ENH make section headers one line and pretty
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@ -974,7 +974,7 @@ using retro- or lentiviral vectors as their means of gene-editing must be tested
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for replication competent vectors\cite{Wang2013} and for contamination via
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bacteria or other pathogens.
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\subsection{T cell Activation Methods}\label{sec:background_activation}
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\subsection{T Cell Activation Methods}\label{sec:background_activation}
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In order for T cells to be expanded \exvivo{} they must be activated with a
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stimulatory signal (Signal 1) and a costimulatory signal (Signal 2). \Invivo{},
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@ -2067,7 +2067,7 @@ water prior to adding it to the microcarrier suspension (which itself is in
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\label{fig:dms_kinetics}
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\end{figure*}
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\subsection{Reaction Kinetics for Coating the DMSs}
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\subsection{DMS Process Has Defined Reaction Kinetics}
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We investigated the reaction kinetics of all three coating steps (accompanying
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MATLAB codes are provided in \cref{sec:appendix_binding}). To quantify the
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@ -2154,7 +2154,7 @@ one that could be optimized).
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MATLAB code and output for all the wash step calculations are given in
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\cref{sec:appendix_washing}.
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\subsection{DMSs can efficiently expand T cells compared to beads}
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\subsection{DMSs Can Efficiently Expand T Cells Compared to Beads}
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\begin{figure*}[ht!]
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\begingroup
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@ -2284,7 +2284,6 @@ expansion by lowering apoptosis of the cells in culture.
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\input{../tables/inside_fraction_regression.tex}
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\end{table}
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% RESULT state the CI of what are inside the carriers
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We also asked how many cells were inside the \glspl{dms} vs. free-floating in
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suspension and/or loosely attached to the surface. We qualitatively verified the
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presence of cells inside the \glspl{dms} using a \gls{mtt} stain to opaquely
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@ -2299,7 +2298,7 @@ regression on this data revealed that the percentage of T cells inside the
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\glspl{dms} does not depend on the initial starting cell density (at least when
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harvested after \SI{14}{\day}) (\cref{tab:inside_regression}).
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\subsection{DMSs lead to greater expansion and memory and CD4+ phenotypes}
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\subsection{DMSs Lead to Greater Expansion and High-Quality Phenotypes}
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\begin{figure*}[ht!]
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\begingroup
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@ -2388,7 +2387,7 @@ indicate the \gls{dms} platform has the capacity to expand higher numbers and
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percentages of highly potent \ptmem{} and \pth{} T cells compared to
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state-of-the-art bead technology.
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\subsection*{DMSs can be used to produce functional CAR T cells}
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\subsection{DMSs Produce Functional CAR T Cells}
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After optimizing for naïve/memory and CD4 yield, we sought to determine if the
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\glspl{dms} were compatible with lentiviral transduction protocols used to
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@ -2489,7 +2488,7 @@ for bead (\cref{fig:car_bcma_total}).
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\label{fig:car_bcma}
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\end{figure*}
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\subsection{DMSs efficiently expand T cells in Grex bioreactors}
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\subsection{DMSs Efficiently Expand T Cells in Grex Bioreactors}
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\begin{figure*}[ht!]
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\begingroup
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@ -2556,7 +2555,7 @@ in Grex bioreactors, although more optimization may be necessary to maximize the
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media feed rate and growth area to get comparable results to those seen in
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tissue-culture plates.
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\subsection{DMSs do not leave antibodies attached to cell product}
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\subsection{DMSs Do Not Leave Antibodies Attached to Cell Product}
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\begin{figure*}[ht!]
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\begingroup
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@ -2581,8 +2580,7 @@ not detect the presence of either \ahcd{3} or \ahcd{28} \glspl{mab} (both of
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which were \gls{igg}) on the final T cell product after \SI{14}{\day} of
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expansion (\cref{fig:nonstick}).
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\subsection{DMSs consistently outperform bead-based expansion compared to
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beads in a variety of conditions}
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\subsection{DMSs Outperform Beads in a Variety of Conditions}
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In order to establish the robustness of our method, we combined all experiments
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performed in our lab using beads or \glspl{dms} and combined them into one
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@ -2915,7 +2913,7 @@ additional samples (\cref{fig:mod_overview_doe}). Process parameters for the
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\si{\IU\per\ml}), \pdms{} (500, 1000, 1500, 2000, 2500, 3000, 3500
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\si{\dms\per\ml}), and \pmab{} (\SI{100}{\percent}) (\cref{fig:mod_overview}).
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\subsection{DMS fabrication}
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\subsection{DMS Fabrication}
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\glspl{dms} were fabricated as described in \cref{sec:dms_fab} with the
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following modifications in order to obtain a variable functional \gls{mab}
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@ -3119,7 +3117,7 @@ a Venn diagram from the \inlinecode{venn} R package.
