ENH proof aim 3

tex/thesis.tex

@@ -380,6 +380,7 @@
 \newcommand{\inlinecode}{\texttt}
 \newcommand{\subcap}[2]{\subref{#1}) #2}
 \newcommand{\sigkey}{Significance test key: *p<0.1; **p < 0.05; ***p<0.01}
+\newcommand{\nVI}{NALM-6}
 
 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
 % ditto for environments
@@ -587,7 +588,7 @@ The goal of this dissertation was to develop a microcarrier-based \gls{dms} T
 cell expansion system and determine biologically-meaningful \glspl{cqa} and
 \glspl{cpp} that could be used to optimize for highly-potent T cells. In
 \cref{aim1}, we developed and characterized the \gls{dms} system, including
-quality control steps. We also demonstrated the feasibility of expanding
+\gls{qc} steps. We also demonstrated the feasibility of expanding
 high-quality T cells. In \cref{aim2a,aim2b}, we used \gls{doe} methodology to
 optimize the \gls{dms} platform, and we developed a computational pipeline to
 identify and model the effects of measurable \glspl{cqa} and \glspl{cpp} on the
@@ -687,7 +688,7 @@ The specific aims of this dissertation are outlined in
   mimics key components of the lymph nodes}
 
 In this first aim, we demonstrated the process for manufacturing \glspl{dms},
-including quality control steps that are necessary for translation of this
+including \gls{qc} steps that are necessary for translation of this
 platform into a scalable manufacturing setting. We also demonstrated that the
 \gls{dms} platform leads to higher overall expansion of T cells and higher
 overall fractions of potent memory and CD4+ subtypes desired for T cell
@@ -1038,8 +1039,8 @@ Matrigel\cite{Rio2018} or 3d-printed lattices\cite{Delalat2017}, ellipsoid
 beads\cite{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms}
 beads\cite{Lambert2017} that respectively recapitulate the cellular membrane,
 large interfacial contact area, 3D-structure, or soft surfaces T cells normally
-experience \textit{in vivo}. None of these have been shown to expand high
+experience \invivo{}. None of these have been shown to expand high quality T
-quality T cells as outlined in \cref{sec:background_quality}.
+cells as outlined in \cref{sec:background_quality}.
 
 \subsection{Microcarriers in Bioprocessing}
 
@@ -1672,7 +1673,7 @@ to secondary controls (\gls{pe}-\gls{stp} with no \gls{ptnl}).
 was added to tubes analogously to \gls{ptnl} and incubated for \SI{45}{\minute}
 prior to analyzing on a \bd{} Accuri
 
-\subsection{CAR Plasmid and Lentiviral Transduction}
+\subsection{CAR Plasmid and Lentiviral Transduction}\label{sec:transduction}
 
 The anti-CD19-CD8-CD137-CD3$\upzeta$ \gls{car} sequence with the EF1$\upalpha$
 promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a
@@ -4115,27 +4116,6 @@ results on expansion and memory phenotype. Essentially this would turn the
 \glspl{dms} into stromal cells that present \il{15}, as seen to be important in
 the early work with \il{15} in mice\cite{Lodolce1998}.
 
-% DISCUSSION 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}
@@ -4156,44 +4136,47 @@ lower-differentiated T cells with higher potency\cite{Ghassemi2018}.
 
 \section{Methods}
 
-\subsection{CD19-CAR T Cell Generation}
-
 \subsection{T Cell Culture}
 
 T cells were grown as described in \cref{sec:tcellculture}.
 
