From 830702d95fcb43b73ddb32104a60340aa73f9848 Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Wed, 28 Jul 2021 17:59:19 -0400 Subject: [PATCH] ADD result for spade stuff --- tex/thesis.tex | 72 +++++++++++++++++++++++++++++++++++++++++++++++++- 1 file changed, 71 insertions(+), 1 deletion(-) diff --git a/tex/thesis.tex b/tex/thesis.tex index 5d60c10..de1da9a 100644 --- a/tex/thesis.tex +++ b/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