ADD result for spade stuff

tex/thesis.tex

@@ -40,6 +40,8 @@
 \renewcommand{\glossarysection}[2][]{} % remove glossary title
 \makeglossaries
 \newacronym{act}{ACT}{adoptive cell therapies}
+\newacronym{tcm}{T\textsubscript{cm}}{central memory T cell}
+\newacronym{tscm}{T\textsubscript{scm}}{stem-memory T cell}
 \newacronym{car}{CAR}{chimeric antigen receptor}
 \newacronym[longplural={monoclonal antibodies}]{mab}{mAb}{monoclonal antibody}
 \newacronym{ecm}{ECM}{extracellular matrix}
@@ -2083,7 +2085,7 @@ provide these benefits.
 \section{introduction}
 \section{methods}
 
-\subsection{collagenase digestion}
+\subsection{DMSs temporal modulation}
 
 % TODO The concentration for the surface marker cleavage experiment was much
 % higher, if that matters
@@ -2097,6 +2099,10 @@ media normally used to feed the cells during the regular media addition cycle at
 day 4. Cultures were then incubated as described in \cref{sec:tcellculture}, and
 the \glspl{dms} were verified to have been digested after \SI{24}{\hour}.
 
+Adding \gls{dms} was relatively much simpler; the number of \gls{dms} used per
+area on day 0 was scaled up by 3 on day 4 to match the change from a 96 well
+plate to a 24 well plate, effectively producing a constant activation signal.
+
 \subsection{mass cytometry and clustering analysis}
 
 T cells were stained using a \product{34 \gls{cytof} marker
@@ -2144,6 +2150,22 @@ To block soluble \gls{il15}, we supplemented analogously with
 
 \subsection{adding or removing DMSs alters expansion and phenotype}
 
+% TODO state what collagenase actually targets
+We hypothesized that adding or removing \gls{dms} in the middle of an active
+culture would alter the activation signal and hence the growth trajectory and
+phenotype of T cells. While adding \glspl{dms} was simple, the easiest way to
+remove \glspl{dms} was to use enzymatic digestion. Collagenase is an enzyme that
+specifically targets the blabla domain on collagen. Since our \glspl{dms} are
+composed of porcine-derived collagen, this enzyme should target the \gls{dms}
+while sparing the cells. We tested this specific hypothesis using either
+\gls{colb}, \gls{cold} or \gls{hbss}, and stained the cells using a typical
+marker panel to assess if any of the markers were cleaved off by the enzyme
+which would bias our final readout. We observed that the marker histograms in
+the \gls{cold} group were similar to that of the buffer group, while the
+\gls{colb} group visibly lowered CD62L and CD4, indicating partial
+enzymatic cleavage (\cref{fig:collagenase_fx}). Based on this result, we used
+\gls{cold} moving forward.
+
 % TODO this figure is tall and skinny like me
 \begin{figure*}[ht!]
   \begingroup
@@ -2157,6 +2179,22 @@ To block soluble \gls{il15}, we supplemented analogously with
   \label{fig:collagenase_fx}
 \end{figure*}
 
+When either adding more \glspl{dms}, removing \glspl{dms} using \gls{cold}, or
+doing nothing, we observed that, counterintuitively, cell growth seemed to be
+inhibited in the \textit{added} group while the cells seemed to grow faster in
+the \textit{removed} group relative to the \textit{no change} group
+(\cref{fig:add_rem_growth}). Additionally, the \textit{removed} group seemed to
+have a negative growth rate in the final \SI{4}{\day} of culture, indicating
+that either the lack activation signal had slowed the cell growth down or that
+the cells were growing fast enough to outpace the media feeding schedule. The
+viability was the same between all groups, indicating that this negative growth
+rate and the lower growth rate in the \textit{added} group were likely not due
+to cell death (\cref{fig:add_rem_viability}). Interestingly, the \textit{added}
+group had significantly higher \pth{} cells compared to the \textit{no change}
+group, and the inverse was true for the \textit{removed} group
+(\cref{fig:add_rem_cd4}). These results show that the growth rate and phenotype
+are fundamentally altered by changing the number of \glspl{dms} temporally.
+
 % TODO this figure still says "carrier"
 \begin{figure*}[ht!]
   \begingroup
@@ -2178,7 +2216,39 @@ To block soluble \gls{il15}, we supplemented analogously with
   \label{fig:add_rem}
 \end{figure*}
 
+We next asked what the effect of removing the \glspl{dms} would have on other
+phenotypes, specifically \gls{tcm} and \gls{tscm} cells. To this end we stained
+cells using a 34-marker mass cytometry panel and analyzed them using a Fluidigm
+Helios. After pooling the \gls{fcs} file events from each group and analyzing
+them via \gls{spade} we see that there is a strong bifurcation of CD4 and CD8 T
+cells. We also observe that among CD27, CD45RA, and CD45RO (markers commonly
+used to identify \gls{tcm} and \gls{tscm} subtypes) we see clear `metaclusters'
+composed of individual \gls{spade} clusters which are high for that marker
+(\cref{fig:spade_msts}). We then gated each of these metaclusters according to
+their marker levels and assigned them to one of three phenotypes for both the
+CD4 and CD8 compartments: \gls{tcm} (high CD45RO, low CD45RA, high CD27),
+\gls{tscm} (low CD45RO, high CD45RA, high CD27), and `transitory' \gls{tscm}
+cells (mid CD45RO, mid CD45RA, high CD27). Together these represent low
+differentiated cells which should be highly potent as anti-tumor therapies.
+
+When quantifying the number of cells from each experimental group in these
+phenotypes, we clearly see that the number of lower differentiated cells is much
+higher in the \textit{no change} or \textit{removed} groups compared to the
+\textit{added} group (\cref{fig:spade_quant}). Furthermore, the \textit{removed}
+group had a much higher fraction of \gls{tscm} cells compared to the \textit{no
+  change} group, which had more `transitory \gls{tscm} cells'. The majority of
+these cells were \cdp{8} cells. When analyzing the same data using \gls{tsne},
+we observe a higher fraction of CD27 and lower fraction of CD45RO in the the
+\textit{removed} group (\cref{fig:spade_tsne_all}). When manually gating on the
+CD27+CD45RO- population, we see there is higher density in the \textit{removed}
+group, indicating more of this population (\cref{fig:spade_tsne_stem}).
+Together, these data indicate that removing \glspl{dms} at lower timepoints
+leads to potentially higher expansion, lower \pthp{}, and higher fraction of
+lower differentiated T cells such as \gls{tscm}, and adding \gls{dms} seems to
+do the inverse.
+
 % TODO this needs some better annotations
+% TODO put the quant graph before the tsne stuff
 \begin{figure*}[ht!]
   \begingroup