diff --git a/tex/thesis.tex b/tex/thesis.tex index 7f244a2..933fb24 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -974,7 +974,7 @@ using retro- or lentiviral vectors as their means of gene-editing must be tested for replication competent vectors\cite{Wang2013} and for contamination via bacteria or other pathogens. -\subsection{T cell Activation Methods}\label{sec:background_activation} +\subsection{T Cell Activation Methods}\label{sec:background_activation} In order for T cells to be expanded \exvivo{} they must be activated with a stimulatory signal (Signal 1) and a costimulatory signal (Signal 2). \Invivo{}, @@ -2067,7 +2067,7 @@ water prior to adding it to the microcarrier suspension (which itself is in \label{fig:dms_kinetics} \end{figure*} -\subsection{Reaction Kinetics for Coating the DMSs} +\subsection{DMS Process Has Defined Reaction Kinetics} We investigated the reaction kinetics of all three coating steps (accompanying MATLAB codes are provided in \cref{sec:appendix_binding}). To quantify the @@ -2154,7 +2154,7 @@ one that could be optimized). MATLAB code and output for all the wash step calculations are given in \cref{sec:appendix_washing}. -\subsection{DMSs can efficiently expand T cells compared to beads} +\subsection{DMSs Can Efficiently Expand T Cells Compared to Beads} \begin{figure*}[ht!] \begingroup @@ -2284,7 +2284,6 @@ expansion by lowering apoptosis of the cells in culture. \input{../tables/inside_fraction_regression.tex} \end{table} -% RESULT state the CI of what are inside the carriers We also asked how many cells were inside the \glspl{dms} vs. free-floating in suspension and/or loosely attached to the surface. We qualitatively verified the presence of cells inside the \glspl{dms} using a \gls{mtt} stain to opaquely @@ -2299,7 +2298,7 @@ regression on this data revealed that the percentage of T cells inside the \glspl{dms} does not depend on the initial starting cell density (at least when harvested after \SI{14}{\day}) (\cref{tab:inside_regression}). -\subsection{DMSs lead to greater expansion and memory and CD4+ phenotypes} +\subsection{DMSs Lead to Greater Expansion and High-Quality Phenotypes} \begin{figure*}[ht!] \begingroup @@ -2388,7 +2387,7 @@ indicate the \gls{dms} platform has the capacity to expand higher numbers and percentages of highly potent \ptmem{} and \pth{} T cells compared to state-of-the-art bead technology. -\subsection*{DMSs can be used to produce functional CAR T cells} +\subsection{DMSs Produce Functional CAR T Cells} After optimizing for naïve/memory and CD4 yield, we sought to determine if the \glspl{dms} were compatible with lentiviral transduction protocols used to @@ -2489,7 +2488,7 @@ for bead (\cref{fig:car_bcma_total}). \label{fig:car_bcma} \end{figure*} -\subsection{DMSs efficiently expand T cells in Grex bioreactors} +\subsection{DMSs Efficiently Expand T Cells in Grex Bioreactors} \begin{figure*}[ht!] \begingroup @@ -2556,7 +2555,7 @@ in Grex bioreactors, although more optimization may be necessary to maximize the media feed rate and growth area to get comparable results to those seen in tissue-culture plates. -\subsection{DMSs do not leave antibodies attached to cell product} +\subsection{DMSs Do Not Leave Antibodies Attached to Cell Product} \begin{figure*}[ht!] \begingroup @@ -2581,8 +2580,7 @@ not detect the presence of either \ahcd{3} or \ahcd{28} \glspl{mab} (both of which were \gls{igg}) on the final T cell product after \SI{14}{\day} of expansion (\cref{fig:nonstick}). -\subsection{DMSs consistently outperform bead-based expansion compared to -beads in a variety of conditions} +\subsection{DMSs Outperform Beads in a Variety of Conditions} In order to establish the robustness of our method, we combined all experiments performed in our lab using beads or \glspl{dms} and combined them into one @@ -2915,7 +2913,7 @@ additional samples (\cref{fig:mod_overview_doe}). Process parameters for the \si{\IU\per\ml}), \pdms{} (500, 1000, 1500, 2000, 2500, 3000, 3500 \si{\dms\per\ml}), and \pmab{} (\SI{100}{\percent}) (\cref{fig:mod_overview}). -\subsection{DMS fabrication} +\subsection{DMS Fabrication} \glspl{dms} were fabricated as described in \cref{sec:dms_fab} with the following modifications in order to obtain a variable functional \gls{mab} @@ -3119,7 +3117,7 @@ a Venn diagram from the \inlinecode{venn} R package. \section{Results} -\subsection{T Cells Can be Grown on DMSs with Lower IL2 Concentrations} +\subsection{DMSs Grow T Cells With Lower IL2 Concentrations} Prior to the main experiments in this aim, we performed a preliminary experiment to assess the effect of lowering the \gls{il2} concentration on the T cells @@ -3176,7 +3174,7 @@ advantage at lower \gls{il2} concentrations compared to beads. For this reason, we decided to investigate the lower range of \gls{il2} concentrations starting at \SI{10}{\IU\per\ml} throughout the remainder of this aim. -\subsection{DOE Shows Optimal Conditions for Expanded Potent T Cells} +\subsection{DOE Shows Optimal Conditions for Potent T Cells} % TABLE not all of these were actually used, explain why by either adding columns % or marking with an asterisk @@ -3364,8 +3362,7 @@ combinations at and around this optimum were tested, the model nonetheless showed that there were no other optimal values or regions elsewhere in the model. -\subsection{Modeling With Artificial Intelligence Methods Reveals Potential - CQAs} +\subsection{Modeling with Machine Learning Reveals Putative CQAs} Due to the heterogeneity of the multivariate data collected and knowing that no single model is perfect for all applications, we implemented an agnostic @@ -3686,7 +3683,7 @@ Adding \glspl{dms} was relatively much simpler; the number of \gls{dms} used per area on day 0 was scaled up by 3 on day 4 to match the change from a 96 well plate to a 24 well plate, effectively producing a constant activation signal. -\subsection{Mass Wytometry and Clustering Analysis} +\subsection{Mass Cytometry and Clustering Analysis} T cells were stained using a \product{34 \gls{cytof} marker panel}{Fluidigm}{201322} and \product{cisplatin}{Fluidigm}{201064} which were @@ -3867,7 +3864,7 @@ leads to potentially higher expansion, lower \pthp{}, and higher fraction of lower differentiated T cells such as \gls{tscm}, and adding \gls{dms} seems to do the inverse. -\subsection{Blocking Integrin Binding Does not Alter Expansion or Phenotype} +\subsection{Blocking Integrin Does Not Alter Expansion or Phenotype} One of the reasons the \gls{dms} platform might perform better than the beads is the fact that they are composed of gelatin, which is a collagen derivative. The @@ -3957,7 +3954,7 @@ CD4, or CD8) were statistically different between groups Taken together, these data suggest that the advantage of the \gls{dms} platform is not due to signaling through \gls{a2b1} or \gls{a2b2}. -\subsection{Blocking IL15 Signaling does not Alter Expansion or Phenotype} +\subsection{Blocking IL15 Does Not Alter Expansion or Phenotype} \gls{il15} is a cytokine responsible for memory T cell survival and maintenance. Furthermore, we observed in other experiments that it is secreted to a much @@ -4175,7 +4172,6 @@ lower-differentiated T cells with higher potency\cite{Ghassemi2018}. T cells were grown as described in \cref{sec:tcellculture}. - \subsection{\Invivo{} Therapeutic Efficacy in NSG Mice Model} % METHOD describe how the luciferase cells were generated (eg the kwong lab) @@ -4226,8 +4222,7 @@ between survival groups. \input{../tables/mouse_dose_car.tex} \end{table} -\subsection{DMS-expanded T Cells Show Greater Anti-Tumor Activity \invivo{} - Compared to Beads} +\subsection{DMSs Lead to Greater \invivo{} Anti-Tumor Activity} \begin{figure*}[ht!] \begingroup @@ -4273,7 +4268,7 @@ between survival groups. \subcap{fig:mouse_dosing_ivis_survival_full}{The same data as \subref{fig:mouse_dosing_ivis_survival} except showing the full time until euthanasia for all mice (including those that died via \gls{gvhd}). Survival - curves were statistically analyzed using the logrank test in GraphPad + curves were statistically analyzed using the Mantel-Cox test in GraphPad Prism.} } \label{fig:mouse_dosing_ivis} @@ -4693,7 +4688,7 @@ 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} +\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 @@ -4820,7 +4815,6 @@ potential mitigation strategies: due to its automated nature. \end{description} - \subsubsection{Surface Stiffness} The beads and \gls{dms} are composed of different materials: iron/polymer in the