diff --git a/tex/thesis.tex b/tex/thesis.tex index e760269..4749004 100644 --- a/tex/thesis.tex +++ b/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 -quality T cells as outlined in \cref{sec:background_quality}. +experience \invivo{}. None of these have been shown to expand high quality T +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 -luciferase-expressing \product{Nalm-6 cells}{ATCC}{CRL-3273} suspended in -ice-cold \gls{pbs} were injected via tail vein into each mouse. At day 7, saline -or \gls{car} T cells at the indicated doses from either bead or -\gls{dms}-expanded T cell cultures (for \SI{14}{\day}) were injected into each -mouse via tail vein. Tumor burden was quantified longitudinally via an -\gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug} luciferin at -\SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally under isoflurane -anesthesia into each mouse and allowed to circulate for at least -\SI{10}{\minute} before imaging. Mice were anesthetized again and imaged using -the \gls{ivis}. Mice from each treatment group/dose were anesthetized, injected, -and imaged together, and exposure time of the \gls{ivis} was limited to avoid -saturation based on the signal from the saline group. \gls{ivis} images were -processed by normalizing them to common minimum and maximum photon counts and -total flux was estimated in terms of photons/second. Endpoint for each mouse was -determined by \gls{iacuc} euthanasia criteria (hunched back, paralysis, +day 0 (\SI{-7}{\day} relative to T cell injection), \num{1e6} firefly +luciferase-expressing\footnote{luciferase transduction was performed and + verified by Ian Miller in the Kwong Lab at Georgia Tech} \product{\nVI{} + cells}{ATCC}{CRL-3273} suspended in ice-cold \gls{pbs} were injected via tail +vein into each mouse. At day 7, saline or \gls{car} T cells at the indicated +doses from either bead or \gls{dms}-expanded T cell cultures (for \SI{14}{\day}) +were injected into each mouse via tail vein. Tumor burden was quantified +longitudinally via an \gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug} +luciferin at \SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally +under isoflurane anesthesia into each mouse and allowed to circulate for at +least \SI{10}{\minute} before imaging. Mice were anesthetized again and imaged +using the \gls{ivis}. Mice from each treatment group/dose were anesthetized, +injected, and imaged together; exposure time of the \gls{ivis} was limited to +avoid saturation based on the signal from the saline group. \gls{ivis} images +were scaled to common minimum and maximum photon counts. Endpoint for each mouse +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 -analyzed using GraphPad Prism using the Mantel-Cox test to assess significance -between survival groups. +Survival curves were created and statistically analyzed using GraphPad Prism +using the Mantel-Cox test to assess significance 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 -\glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the \pthp{} of -the final product was higher in \gls{dms} (\cref{fig:mouse_dosing_qc_cd4}). The -\gls{dms} T cells also expanded more robustly than the beads -(\cref{fig:mouse_dosing_qc_growth}). +fraction of \cdp{45RA} cells, which is present on lower-differentiated +\glspl{tn} and \glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the +\pthp{} of the final product was higher in \gls{dms} +(\cref{fig:mouse_dosing_qc_cd4}). The \gls{dms} T cells also expanded more +robustly than the beads (\cref{fig:mouse_dosing_qc_growth}). -In the Nalm-6/\gls{nsg} xenograft model, we observed lower tumor burden and -significantly longer survival of bead and \gls{dms}-treated mice at all doses -compared to the saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at -each dose we observed that the \gls{dms}-treated mice had much lower tumor -burden and significantly higher survival than their bead-treated counterparts +In the \nVI{}/\gls{nsg} xenograft model, bead and \gls{dms}-treated mice at all +doses had lower tumor burden and significantly longer survival compared to the +saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at each dose the +\gls{dms}-treated mice had much lower tumor burden and significantly higher +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 -\gls{dms} dose (which had similar total \gls{car} T cells compared to the bead -medium dose and less than the bead high dose) is significantly higher than that -of both the bead medium dose and the bead high dose +in each dose that expressed the \gls{car}, survival of the low \gls{dms} dose +(which had similar total \gls{car} T cells compared to the bead medium dose and +less than the bead high dose) was significantly higher than that of both the +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 -to day 46 (\cref{fig:mouse_dosing_ivis_survival_full}) where most of the mice -euthanized from day 40 through day 46 from \gls{dms} groups showed no or very -small fragment of spleen which was due to \gls{gvhd} responses. Similar -\gls{gvhd} responses were reported earlier in \gls{nsg} mice where the mice -injected with human \gls{pbmc} exhibited acute \gls{gvhd} between -\SIrange{40}{50}{\day} post intravenous injection\cite{Ali2012}. Notably, both -survival analyses (up to day 40 in \cref{fig:mouse_dosing_ivis_survival} and up -to day 46 in \cref{fig:mouse_dosing_ivis_survival_full}) confirmed that -\gls{dms}-expanded groups outperformed bead-expanded groups in terms of -prolonging survival of Nalm-6 tumor challenged \gls{nsg} mice. +elsewhere\cite{Fraietta2018}. However, most of the mice euthanized from day 40 +through day 46 from \gls{dms} groups showed no or very small fragment of spleen +which was due to \gls{gvhd} responses +(\cref{fig:mouse_dosing_ivis_survival_full}). Similar \gls{gvhd} responses +\SIrange{40}{50}{\day} after injection have been reported by others in \gls{nsg} +mice injected with human \gls{pbmc}\cite{Ali2012}. Both survival analyses (up to +day 40 in \cref{fig:mouse_dosing_ivis_survival} and up to day 46 in +\cref{fig:mouse_dosing_ivis_survival_full}) confirmed that \gls{dms}-expanded +groups outperformed bead-expanded groups in terms of prolonging survival of +\nVI{} 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 prior to injecting into the mice, it seems that this advantage is either -due to the higher \pthp{} or the overall fitness of the T cells given the higher -expansion in the case of \gls{dms} +cells are effective at lower doses. Given the \gls{qc} data of T cells prior to +injection, it seems that this advantage for \gls{dms} groups was either due to +higher \pthp{} or greater overall fitness (implied by higher fold change) (\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 -case of beads (\cref{fig:mouse_dosing_qc_mem}). +due to memory phenotype given that this was actually slightly higher for the +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 -when harvested at earlier timepoints\cite{Ghassemi2018}. We performed the same +We then asked how T cells activated using beads or \gls{dms} performed when +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 -\glspl{dms} expanded much more efficiently compared to those expanded with beads -(\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} of T -cells harvested at each timepoint, we noted that the bead group had much higher -\ptcar{} expression at earlier timpoints compared to \gls{dms}, while they -equalized at later timepoints (\cref{fig:mouse_timecourse_qc_car}). In addition, -overall \ptcar{} expression decreased at later timepoints, indicating that -\gls{car} transduced T cells either grow slower or died faster compared to -untransduced cells. The \pthp{} of the harvested T cells was higher overall in -\gls{dms} expanded T cells but decreased with increasing timepoints -(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6 -between bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{} -at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere -(\cref{fig:mouse_timecourse_qc_mem})\cite{Ghassemi2018}. +As was the case with the first \invivo{} experiment, \gls{dms} cultures expanded +much more efficiently than bead cultures +(\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} at +each timepoint, the bead group had much higher \ptcar{} expression at earlier +timpoints compared to \gls{dms}, while they equalized at later timepoints +(\cref{fig:mouse_timecourse_qc_car}). In addition, overall \ptcar{} expression +decreased at later timepoints, indicating that transduced cells either grew +slower or died faster compared to untransduced cells. The \pthp{} was higher +overall in \gls{dms} groups but decreased with increasing timepoints +(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6 between +bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{} at day 14 +despite the overall \ptmemp{} decreasing with time +(\cref{fig:mouse_timecourse_qc_mem}). \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 -received T cells from any group performed better than those that received only -saline (\cref{fig:mouse_timecourse_ivis}). Note that unlike the previous -experiment, many of the mice survived until day 40 at which point \gls{gvhd} -began to take effect (after euthanizing the mice at day 42, most had small or no -spleen). When comparing bead and \gls{dms} groups, the \gls{dms} T cells still -seemed superior to the bead group, at least initially (note that in this case -they had similar numbers of \ptcar{} cells). At day 6, both \gls{dms} and bead -groups seemed to eradicate the tumor initially, after which it came back after -day 21 for the bead and day 28 for the \gls{dms} group. The day 10 groups -performed somewhere in between, where they increased linearly unlike the day 6 -groups but not as quickly as the day 14 groups. In the case of the \gls{dms} day -10 group, it also appeared like a few mice actually performed better than all -other groups in regard to the final tumor burden. +Analyzing the tumor burden using \gls{ivis} showed that mice who received T +cells from any group had less tumor than those that received only saline +(\cref{fig:mouse_timecourse_ivis}). Unlike the previous experiment, most mice +survived until day 40 after which \gls{gvhd} began to take effect (upon +euthanization at day 42, most had little or no spleen). When comparing bead and +\gls{dms} groups, the \gls{dms} groups had lower tumor than the bead group, at +least initially (note that in this experiment they had similar numbers of +\ptcar{} cells). For day 6 groups, both treatments seemed to eradicate the tumor +initially, then it came back after \SI{21}{\day} for the beads and \SI{28}{\day} +for \glspl{dms}. The day 10 groups performed somewhere in