+\subsection{CD19-CAR T Cell Generation}
+
+T cells were grown as described in \cref{sec:transduction}.
+
 \subsection{\Invivo{} Therapeutic Efficacy in NSG Mice Model}
 
 % METHOD describe how the luciferase cells were generated (eg the kwong lab)
 % METHOD use actual product numbers for mice
 All mice in this study were male \gls{nsg} mice from Jackson Laboratories. At
-day 0 (\SI{-7}{\day} relative to T cell injection), 1e6 firefly
+day 0 (\SI{-7}{\day} relative to T cell injection), \num{1e6} firefly
-luciferase-expressing \product{Nalm-6 cells}{ATCC}{CRL-3273} suspended in
+luciferase-expressing\footnote{luciferase transduction was performed and
-ice-cold \gls{pbs} were injected via tail vein into each mouse. At day 7, saline
+  verified by Ian Miller in the Kwong Lab at Georgia Tech} \product{\nVI{}
-or \gls{car} T cells at the indicated doses from either bead or
+  cells}{ATCC}{CRL-3273} suspended in ice-cold \gls{pbs} were injected via tail
-\gls{dms}-expanded T cell cultures (for \SI{14}{\day}) were injected into each
+vein into each mouse. At day 7, saline or \gls{car} T cells at the indicated
-mouse via tail vein. Tumor burden was quantified longitudinally via an
+doses from either bead or \gls{dms}-expanded T cell cultures (for \SI{14}{\day})
-\gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug} luciferin at
+were injected into each mouse via tail vein. Tumor burden was quantified
-\SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally under isoflurane
+longitudinally via an \gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug}
-anesthesia into each mouse and allowed to circulate for at least
+luciferin at \SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally
-\SI{10}{\minute} before imaging. Mice were anesthetized again and imaged using
+under isoflurane anesthesia into each mouse and allowed to circulate for at
-the \gls{ivis}. Mice from each treatment group/dose were anesthetized, injected,
+least \SI{10}{\minute} before imaging. Mice were anesthetized again and imaged
-and imaged together, and exposure time of the \gls{ivis} was limited to avoid
+using the \gls{ivis}. Mice from each treatment group/dose were anesthetized,
-saturation based on the signal from the saline group. \gls{ivis} images were
+injected, and imaged together; exposure time of the \gls{ivis} was limited to
-processed by normalizing them to common minimum and maximum photon counts and
+avoid saturation based on the signal from the saline group. \gls{ivis} images
-total flux was estimated in terms of photons/second. Endpoint for each mouse was
+were scaled to common minimum and maximum photon counts. Endpoint for each mouse
-determined by \gls{iacuc} euthanasia criteria (hunched back, paralysis,
+was determined by \gls{iacuc} euthanasia criteria (hunched back, paralysis,
 blindness, lethargy, and weight loss). Mice were euthanized according to these
 endpoint criteria using carbon dioxide asphyxiation.
 
 \subsection{Statistics}
 
-For the \invivo{} model, the survival curves were created and statistically
+Survival curves were created and statistically analyzed using GraphPad Prism
-analyzed using GraphPad Prism using the Mantel-Cox test to assess significance
+using the Mantel-Cox test to assess significance between survival groups.
-between survival groups.
 
 \section{Results}
 
+\subsection{DMSs Lead to Greater \invivo{} Anti-Tumor Activity}
+
 \begin{figure*}[ht!]
   \begingroup
 
@@ -4212,7 +4195,6 @@ between survival groups.
   \input{../tables/mouse_dose_car.tex}
 \end{table}
 
-\subsection{DMSs Lead to Greater \invivo{} Anti-Tumor Activity}
 
 \begin{figure*}[ht!]
   \begingroup
@@ -4248,7 +4230,7 @@ between survival groups.
   \caption[Mouse Dosing IVIS and Survival Results]
   {T cells expanded with \glspl{dms} confer greater anti-tumor potency \invivo{}
     even at lower doses.
-    \subcap{fig:mouse_dosing_ivis_images}{IVIS images of Nalm-6 tumor-bearing
+    \subcap{fig:mouse_dosing_ivis_images}{IVIS images of \nVI{} tumor-bearing
       \gls{nsg} mice injected with varying doses of T cells}
     \subcap{fig:mouse_dosing_ivis_plots}{Plots showing quantified photon counts
       of the results from (\subref{fig:mouse_dosing_ivis_plots}).}
@@ -4266,11 +4248,11 @@ between survival groups.
 
