ENH integrate some of the new figures
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tex/thesis.tex
192
tex/thesis.tex
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@ -1390,7 +1390,7 @@ model, the \gls{mab} binding reaction should be complete within \SI{15}{\minute}
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under the conditions used for our protocol (\cref{fig:dms_mab_per_time}). Note
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that our unoptimized coated steps were done in \SI{45}{\minute}, which seemed
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reasonable given the slightly larger hydrodynamic radius of \glspl{mab} compared
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to \gls{stp} which was shown to react in \SI{30}{\minutes} experimentally. The
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to \gls{stp} which was shown to react in \SI{30}{\minute} experimentally. The
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results of this model should be experimentally verified.
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% TODO find the actual numbers for this
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@ -1415,21 +1415,6 @@ far less severe than that of \gls{snb}.
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\subsection{DMSs can efficiently expand T cells compared to beads}
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% RESULT add other subfigures here
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We next sought to determine how our \glspl{dms} could expand T cells compared to
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state-of-the-art methods used in industry. All bead expansions were performed as
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per the manufacturer’s protocol, with the exception that the starting cell
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densities were matched between the beads and carriers to
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~\SI{2.5e6}{\cell\per\ml}. Throughout the culture we observed that T cells in
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\gls{dms} culture grew in tight clumps on the surface of the \glspl{dms} as well
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as inside the pores of the \glspl{dms}
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(\cref{fig:dms_cells_phase,fig:dms_cells_fluor}). Furthermore, we observed that
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the \glspl{dms} conferred greater expansion compared to traditional beads, and
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significantly greater expansion after \SI{12}{\day} of culture {Figure X}. We
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also observed no T cell expansion using \glspl{dms} coated with an isotype
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control mAb compared to \glspl{dms} coated with \acd{3}/\acd{28} \glspl{mab}
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{Figure X}, confirming specificity of the expansion method.
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% FIGURE make sure the day on these is correct
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\begin{figure*}[ht!]
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\begingroup
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@ -1450,7 +1435,6 @@ control mAb compared to \glspl{dms} coated with \acd{3}/\acd{28} \glspl{mab}
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\label{fig:dms_cells}
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\end{figure*}
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% RESULT for this figure
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\begin{figure*}[ht!]
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\begingroup
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@ -1470,26 +1454,23 @@ control mAb compared to \glspl{dms} coated with \acd{3}/\acd{28} \glspl{mab}
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\label{fig:dms_expansion}
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\end{figure*}
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% RESULT talk about this table somewhere
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\begin{table}[!h] \centering
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\caption{Regression for fraction of cells in \gls{dms} at day 14}
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\label{tab:inside_regression}
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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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mark cells and enable visualization on a brightfield microscope
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(\cref{fig:dms_inside_bf}). After seeding \glspl{dms} at different densities and
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expanding for \SI{14}{\day}, we filtered the \glspl{dms} out of the cell
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suspension and digested them using dispase to free any cells attached on the
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inner surface. We observed that approximately \SI{15}{\percent} of the total
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cells after \SI{14}{\day} were on the interior surface of the \glspl{dms}
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(\cref{fig:dms_inside_regression}).
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%, and this did not significantly change with initial seeding density (Supplemental Table 1).
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% DISCUSSION krish seems concerned about this isotype control figure, add some
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% discussion saying that IL2 does not spontaneously activate T cells to appease
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% him
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We next sought to determine how our \glspl{dms} could expand T cells compared to
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state-of-the-art methods used in industry. All bead expansions were performed as
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per the manufacturer’s protocol, with the exception that the starting cell
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densities were matched between the beads and carriers to
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~\SI{2.5e6}{\cell\per\ml}. Throughout the culture we observed that T cells in
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\gls{dms} culture grew in tight clumps on the surface of the \glspl{dms} as well
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as inside the pores of the \glspl{dms}
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(\cref{fig:dms_cells_phase,fig:dms_cells_fluor}). Furthermore, we observed that
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the \glspl{dms} conferred greater expansion compared to traditional beads, and
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significantly greater expansion after \SI{12}{\day} of culture
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(\cref{fig:dms_expansion_bead}). We also observed no T cell expansion using
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\glspl{dms} coated with an isotype control mAb compared to \glspl{dms} coated
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with \acd{3}/\acd{28} \glspl{mab} (\cref{fig:dms_expansion_isotype}), confirming
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specificity of the expansion method.
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\begin{figure*}[ht!]
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\begingroup
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@ -1509,11 +1490,34 @@ cells after \SI{14}{\day} were on the interior surface of the \glspl{dms}
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CellEvent dye.}
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\subcap{fig:apoptosis_bcl2}{Quantification of BCL-2 expression using
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\gls{elisa}. All statistical tests shown are two-tailed homoschodastic
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t-tests.}
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t-tests. All cells were harvested at day 8.}
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}
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\label{fig:dms_flowchart}
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\label{fig:dms_apoptosis}
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\end{figure*}
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Given that the \gls{dms} system seemed to expand T cells more effectively, we
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asked if this difference was due to a reduction in apoptosis or an increase in
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proliferation rate (or both). We assessed the apoptotic state of T cells grown
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using either bead or \gls{dms} harvested on day 8 using \gls{pi} and \gls{anv}.
