From b0dfe664c36745752d75647f3b3e59021a447974 Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Tue, 7 Sep 2021 13:56:08 -0400 Subject: [PATCH] ENH split Cd19 car fig --- figures/{car_production.svg => car_cd19.svg} | 2809 +--------------- figures/car_degranulation.svg | 3047 ++++++++++++++++++ tex/thesis.tex | 46 +- 3 files changed, 3081 insertions(+), 2821 deletions(-) rename figures/{car_production.svg => car_cd19.svg} (52%) create mode 100644 figures/car_degranulation.svg diff --git a/figures/car_production.svg b/figures/car_cd19.svg similarity index 52% rename from figures/car_production.svg rename to figures/car_cd19.svg index c5f448a..f25cd04 100644 --- a/figures/car_production.svg +++ b/figures/car_cd19.svg @@ -8,7 +8,7 @@ version="1.1" id="svg8" inkscape:version="1.1 (c4e8f9ed74, 2021-05-24)" - sodipodi:docname="car_production.svg" + sodipodi:docname="car_cd19.svg" xmlns:inkscape="http://www.inkscape.org/namespaces/inkscape" xmlns:sodipodi="http://sodipodi.sourceforge.net/DTD/sodipodi-0.dtd" xmlns="http://www.w3.org/2000/svg" @@ -277,15 +277,15 @@ inkscape:pageopacity="1" inkscape:pageshadow="2" inkscape:zoom="0.98994949" - inkscape:cx="125.76399" - inkscape:cy="272.23611" + inkscape:cx="125.25892" + inkscape:cy="271.22596" inkscape:document-units="px" inkscape:current-layer="layer1" showgrid="true" units="in" inkscape:window-width="1920" - inkscape:window-height="1060" - inkscape:window-x="3840" + inkscape:window-height="1036" + inkscape:window-x="1920" inkscape:window-y="20" inkscape:window-maximized="0" inkscape:pagecheckerboard="0"> @@ -335,26 +335,6 @@ y="14.927912" x="343.55347" sodipodi:role="line">b. - d. - c. - CAR- - CAR+ - CD107a - APC - - - K562-CD19 - - K562-wt - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 10 - - - 2 - - - - - - - - - - - - 10 - - - 3 - - - - - - - - - - - - 10 - - - 4 - - - - - - - - - - - - 10 - - - 5 - - - - - - - - - - - - - - - - - - - 0 - - - - - - - - - - - - - - - 50 - - - - - - - - - - - - - - 100 - - - - - - - - - - - - - - 150 - - - - - - - - - - - - - - 200 - - - - - - - - - - - - - - - - - CD107a+ - 4.24% - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 0 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 10 - - - 2 - - - - - - - - - - - - 10 - - - 3 - - - - - - - - - - - - 10 - - - 4 - - - - - - - - - - - - 10 - - - 5 - - - - - - - - - - - - - - - - - - - 0 - - - - - - - - - - - - - - - 50 - - - - - - - - - - - - - - 100 - - - - - - - - - - - - - - 150 - - - - - - - - - - - - - - 200 - - - - - - - - - - CD107a+ - 16.7% - - - - - - - - - - - - - - - - - - - - - - - - - - ● - ● - ● - ● - ● - ● - ● - ● - - - - - - - - - - - - - - - - - - - - - 4.0% - 8.0% - 12.0% - 16.0% - - - - - - - - - - - - - - - - - - - - - - - - - - CAR− - CAR+ - - - - - - - - - - CD8+CD107A+ diff - (CD19 − wt) - - - - - - - - - - - - - - - - Method - - - - - - - ● - - - - - - ● - - - - - - Bead - - - - - - - DMS - - - - - - - - - - - - - - - - - - - - SumSq.DFF-valuep-value - - - - Method0.000410.560.497CAR0.0260140.030.003Method:CAR0.000110.200.678Residuals0.00264 - - - - - - - - - - - - - - - - - - - - - - - - - - - ● - ● - ● - ● - ● - ● - ● - ● - ● - ● - ● - ● - - - - - - - - - - - - - - - - - - - - - 2.5% - 5.0% - 7.5% - 10.0% - 12.5% - - - - - - - - - - - - - - - - - - - - - - - - - - - CAR− - CAR+ - - - - - - - - - - Migration - (background corrected) - - - - - - - - - - - - - - - - Method - - - - - - - ● - - - - - - ● - - - - - - Bead - - - - - - - DMS - - - - - - - - - - - - - - - - e. - - - - - SumSq.DFF-valuep-value - - - - CAR0.000712.330.165Method0.0052118.270.003CAR:Method0.001314.450.068Residuals0.00238 - - - - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + image/svg+xml + + + + + + b. + a. + CAR- + CAR+ + CD107a - APC + + + K562-CD19 + + K562-wt + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 0 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 10 + + + 2 + + + + + + + + + + + + 10 + + + 3 + + + + + + + + + + + + 10 + + + 4 + + + + + + + + + + + + 10 + + + 5 + + + + + + + + + + + + + + + + + + + 0 + + + + + + + + + + + + + + + 50 + + + + + + + + + + + + + + 100 + + + + + + + + + + + + + + 150 + + + + + + + + + + + + + + 200 + + + + + + + + + + + + + + + + + CD107a+ + 4.24% + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 0 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 10 + + + 2 + + + + + + + + + + + + 10 + + + 3 + + + + + + + + + + + + 10 + + + 4 + + + + + + + + + + + + 10 + + + 5 + + + + + + + + + + + + + + + + + + + 0 + + + + + + + + + + + + + + + 50 + + + + + + + + + + + + + + 100 + + + + + + + + + + + + + + 150 + + + + + + + + + + + + + + 200 + + + + + + + + + + CD107a+ + 16.7% + + + + + + + + + + + + + + + + + + + + + + + + + + ● + ● + ● + ● + ● + ● + ● + ● + + + + + + + + + + + + + + + + + + + + + 4.0% + 8.0% + 12.0% + 16.0% + + + + + + + + + + + + + + + + + + + + + + + + + + CAR− + CAR+ + + + + + + + + + + CD8+CD107A+ diff + (CD19 − wt) + + + + + + + + + + + + + + + + Method + + + + + + + ● + + + + + + ● + + + + + + Bead + + + + + + + DMS + + + + + + + + + + + + + + + + + + + + SumSq.DFF-valuep-value + + + + Method0.000410.560.497CAR0.0260140.030.003Method:CAR0.000110.200.678Residuals0.00264 + + + + + + + + + + + + + + + + + + + + + + + + + + + ● + ● + ● + ● + ● + ● + ● + ● + ● + ● + ● + ● + + + + + + + + + + + + + + + + + + + + + 2.5% + 5.0% + 7.5% + 10.0% + 12.5% + + + + + + + + + + + + + + + + + + + + + + + + + + + CAR− + CAR+ + + + + + + + + + + Migration + (background corrected) + + + + + + + + + + + + + + + + Method + + + + + + + ● + + + + + + ● + + + + + + Bead + + + + + + + DMS + + + + + + + + + + + + + + + + c. + + + + + SumSq.DFF-valuep-value + + + + CAR0.000712.330.165Method0.0052118.270.003CAR:Method0.001314.450.068Residuals0.00238 + + + + + + diff --git a/tex/thesis.tex b/tex/thesis.tex index 2d7f1ad..0f52ca6 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -1937,7 +1937,6 @@ appeared that the \gls{mab} binding was quadratically related to biotin binding critical to controlling the amount and \glspl{mab} and thus the amount of signal the T cells receive downstream. -% FIGURE combine this DOE figure into one interaction plot \begin{figure*}[ht!] \begingroup @@ -2367,7 +2366,7 @@ generate \gls{car} T cells\cite{Tumaini2013, Lamers2014}. We added a \SI{24}{\hour} transduction step on day 1 of the \SI{14}{\day} expansion to insert an anti-CD19 \gls{car}\cite{Milone2009} and subsequently measured the surface expression of the \gls{car} on day 14 -(\cref{fig:car_production_flow_pl,fig:car_production_endpoint_pl}). We noted +(\cref{fig:car_cd19_flow,fig:car_cd19_endpoint}). We noted that there was robust \gls{car} expression in over \SI{25}{\percent} of expanded T cells, and there was no observable difference in \gls{car} expression between beads and \glspl{dms}. @@ -2380,7 +2379,7 @@ appearance of CD107a on CD8+ T cells. CD107a is found on the inner-surface of cytotoxic granules and will emerge on the surface after cytotoxic T cells are activated and