 We asked if the higher memory/naive phenotype and more balanced CD4/CD8 ratio of
 our \gls{dms}-expanded \gls{car} T cells would lead to better anti-tumor potency
-in vivo compared to bead-expanded \gls{car} T cells. We also asked if this
+\invivo{} compared to bead-expanded \gls{car} T cells. We also asked if this
 superior anti-tumor potency would hold true at lower doses of \gls{car}
 expressing T cells in the DMS group vs the bead group. To test this, we used a
 human xenograft model of B cell \gls{all} by intravenously injecting \gls{nsg}
-mice with \num{1e6} Nalm-6 tumor cells expression firefly
+mice with \num{1e6} \nVI{} tumor cells expressing firefly
 luciferase\cite{Fraietta2018}. After \SI{7}{\day} of tumor cell growth
 (\cref{fig:mouse_dosing_overview}), we intravenously injected saline or three
 doses (high, medium, and low) of \gls{car} T cells from either bead or \gls{dms}
@@ -4280,57 +4262,55 @@ groups using the \gls{ptnl} assay (\cref{tab:mouse_dosing_results}).
 Before injecting the T cells into the mice, we quantified their phenotype and
 growth. We observed that for this expansion, the bead and \gls{dms} T cells
 produced similar numbers of \ptmem{} T cells, and the beads even had a higher
-fraction of CD45RA, which is present on lower-differentiated \glspl{tn} and
+fraction of \cdp{45RA} cells, which is present on lower-differentiated
-\glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the \pthp{} of
+\glspl{tn} and \glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the
-the final product was higher in \gls{dms} (\cref{fig:mouse_dosing_qc_cd4}). The
+\pthp{} of the final product was higher in \gls{dms}
-\gls{dms} T cells also expanded more robustly than the beads
+(\cref{fig:mouse_dosing_qc_cd4}). The \gls{dms} T cells also expanded more
-(\cref{fig:mouse_dosing_qc_growth}).
+robustly than the beads (\cref{fig:mouse_dosing_qc_growth}).
 
-In the Nalm-6/\gls{nsg} xenograft model, we observed lower tumor burden and
+In the \nVI{}/\gls{nsg} xenograft model, bead and \gls{dms}-treated mice at all
-significantly longer survival of bead and \gls{dms}-treated mice at all doses
+doses had lower tumor burden and significantly longer survival compared to the
-compared to the saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at
+saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at each dose the
-each dose we observed that the \gls{dms}-treated mice had much lower tumor
+\gls{dms}-treated mice had much lower tumor burden and significantly higher
-burden and significantly higher survival than their bead-treated counterparts
+survival than their bead-treated counterparts
 (\cref{fig:mouse_dosing_ivis_survival}). When factoring the percentage T cells
-in each dose that expressed the \gls{car}, we note that survival of the low
+in each dose that expressed the \gls{car}, survival of the low \gls{dms} dose
-\gls{dms} dose (which had similar total \gls{car} T cells compared to the bead
+(which had similar total \gls{car} T cells compared to the bead medium dose and
-medium dose and less than the bead high dose) is significantly higher than that
+less than the bead high dose) was significantly higher than that of both the
-of both the bead medium dose and the bead high dose
+bead medium dose and the bead high dose
 (\cref{fig:mouse_dosing_ivis_survival_comp}). Overall, the Kaplan-Meier survival
-of Nalm-6 tumor bearing \gls{nsg} mice shown in the
+of \nVI{} tumor bearing \gls{nsg} mice shown in the
 \cref{fig:mouse_dosing_ivis_survival} was up to day 40 as reported
-elsewhere\cite{Fraietta2018}. However, we also included a Kaplan-Meier figure up
+elsewhere\cite{Fraietta2018}. However, most of the mice euthanized from day 40
-to day 46 (\cref{fig:mouse_dosing_ivis_survival_full}) where most of the mice
+through day 46 from \gls{dms} groups showed no or very small fragment of spleen
-euthanized from day 40 through day 46 from \gls{dms} groups showed no or very
+which was due to \gls{gvhd} responses
-small fragment of spleen which was due to \gls{gvhd} responses. Similar
+(\cref{fig:mouse_dosing_ivis_survival_full}). Similar \gls{gvhd} responses
-\gls{gvhd} responses were reported earlier in \gls{nsg} mice where the mice
+\SIrange{40}{50}{\day} after injection have been reported by others in \gls{nsg}
-injected with human \gls{pbmc} exhibited acute \gls{gvhd} between
+mice injected with human \gls{pbmc}\cite{Ali2012}. Both survival analyses (up to
-\SIrange{40}{50}{\day} post intravenous injection\cite{Ali2012}. Notably, both
+day 40 in \cref{fig:mouse_dosing_ivis_survival} and up to day 46 in
-survival analyses (up to day 40 in \cref{fig:mouse_dosing_ivis_survival} and up
+\cref{fig:mouse_dosing_ivis_survival_full}) confirmed that \gls{dms}-expanded
-to day 46 in \cref{fig:mouse_dosing_ivis_survival_full}) confirmed that
+groups outperformed bead-expanded groups in terms of prolonging survival of
-\gls{dms}-expanded groups outperformed bead-expanded groups in terms of
+\nVI{} tumor challenged \gls{nsg} mice.
-prolonging survival of Nalm-6 tumor challenged \gls{nsg} mice.
 