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\gls{anv} is a marker which stains phospholipid phosphatidylserine, which is
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usually present only on the cytoplasmic surface of the cell membrane, but flips
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to the outside when the cell becomes apoptotic. \gls{pi} stains the nucleus of
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the cell, but only penetrates necrotic cells which have a perforated cell
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membrane. When staining for these two markers and assessing via flow cytometry,
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we observe that the \gls{dms}-expanded T cells have lower frequencies of
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apoptotic and necrotic cells (\cref{fig:apoptosis_annV}). Furthermore, we
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stained our cultures with CellEvent dye, which is an indicator of \gls{cas37},
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which is activated in apoptotic cells {\#}{cas37 activation}. In line with the
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\gls{pi}/\gls{anv} results, we observed that the \gls{dms} T cells had lower
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frequency of \gls{cas37} expression, indicating less apoptosis for our method
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(\cref{fig:apoptosis_cas}). Finally, we lysed our cells and stained for
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\gls{bcl2}, which is also upregulated in apoptosis. In this case, some (but not
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all) of the bead-expanded cultures showed higher \gls{bcl2} expression, which
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could indicate more apoptosis in those groups (\cref{fig:apoptosis_bcl2}). None
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of the \gls{dms} cultures showed similar heightened expression. Taken together,
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these data suggest that the \gls{dms} platform at least in part achieves higher
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expansion by lowering apoptosis of the cells in culture.
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% FIGURE double check the timing of this experiment (it might not be day 14)
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\begin{figure*}[ht!]
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\begingroup
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@ -1535,31 +1539,29 @@ cells after \SI{14}{\day} were on the interior surface of the \glspl{dms}
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\label{fig:dms_inside}
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\end{figure*}
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\begin{table}[!h] \centering
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\caption{Regression for fraction of cells in \gls{dms} at day 14}
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\label{tab:inside_regression}
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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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mark cells and enable visualization on a brightfield microscope
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(\cref{fig:dms_inside_bf}). After seeding \glspl{dms} at different densities and
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expanding for \SI{14}{\day}, we filtered the \glspl{dms} out of the cell
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suspension and digested them using dispase to free any cells attached on the
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inner surface. We observed that approximately \SI{15}{\percent} of the total
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cells after \SI{14}{\day} were on the interior surface of the \glspl{dms}
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(\cref{fig:dms_inside_regression,tab:inside_regression}). Performing linear
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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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After observing differences in expansion, we further hypothesized that the
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\gls{dms} cultures could lead to a different T cell phenotype. In particular, we
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were interested in the formation of naïve and memory T cells, as these represent
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a subset with higher replicative potential and therefore improved clinical
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prognosis\cite{Gattinoni2011, Wang2018}. We measured naïve and memory T cell
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frequency staining for CCR7 and CD62L (both of which are present on lower
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differentiated T cells such as naïve, central memory, and stem memory
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cells\cite{Gattinoni2012}). Using three donors, we noted again \glspl{dms}
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produced more T cells over a \SI{14}{\day} expansion than beads, with
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significant differences in number appearing as early after \SI{5}{\day}
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(\cref{fig:dms_exp_fold_change}). Furthermore, we noted that \glspl{dms}
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produced more memory/naïve cells after \SI{14}{\day} when compared to beads for
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all donors (\cref{fig:dms_exp_mem,fig:dms_exp_cd4}) showing that the \gls{dms}
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platform is able to selectively expand potent, early differentiation T cells.
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Of additional interest was the preservation of the CD4+ compartment. In healthy
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donor samples (such as those used here), the typical CD4:CD8 ratio is 2:1. We
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noted that \glspl{dms} produced more CD4+ T cells than bead cultures as well as
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naïve/memory, showing that the \gls{dms} platform can selectively expand CD4 T
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cells to a greater degree than beads (Figure 2c). The trends held true when
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observing the CD4+ and CD8+ fractions of the naïve/memory subset (CD62L+CCR7+)
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(\cref{fig:dms_exp_mem4,fig:dms_exp_mem8}).
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\begin{figure*}[ht!]
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\begingroup
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@ -1587,7 +1589,28 @@ observing the CD4+ and CD8+ fractions of the naïve/memory subset (CD62L+CCR7+)
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\label{fig:dms_exp}
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\end{figure*}
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% RESULT add a paragraph for this figure
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After observing differences in expansion, we further hypothesized that the
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\gls{dms} cultures could lead to a different T cell phenotype. In particular, we
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were interested in the formation of naïve and memory T cells, as these represent
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a subset with higher replicative potential and therefore improved clinical
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prognosis\cite{Gattinoni2011, Wang2018}. We measured naïve and memory T cell
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frequency staining for CCR7 and CD62L (both of which are present on lower
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differentiated T cells such as naïve, central memory, and stem memory
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cells\cite{Gattinoni2012}). Using three donors, we noted again \glspl{dms}
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produced more T cells over a \SI{14}{\day} expansion than beads, with
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significant differences in number appearing as early after \SI{5}{\day}
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(\cref{fig:dms_exp_fold_change}). Furthermore, we noted that \glspl{dms}
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produced more memory/naïve cells after \SI{14}{\day} when compared to beads for
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all donors (\cref{fig:dms_exp_mem,fig:dms_exp_cd4}) showing that the \gls{dms}
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platform is able to selectively expand potent, early differentiation T cells.