between, where they +increased linearly unlike the day 6 groups but not as quickly as the day 14 +groups. In the case of the \gls{dms} day 10 group, a few mice actually had less +tumor burden overall than all other groups. \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 - \subcap{fig:mouse_summary_1}{the first mouse experiment} and - \subcap{fig:mouse_summary_2}{the second mouse experiment}. The y axis - maximum is set to the maximum number of cells injected between both - experiments (\num{1.25e6}). Note that the \gls{car} was quantified using a - separate panel than the rest of the markers. - } + {Summary of T cells injected into mice for the + \subcap{fig:mouse_summary_1}{first} and \subcap{fig:mouse_summary_2}{second} + experiments. The y-axis maximum is set to the maximum cell number + injected between both experiments (\num{1.25e6}). NOTE: the \gls{car} was + quantified using a separate panel from the other markers. } \label{fig:mouse_summary} \end{figure*} -The total number of T cells injected for each \invivo{} experiment are shown in -\cref{fig:mouse_summary}. +When we tested bead- and \gls{dms}-expanded \gls{car} T cells, the latter +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 -\gls{dms} expanded \gls{car} T cells outperformed bead groups in prolonging -survival of Nalm-6 tumor challenged (intravenously injected) \gls{nsg} mice. -\gls{dms} expanded CAR-T cells were very effective in clearing tumor cells as -early as \SI{7}{\day} post \gls{car} T injection even at low total T cell dose -compared to the bead groups where tumor burden was higher than \gls{dms} groups -across all the total T cell doses tested here. More interestingly, when only -\gls{car}-expressing T cell doses between bead and \gls{dms} groups were -compared, \gls{dms} group had significantly higher survival effects over similar -or higher CAR expression T cell doses from bead group. All these results suggest -that the T cells in \gls{dms} groups (compared to bead group) resulted in highly -effective \gls{car} T cells that can efficiently kill tumor cells. +When comparing total number of injected T cells with different phenotypes, the +number of \ptmem{} (both with and without CD45RA) cells was lower in the +low-dose \gls{dms} group compared to the med-dose bead group (which had similar +numbers of \gls{car} T cells) (\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 \nVI{} 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. -When comparing the total number of T cells of different phenotypes, we observed -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 +We can 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 -\gls{all} model, previous groups have shown that \pthp{} T cells are important -for response\cite{Wang2018}. +that favors the \gls{dms} group. Previous groups have shown that \pthp{} T cells +are important for response (albeit for a glioblastoma model and not a B-cell +\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} -treatments seemed to work as well as they did in the first experiment. However, -the total number of \gls{car} T cells was generally much lower in this second -experiment relative to the first (\cref{fig:mouse_summary}). Only the day 6 -group had \gls{car} T cell numbers comparable to the weakest dose of bead cells -given in the first experiment, and these T cells were harvested at earlier -timepoints than the first mouse experiment and thus may not be safely -comparable. Furthermore, the \ptcarp{} decreased over time, which suggested that -the transduced T cells grew slower. This has been observed elsewhere and could -be due to tonic signaling\cite{GomesSilva2017}. The lower overall \gls{car} -doses may explain why at best, the tumor seemed to be in remission only -temporarily. Even so, the \gls{dms} group seemed to perform better at day 6 as -it held off the tumor longer, and also slowed the tumor progression relative to -the bead group at day 14 (\cref{fig:mouse_timecourse_ivis_plots}). +in the first \invivo{} experiment, none of the \gls{car} treatments seemed to +work as well as they did in the first experiment. However, the total number of +\gls{car} T cells was generally much lower in this second experiment relative to +the first (\cref{fig:mouse_summary}). Only the day 6 group had \gls{car} T cell +numbers comparable to the weakest dose of bead cells given in the first +experiment, and these T cells were harvested at earlier timepoints than the +first mouse experiment and thus are not directly comparable. Furthermore, the +\ptcarp{} decreased over time, which suggested that the transduced T cells grew +slower. This has been observed elsewhere and could be due to tonic +signaling\cite{GomesSilva2017}. The lower overall \gls{car} doses may explain +why at best, the tumor seemed to be in remission only temporarily. Even so, the +\gls{dms} group seemed to perform better at day 6 as it held off the tumor +longer, and also slowed the tumor progression relative to the bead group at day +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 -performed to determine the precise phenotype responsible for these responses in -our hands. +the \ptcarp{} of the final product. Followup experiments are needed to determine +the precise phenotype responsible for these results. \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}). 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