 Together, these data suggested that \glspl{dms} produce T cells that are not
 only more potent that bead-expanded T cells (even when accounting for
 differences in \gls{car} expression) but also showed that \gls{dms} expanded T
-cells are effective at lower doses. Given the quality control data of the T
+cells are effective at lower doses. Given the \gls{qc} data of T cells prior to
-cells prior to injecting into the mice, it seems that this advantage is either
+injection, it seems that this advantage for \gls{dms} groups was either due to
-due to the higher \pthp{} or the overall fitness of the T cells given the higher
+higher \pthp{} or greater overall fitness (implied by higher fold change)
-expansion in the case of \gls{dms}
 (\cref{fig:mouse_dosing_qc_cd4,fig:mouse_dosing_qc_growth}). It was likely not
-due to the memory phenotype given that it was actually slightly higher in the
+due to memory phenotype given that this was actually slightly higher for the
-case of beads (\cref{fig:mouse_dosing_qc_mem}).
+bead culture (\cref{fig:mouse_dosing_qc_mem}).
 
 \subsection{Beads and DMSs Perform Similarly at Earlier Timepoints}
 
-We then asked how T cells harvested using either beads or \gls{dms} performed
+We then asked how T cells activated using beads or \gls{dms} performed when
-when harvested at earlier timepoints\cite{Ghassemi2018}. We performed the same
+harvested at earlier timepoints\cite{Ghassemi2018}. We performed the same
 experiments as described in \cref{fig:mouse_dosing_overview} with the
-modification that T cells were only grown and harvested after \SI{6}{\day},
+modification that T cells were only expanded and harvested after \SI{6}{\day},
 \SI{10}{\day}, or \SI{14}{\day} of expansion
 (\cref{fig:mouse_timecourse_overview}). T cells were frozen after harvest, and
-all timepoints were thawed at the same time prior to injection. The dose of T
+all timepoints were thawed simultaneously prior to injection. The dose of T
 cells injected was \num{1.25e6} cells per mouse (the same as the high dose in
 the first experiment). All other characteristics of the experiment were the
 same.
@@ -4348,20 +4328,19 @@ same.
   \label{fig:mouse_timecourse_overview}
 \end{figure*}
 
