ADD spade gating figure

tex/thesis.tex

@@ -2723,8 +2723,6 @@ T cells were stained using a \product{34 \gls{cytof} marker
 used according to the manufacturer’s instructions. \numrange{2e6}{3e6} stained
 cells per group were analyzed on a Fluidigm Helios.
 
-% FIGURE add the spade gating diagram from the paper
-
 Unbiased cell clusters were obtained using \gls{spade} analysis by pooling three
 representative \gls{fcs} files and running the \gls{spade} pipeline with k-means
 clustering (k = 100), arcsinh transformation with cofactor 5, density
@@ -2829,36 +2827,18 @@ are fundamentally altered by changing the number of \glspl{dms} temporally.
   \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
+\begin{figure*}[ht!]
-phenotypes, we clearly see that the number of lower differentiated cells is much
+  \begingroup
-higher in the \textit{no change} or \textit{removed} groups compared to the
+
-\textit{added} group (\cref{fig:spade_quant}). Furthermore, the \textit{removed}
+  \includegraphics{../figures/spade_gates.png}
-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
+  \endgroup
-these cells were \cdp{8} cells. When analyzing the same data using \gls{tsne},
+  \caption[SPADE Gating Strategy]
-we observe a higher fraction of CD27 and lower fraction of CD45RO in the the
+  {Gating strategy for quantifying early-differentiated T cells via
-\textit{removed} group (\cref{fig:spade_tsne_all}). When manually gating on the
+    \gls{spade}.}
-CD27+CD45RO- population, we see there is higher density in the \textit{removed}
+  \label{fig:spade_gates}
-group, indicating more of this population (\cref{fig:spade_tsne_stem}).
+\end{figure*}
-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
@@ -2890,6 +2870,38 @@ do the inverse.
   \label{fig:spade}
 \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,fig:spade_gates}). 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.
+
 \subsection{blocking integrin binding does not alter expansion or phenotype}
 
 % BACKGROUND add background into why integrins are important