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Of additional interest was the preservation of the CD4+ compartment. In healthy
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donor samples (such as those used here), the typical CD4:CD8 ratio is 2:1. We
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noted that \glspl{dms} produced more CD4+ T cells than bead cultures as well as
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naïve/memory, showing that the \gls{dms} platform can selectively expand CD4 T
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cells to a greater degree than beads (Figure 2c). The trends held true when
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observing the CD4+ and CD8+ fractions of the naïve/memory subset (\ptmem{})
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(\cref{fig:dms_exp_mem4,fig:dms_exp_mem8}).
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% FIGURE this figure has weird proportions
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\begin{figure*}[ht!]
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@ -1608,6 +1631,25 @@ observing the CD4+ and CD8+ fractions of the naïve/memory subset (CD62L+CCR7+)
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\label{fig:dms_phenotype}
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\end{figure*}
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We also observed that, at least with the donors and conditions tested in these
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experiments\footnote{these results were not always consistent, see the
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metaanalysis at the end of this aim for an in-depth quantification of this
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observation} that the fraction of \ptmem{} and \pth{} T cells was higher in
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the \gls{dms} groups compared to the bead groups (\cref{fig:dms_phenotype}).
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This result was seen for multiple donors. We should not that in the case of
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\pthp{}, the donors we used had an initial \pthp{} that was much higher (healthy
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donors generally have a CD4:CD8 ratio of 2:1), so the proper interpretation of
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this is that the \pthp{} decreases less over the culture period with the
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\gls{dms} platform as opposed to the beads (or alternatively, the \gls{dms} has
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less preferential expansion for CD8 T cells). We cannot say the same about
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the \ptmemp{} since we did not have the initial data for this phenotype;
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however (although it should be the vast majority of cells given that
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cryopreserved T cells from a healthy donor should generally be composed of
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circulated memory and naive T cells). Taken together, these data indicate the
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\gls{dms} platform has the capacity to expand higher numbers and percentages of
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highly potent \ptmem{} and \pth{} T cells compared to state-of-the-art bead
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technology.
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\subsection*{DMSs can be used to 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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@ -3077,8 +3119,6 @@ them to grow better in the \gls{dms} system.
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\label{fig:integrin_1}
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\end{figure*}
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% RESULT alude to these tables
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\begin{table}[!h] \centering
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\caption{Linear regression for day 14 phenotype shown in \cref{fig:integrin_1}}
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\label{tab:integrin_1_reg}
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@ -3092,9 +3132,9 @@ These block \glspl{mab} were added at day 6 of culture when \gls{a2b1} and
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identical in all the blocked groups vs the unblocked control group
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(\cref{fig:inegrin_1_fc}). Furthermore, we observed that the \ptmemp{} (total
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and across the CD4/CD8 compartments) was not significantly different between any
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of the groups (\cref{fig:inegrin_1_mem}). We also noted that \gls{a2b1} and
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\gls{a2b2} were present on the surface of a significant subset of T cells at day
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6, showing that the target we wished to block was present
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of the groups (\cref{fig:inegrin_1_mem,tab:integrin_1_reg}). We also noted that
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\gls{a2b1} and \gls{a2b2} were present on the surface of a significant subset of
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T cells at day 6, showing that the target we wished to block was present
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(\cref{fig:inegrin_1_cd49}).
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\begin{figure*}[ht!]
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@ -3131,22 +3171,22 @@ its maximum. Once again, we observed no difference between any of the blocked
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conditions and the unblocked controls in regard to expansion
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(\cref{fig:inegrin_2_fc}). Furthermore, none of the \ptmemp{} readouts (total,
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CD4, or CD8) were statistically different between groups
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(\cref{fig:inegrin_2_mem}).
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(\cref{fig:inegrin_2_mem,tab:integrin_2_reg}).
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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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% RESULT cite the luminex data
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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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greater extend in \gls{dms} compared to bead cultures. One of our driving
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hypotheses in designing the \gls{dms} system was that the higher cell density
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would lead to greater local signaling. Since we observed higher \ptmemp{} across
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many conditions, we hypothesized that \gls{il15} may be responsible for this,
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and further that the unique \textit{cis/trans} activity of \gls{il15} may be
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more active in the \gls{dms} system due to higher cell density.
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greater extend in \gls{dms} compared to bead cultures (\cref{fig:doe_luminex}).
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One of our driving hypotheses in designing the \gls{dms} system was that the
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higher cell density would lead to greater local signaling. Since we observed
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higher \ptmemp{} across many conditions, we hypothesized that \gls{il15} may be
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responsible for this, and further that the unique \textit{cis/trans} activity of
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\gls{il15} may be more active in the \gls{dms} system due to higher cell
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density.
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\begin{figure*}[ht!]
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\begingroup
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