-As was the case with the first \invivo{} experiment, T cells activated with
+As was the case with the first \invivo{} experiment, \gls{dms} cultures expanded
-\glspl{dms} expanded much more efficiently compared to those expanded with beads
+much more efficiently than bead cultures
-(\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} of T
+(\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} at
-cells harvested at each timepoint, we noted that the bead group had much higher
+each timepoint, the bead group had much higher \ptcar{} expression at earlier
-\ptcar{} expression at earlier timpoints compared to \gls{dms}, while they
+timpoints compared to \gls{dms}, while they equalized at later timepoints
-equalized at later timepoints (\cref{fig:mouse_timecourse_qc_car}). In addition,
+(\cref{fig:mouse_timecourse_qc_car}). In addition, overall \ptcar{} expression
-overall \ptcar{} expression decreased at later timepoints, indicating that
+decreased at later timepoints, indicating that transduced cells either grew
-\gls{car} transduced T cells either grow slower or died faster compared to
+slower or died faster compared to untransduced cells. The \pthp{} was higher
-untransduced cells. The \pthp{} of the harvested T cells was higher overall in
+overall in \gls{dms} groups but decreased with increasing timepoints
-\gls{dms} expanded T cells but decreased with increasing timepoints 
+(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6 between
-(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6
+bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{} at day 14
-between bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{}
+despite the overall \ptmemp{} decreasing with time
-at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere
+(\cref{fig:mouse_timecourse_qc_mem}).
-(\cref{fig:mouse_timecourse_qc_mem})\cite{Ghassemi2018}.
 
 \begin{figure*}[ht!]
   \begingroup
@@ -4386,20 +4365,19 @@ at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere
   \label{fig:mouse_timecourse_qc}
 \end{figure*}
 
-We analyzed the tumor burden using \gls{ivis} which showed that mice that
+Analyzing the tumor burden using \gls{ivis} showed that mice who received T
-received T cells from any group performed better than those that received only
+cells from any group had less tumor than those that received only saline
-saline (\cref{fig:mouse_timecourse_ivis}). Note that unlike the previous
+(\cref{fig:mouse_timecourse_ivis}). Unlike the previous experiment, most mice
-experiment, many of the mice survived until day 40 at which point \gls{gvhd}
+survived until day 40 after which \gls{gvhd} began to take effect (upon
-began to take effect (after euthanizing the mice at day 42, most had small or no
+euthanization at day 42, most had little or no spleen). When comparing bead and
-spleen). When comparing bead and \gls{dms} groups, the \gls{dms} T cells still
+\gls{dms} groups, the \gls{dms} groups had lower tumor than the bead group, at
-seemed superior to the bead group, at least initially (note that in this case
+least initially (note that in this experiment they had similar numbers of
-they had similar numbers of \ptcar{} cells). At day 6, both \gls{dms} and bead
+\ptcar{} cells). For day 6 groups, both treatments seemed to eradicate the tumor
-groups seemed to eradicate the tumor initially, after which it came back after
+initially, then it came back after \SI{21}{\day} for the beads and \SI{28}{\day}
-day 21 for the bead and day 28 for the \gls{dms} group. The day 10 groups
+for \glspl{dms}. The day 10 groups performed somewhere in between, where they
-performed somewhere in between, where they increased linearly unlike the day 6
+increased linearly unlike the day 6 groups but not as quickly as the day 14
-groups but not as quickly as the day 14 groups. In the case of the \gls{dms} day
+groups. In the case of the \gls{dms} day 10 group, a few mice actually had less
-10 group, it also appeared like a few mice actually performed better than all
+tumor burden overall than all other groups.
-other groups in regard to the final tumor burden.
 
 \begin{figure*}[ht!]
   \begingroup
@@ -4433,85 +4411,76 @@ other groups in regard to the final tumor burden.
 
   \endgroup
   \caption[Mouse Summary]
-  {Summary of cells injected into mice during for
+  {Summary of T cells injected into mice for the
-    \subcap{fig:mouse_summary_1}{the first mouse experiment} and 
+    \subcap{fig:mouse_summary_1}{first} and \subcap{fig:mouse_summary_2}{second}
-    \subcap{fig:mouse_summary_2}{the second mouse experiment}. The y axis
+    experiments. The y-axis maximum is set to the maximum cell number
-    maximum is set to the maximum number of cells injected between both
+    injected between both experiments (\num{1.25e6}). NOTE: the \gls{car} was
-    experiments (\num{1.25e6}). Note that the \gls{car} was quantified using a
+    quantified using a separate panel from the other markers. }
-    separate panel than the rest of the markers.
-  }
   \label{fig:mouse_summary}
 \end{figure*}
 