degranulate. Indeed, we observed degranulation in T cells expanded with both beads and \glspl{dms}, although not to an observably different degree -(\cref{fig:car_production_flow_degran,fig:car_production_endpoint_degran}). +(\cref{fig:car_degran_flow,fig:car_degran_endpoint}). Taken together, these results indicated that the \glspl{dms} provide similar transduction efficiency compared to beads. @@ -2389,7 +2388,7 @@ for CCL21; since \glspl{dms} produced a larger percentage of naïve and memory T cells (which have CCR7, the receptor for CCL21) we would expect higher migration in \gls{dms}-expanded cells vs.\ their bead counterparts. Indeed, we noted a significantly higher migration percentage for T cells grown using \glspl{dms} -versus beads (\cref{fig:car_production_migration}). Interestingly, there also +versus beads (\cref{fig:car_degran_migration}). Interestingly, there also appeared to be a decrease in CCL21 migration between transduced and untransduced T cells expanded using beads, but this interaction effect was only weakly significant (p = 0.068). No such effect was seen for \gls{dms}-expanded T cells, @@ -2399,25 +2398,38 @@ showing that migration was likely independent of \gls{car} transduction. \begin{figure*}[ht!] \begingroup - \includegraphics{../figures/car_production.png} - \phantomsubcaption\label{fig:car_production_flow_pl} - \phantomsubcaption\label{fig:car_production_endpoint_pl} - \phantomsubcaption\label{fig:car_production_flow_degran} - \phantomsubcaption\label{fig:car_production_endpoint_degran} - \phantomsubcaption\label{fig:car_production_migration} + \includegraphics{../figures/car_cd19.png} + \phantomsubcaption\label{fig:car_cd19_flow} + \phantomsubcaption\label{fig:car_cd19_endpoint} + + \endgroup + \caption[\glspl{dms} lead to efficient CD19 transduction] + {\glspl{dms} lead to efficient CD19 transduction. + \subcap{fig:car_cd19_flow}{Representative flow cytometry plot for + transduced or untransduced T cells stained with \gls{ptnl}.} + \subcap{fig:car_cd19_endpoint}{Endpoint plots with \gls{anova} for + transduced or untransduced T cells stained with \gls{ptnl}.} + All data is from T cells expanded for \SI{14}{\day}. + } + \label{fig:car_production} +\end{figure*} + +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/car_degranulation.png} + \phantomsubcaption\label{fig:car_degran_flow} + \phantomsubcaption\label{fig:car_degran_endpoint} + \phantomsubcaption\label{fig:car_degran_migration} \endgroup \caption[\glspl{dms} produce functional \gls{car} T cells] {\glspl{dms} produce functional \gls{car} T cells. - \subcap{fig:car_production_flow_pl}{Representative flow cytometry plot for - transduced or untransduced T cells stained with \gls{ptnl}.} - \subcap{fig:car_production_endpoint_pl}{Endpoint plots with \gls{anova} for - transduced or untransduced T cells stained with \gls{ptnl}.} - \subcap{fig:car_production_flow_degran}{Representative flow plot for + \subcap{fig:car_degran_flow}{Representative flow plot for degenerating T cells.} - \subcap{fig:car_production_endpoint_degran}{Endpoint plots for transduced or + \subcap{fig:car_degran_endpoint}{Endpoint plots for transduced or untransduced T cells stained with \cd{107a} for the degranulation assay.} - \subcap{fig:car_production_migration}{Endpoint plot for transmigration assay + \subcap{fig:car_degran_migration}{Endpoint plot for transmigration assay with \gls{anova}.} All data is from T cells expanded for \SI{14}{\day}. } \label{fig:car_production}