ADD experiments for finding the mechanism

tex/references.bib

@@ -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:

tex/thesis.tex

@@ -95,7 +95,7 @@
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 \titleformat{\subsection}[block]{\itshape\large}{}{0pt}{\titlecap}
-\titleformat{\subsubsection}[runin]{\bfseries\itshape\/}{}{0pt}{\titlecap}
+\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}