-The total number of T cells injected for each \invivo{} experiment are shown in
+When we tested bead- and \gls{dms}-expanded \gls{car} T cells, the latter
-\cref{fig:mouse_summary}.
+prolonged survival compared to the former in \nVI{} tumor challenged
+(intravenously injected) \gls{nsg} mice. This held true when matching groups for
+absolute \gls{car} dose. Furthermore, \gls{dms}-expanded \gls{car} T cells were
+effective in clearing tumor cells as early as \SI{7}{\day} post T injection even
+at low dose compared to the bead groups where tumor burden was higher than
+\gls{dms} groups across all the total T cell doses tested here. These suggest
+that \glspl{dms} (compared to beads) produced highly effective \gls{car} T cells
+that can efficiently kill tumor cells.
 
-When we tested bead and \gls{dms} expanded \gls{car} T cells, we found that the
+When comparing total number of injected T cells with different phenotypes, the
-\gls{dms} expanded \gls{car} T cells outperformed bead groups in prolonging
+number of \ptmem{} (both with and without CD45RA) cells was lower in the
-survival of Nalm-6 tumor challenged (intravenously injected) \gls{nsg} mice.
+low-dose \gls{dms} group compared to the med-dose bead group (which had similar
-\gls{dms} expanded CAR-T cells were very effective in clearing tumor cells as
+numbers of \gls{car} T cells) (\cref{fig:mouse_summary_1}). This could mean
-early as \SI{7}{\day} post \gls{car} T injection even at low total T cell dose
+several things. First, the \ptmem{} phenotype may have nothing to do with the
-compared to the bead groups where tumor burden was higher than \gls{dms} groups
+results seen here, at least in this model. While this may have been the case in
-across all the total T cell doses tested here. More interestingly, when only
+our hands, this would contradict previous evidence suggesting that \gls{tn} and
-\gls{car}-expressing T cell doses between bead and \gls{dms} groups were
+\gls{tcm} cells work better in almost the same model (the only difference being
-compared, \gls{dms} group had significantly higher survival effects over similar
+Raji cells in place of \nVI{} cells, both of which express
-or higher CAR expression T cell doses from bead group. All these results suggest
+CD19)\cite{Sommermeyer2015}. Second, the distribution of \gls{car} T cells
-that the T cells in \gls{dms} groups (compared to bead group) resulted in highly
+across different subtypes of T cells was different between the \gls{dms} and
-effective \gls{car} T cells that can efficiently kill tumor cells.
+bead groups (with possibly higher correlation of \gls{car} expression and the
+\ptmem{} phenotype). It is hard to assess this without strong assumptions as the
+\gls{car} was quantified using a separate flow panel relative to the other
+markers.
 
-When comparing the total number of T cells of different phenotypes, we observed
+We can make a similar observation for the number of \pth{} T cells injected
-that when comparing low-dose \gls{dms} group to the mid- bead groups (which had
-similar numbers of \gls{car} T cells), the number of \ptmem{} (both with and
-without CD45RA) T cells injected was much lower in the \gls{dms} group
-(\cref{fig:mouse_summary_1}). This could mean several things. First, the
-\ptmem{} phenotype may have nothing to do with the results seen here, at least
-in this model. While this may have been the case in our hands, this would
-contradict previous evidence suggesting that \gls{tn} and \gls{tcm} cells work
-better in almost the same model (the only difference being Raji cells in place
-of Nalm-6 cells, both of which express CD19)\cite{Sommermeyer2015}. Second, the
-distribution of \gls{car} T cells across different subtypes of T cells was
-different between the \gls{dms} and bead groups (with possibly higher
-correlation of \gls{car} expression and the \ptmem{} phenotype). It is hard to
-assess this without strong assumptions as the \gls{car} was quantified using a
-separate flow panel relative to the other markers.
-
-We can also make a similar observation for the number of \pth{} T cells injected
 (\cref{fig:mouse_summary_1}). In this case, either the \pth{} phenotype doesn't
 matter in this model (or the \ptk{} population matters much more), or the
 distribution of \gls{car} is different between CD4 and CD8 T cells in a manner
-that favors the \gls{dms} group. While in a glioblastoma model and not a B-cell
+that favors the \gls{dms} group. Previous groups have shown that \pthp{} T cells
-\gls{all} model, previous groups have shown that \pthp{} T cells are important
+are important for response (albeit for a glioblastoma model and not a B-cell
-for response\cite{Wang2018}.
+\gls{all} model)\cite{Wang2018}.
 
