ENH integrate some of the new figures

tex/thesis.tex

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