ -95,7 +95,7 @@
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\titleformat{\subsubsection}[runin]{\bfseries}{}{0pt}{\titlecap}
\setlist[description]{font=$\bullet$~\textbf\normalfont}
@ -4574,16 +4574,94 @@ animal-origin concerns.
Despite the improved outcomes in terms of expansion and phenotype relative to
beads, we don't have a good understanding of why they \gls{dms} platform works
as well as it does. Several broad areas remain to be investigated, including the
role of the increased cytokine output (including \il{15} which was explored to
some extent in this work), the role of cells on the interior of the \gls{dms}
relative to those outside the \gls{dms}, and the role of the physical surface
properties of the \gls{dms} (including the morphology and the stiffness). One
plausible hypothesis to be tested is that the bumpy microcarrier surface is more
like that of an \gls{apc}, which enhances immunological synapse formation and
thus activation. Another related hypothesis is that the signal strength is
lower than the beads, which leads to increased proliferation, less exhaustion,
and by extension more memory.
as well as it does. The following are several plausible hypotheses and a
proposed experiment for testing them:
\subsubsection{Cytokine Cross-talk}
As hypothesized in the beginning of this work, the \gls{dms} may derive their
advantage through increased cytokine cross-talk. While this work found that
blocking \il{15} did not lead to differences in outcome, other cytokines could
be explored in a similar vein.
An efficient test of this hypothesis would be to simply incubate T cells grown
with either bead or \glspl{dms} with a cocktail of \glspl{mab} each feeding
cycle that target the cytokines seen in \cref{fig:doe_luminex}, assuming that at
least a few of the targeted cytokines will cause a difference. The experiment
should be sized appropriately such that the second order interaction effect can
be resolved (that is, the effect of adding the cocktail conditional on the
activation method). In these terms, we hypothesize that the growth and phenotype
will be more similar between the beads and \glspl{dms} when the cocktail is
added, while the \gls{dms} will have better expansion and phenotype when the
cocktail is not added. If this experiment shows any effects, the cytokines
responsible can be resolved by testing individually (or in small pools).
One caveat with this approach is that it assumes that the \gls{mab} cocktail
will completely quench their target cytokines between each feed cycle. This assumption
can be tested by running luminex with each cocktail addition. If a given
cytokine is undetectable, this indicates that the blocking \gls{mab} completely
quenched all target cytokine at the time of addition and in the time between
feeding cycles.
\subsubsection{Interior cell phenotype}
Unlike the beads, the \glspl{dms} have interior and exterior surfaces. We
demonstrated that some T cell expand on the interior of the \glspl{dms}, and is
plausible that these cells are phenotypically different than those growing on
the exterior or completely detached from the microcarriers, and that this leads
to an asymmetric cytokine cross-talk which accounts for the population-level
differences seen in comparison to the beads.
Experimentally, the first step involves separating the \glspl{dms} from the
loosely or non-adhered T cells and digesting the \glspl{dms} wth \gls{cold}
(concentrations of \SI{10}{\ug\per\ml} will completely the \glspl{dms} within
\SIrange{30}{45}{\min}) isolate the interior T cells. Unfortunately, only
\SIrange{10}{20}{\percent} of all cells will be on the interior, so the interior
group may only have cells on the order of \si{1e3} to \si{1e4} for analysis. A
good first pass experiment would be to analyze both populations with a T cell
differentiation/activation state flow panel first (since flow cytometry is
relatively cheap and doesn't require a large number of cells) to simply
establish if the two groups are different phenotypes or are in a different state
of activation. From there, more in-depth analysis using \gls{cytof} or another
high-dimensionality method may be used to evaluate differential cytokine
expression.
\subsubsection{Antibody Surface Density}
While our \gls{doe} experiments showed a relationship between activating
\gls{mab} density and number of cells, we don't know how the \gls{mab} surface
density of the \gls{dms} compares to that of the beads. In all likelihood, the
\gls{mab} density on the \gls{dms} surface is lower (given the number of total
binding sites on \gls{stp} and the number of \glspl{mab} that actually bind)
which may lead to differences in performance\cite{Lozza2008}.
% TODO make sure this actually is "below"
Before attempting this experiment, it will be vital to improve the \gls{dms}
manufacturing process such that \gls{mab} binding is predictable and
reproducible (see below). Once this is established, we can then determine the
amount of \glspl{mab} that bind to the beads, which could be performed much like
the \gls{mab} binding step is quantified in the \gls{dms} process (eg with
ELISA, \cref{fig:dms_flowchart}). Knowing this, we can vary the
\gls{mab} surface density for both the bead and the \glspl{dms} using a dummy
\gls{mab} as done previously with the \gls{doe} experiments in \cref{aim2a}.
Using varying surface densities that are matched per-area between the beads and
\glspl{dms} we can then activate T cells and assess their growth/phenotype as a
function of surface density and the presentation method.
\subsubsection{Surface Stiffness}
The beads and \gls{dms} are composed of different materials: iron/polymer in the
former case and cross-linked gelatin in the latter. These materials likely have
different stiffnesses, and stiffness could play a role in T cell
activation\cite{Lambert2017}.
This hypotheses will be difficult to test directly, so it is advised to
eliminate other hypothesis before proceeding here. Direct testing could be
performed using a force probe to determine the Young's modulus of each
material\cite{Ju2017}. Since the microcarriers are porous and the cells will be
interacting with the bulk material itself, the void fraction and pore size will
need to be taken into account to find the bulk material properties of the
cross-linked gelatin\cite{Wang1984}.
\subsection{Additional Ligands and Signals on the DMSs}