 When testing \gls{car} T cells at earlier timepoints relative to day 14 as used
-in the first \invivo{} experiment, we noted that none of the \gls{car}
+in the first \invivo{} experiment, none of the \gls{car} treatments seemed to
-treatments seemed to work as well as they did in the first experiment. However,
+work as well as they did in the first experiment. However, the total number of
-the total number of \gls{car} T cells was generally much lower in this second
+\gls{car} T cells was generally much lower in this second experiment relative to
-experiment relative to the first (\cref{fig:mouse_summary}). Only the day 6
+the first (\cref{fig:mouse_summary}). Only the day 6 group had \gls{car} T cell
-group had \gls{car} T cell numbers comparable to the weakest dose of bead cells
+numbers comparable to the weakest dose of bead cells given in the first
-given in the first experiment, and these T cells were harvested at earlier
+experiment, and these T cells were harvested at earlier timepoints than the
-timepoints than the first mouse experiment and thus may not be safely
+first mouse experiment and thus are not directly comparable. Furthermore, the
-comparable. Furthermore, the \ptcarp{} decreased over time, which suggested that
+\ptcarp{} decreased over time, which suggested that the transduced T cells grew
-the transduced T cells grew slower. This has been observed elsewhere and could
+slower. This has been observed elsewhere and could be due to tonic
-be due to tonic signaling\cite{GomesSilva2017}. The lower overall \gls{car}
+signaling\cite{GomesSilva2017}. The lower overall \gls{car} doses may explain
-doses may explain why at best, the tumor seemed to be in remission only
+why at best, the tumor seemed to be in remission only temporarily. Even so, the
-temporarily. Even so, the \gls{dms} group seemed to perform better at day 6 as
+\gls{dms} group seemed to perform better at day 6 as it held off the tumor
-it held off the tumor longer, and also slowed the tumor progression relative to
+longer, and also slowed the tumor progression relative to the bead group at day
-the bead group at day 14 (\cref{fig:mouse_timecourse_ivis_plots}).
+14 (\cref{fig:mouse_timecourse_ivis_plots}).
 
 Taken together, these data suggest that the \gls{dms} platform produces T cells
 that have an advantage \invivo{} over beads. While we may not know the exact
 mechanism, our data suggests that the responses are unsurprisingly influenced by
-the \ptcarp{} of the final product. Followup experiments would need to be
+the \ptcarp{} of the final product. Followup experiments are needed to determine
-performed to determine the precise phenotype responsible for these responses in
+the precise phenotype responsible for these results.
-our hands.
 
 \chapter{CONCLUSIONS AND FUTURE WORK}\label{conclusions}
 
 \section{Conclusions}
 
 This dissertation describes the development of a novel T cell expansion
-platform, including the fabrication, quality control, and biological validation
+platform, including the fabrication, \gls{qc}, and biological validation
 of its performance both \invitro{} and \invivo{}. Development of such a system
 would be meaningful even if it only performed as well as current methods, as
 adding another method to the arsenal of the growing T cell manufacturing
@@ -4724,7 +4693,7 @@ function of surface density and the presentation method.
 
 \subsection{Reducing Ligand Variance}
 
-While we have robust quality control steps to quantify each step of the
+While we have robust \gls{qc} steps to quantify each step of the
 \gls{dms} coating process, we still see high variance across time and personnel
 (\cref{fig:dms_coating}). This is less than ideal for translation.