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\section{Results}
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\subsection{T Cells Can be Grown on DMSs with Lower IL2 Concentrations}
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\subsection{DMSs Grow T Cells With Lower IL2 Concentrations}
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Prior to the main experiments in this aim, we performed a preliminary experiment
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to assess the effect of lowering the \gls{il2} concentration on the T cells
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@ -3176,7 +3174,7 @@ advantage at lower \gls{il2} concentrations compared to beads. For this reason,
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we decided to investigate the lower range of \gls{il2} concentrations starting
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at \SI{10}{\IU\per\ml} throughout the remainder of this aim.
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\subsection{DOE Shows Optimal Conditions for Expanded Potent T Cells}
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\subsection{DOE Shows Optimal Conditions for Potent T Cells}
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% TABLE not all of these were actually used, explain why by either adding columns
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% or marking with an asterisk
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@ -3364,8 +3362,7 @@ combinations at and around this optimum were tested, the model nonetheless
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showed that there were no other optimal values or regions elsewhere in the
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model.
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\subsection{Modeling With Artificial Intelligence Methods Reveals Potential
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CQAs}
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\subsection{Modeling with Machine Learning Reveals Putative CQAs}
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Due to the heterogeneity of the multivariate data collected and knowing that no
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single model is perfect for all applications, we implemented an agnostic
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@ -3686,7 +3683,7 @@ Adding \glspl{dms} was relatively much simpler; the number of \gls{dms} used per
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area on day 0 was scaled up by 3 on day 4 to match the change from a 96 well
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plate to a 24 well plate, effectively producing a constant activation signal.
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\subsection{Mass Wytometry and Clustering Analysis}
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\subsection{Mass Cytometry and Clustering Analysis}
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T cells were stained using a \product{34 \gls{cytof} marker
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panel}{Fluidigm}{201322} and \product{cisplatin}{Fluidigm}{201064} which were
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@ -3867,7 +3864,7 @@ leads to potentially higher expansion, lower \pthp{}, and higher fraction of
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lower differentiated T cells such as \gls{tscm}, and adding \gls{dms} seems to
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do the inverse.
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\subsection{Blocking Integrin Binding Does not Alter Expansion or Phenotype}
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\subsection{Blocking Integrin Does Not Alter Expansion or Phenotype}
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One of the reasons the \gls{dms} platform might perform better than the beads is
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the fact that they are composed of gelatin, which is a collagen derivative. The
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@ -3957,7 +3954,7 @@ CD4, or CD8) were statistically different between groups
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Taken together, these data suggest that the advantage of the \gls{dms} platform
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is not due to signaling through \gls{a2b1} or \gls{a2b2}.
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\subsection{Blocking IL15 Signaling does not Alter Expansion or Phenotype}
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\subsection{Blocking IL15 Does Not Alter Expansion or Phenotype}
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\gls{il15} is a cytokine responsible for memory T cell survival and maintenance.
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Furthermore, we observed in other experiments that it is secreted to a much
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@ -4175,7 +4172,6 @@ lower-differentiated T cells with higher potency\cite{Ghassemi2018}.
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T cells were grown as described in \cref{sec:tcellculture}.
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\subsection{\Invivo{} Therapeutic Efficacy in NSG Mice Model}
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% METHOD describe how the luciferase cells were generated (eg the kwong lab)
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@ -4226,8 +4222,7 @@ between survival groups.
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\input{../tables/mouse_dose_car.tex}
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\end{table}
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\subsection{DMS-expanded T Cells Show Greater Anti-Tumor Activity \invivo{}
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Compared to Beads}
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\subsection{DMSs Lead to Greater \invivo{} Anti-Tumor Activity}
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\begin{figure*}[ht!]
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\begingroup
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@ -4273,7 +4268,7 @@ between survival groups.
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\subcap{fig:mouse_dosing_ivis_survival_full}{The same data as
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\subref{fig:mouse_dosing_ivis_survival} except showing the full time until
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euthanasia for all mice (including those that died via \gls{gvhd}). Survival
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curves were statistically analyzed using the logrank test in GraphPad
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curves were statistically analyzed using the Mantel-Cox test in GraphPad
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Prism.}
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}
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\label{fig:mouse_dosing_ivis}
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@ -4693,7 +4688,7 @@ cytokine is undetectable, this indicates that the blocking \gls{mab} completely
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quenched all target cytokine at the time of addition and in the time between
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feeding cycles.
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\subsubsection{Interior cell phenotype}
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\subsubsection{Interior Cell Phenotype}
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Unlike the beads, the \glspl{dms} have interior and exterior surfaces. We
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demonstrated that some T cell expand on the interior of the \glspl{dms}, and is
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@ -4820,7 +4815,6 @@ potential mitigation strategies:
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due to its automated nature.
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\end{description}
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\subsubsection{Surface Stiffness}
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The beads and \gls{dms} are composed of different materials: iron/polymer in the
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