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+\newacronym{tsne}{tSNE}{t-stochastic neighbor embedding} %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % SI units for uber nerds @@ -718,7 +721,7 @@ entailed adding sterile \gls{pbs} in a 10:1 volumetric ratio, agitating at reaction volume. To coat with \gls{stp}, \SI{40}{\ug\per\mL} \product{\gls{stp}}{Jackson - Immunoresearch}{ 016-000-114} was added and allowed to react for + Immunoresearch}{016-000-114} was added and allowed to react for \SI{60}{\minute} at \SI{700}{RPM} of agitation. After the reaction, supernatant was taken for the \gls{bca} assay, and the carriers were washed analogously to the previous wash step to remove the biotin, except two washes were done and the @@ -1063,6 +1066,8 @@ cells after \SI{14}{\day} were on the interior surface of the \glspl{dms} %, and this did not significantly change with initial seeding density (Supplemental Table 1). +% FIGURE caspase/bcl2 figure + % TODO add this to the methods section % TODO double check the timing of this experiment (it might not be day 14) % TODO provide the regression results and coefficients from this @@ -1553,6 +1558,7 @@ provide these benefits. \subsection{DOE shows optimal conditions for expanded potent T cells} +% FIGURE IL2 modulation figure % TODO not all of these were actually use, explain why by either adding columns % or marking with an asterisk @@ -1642,6 +1648,142 @@ provide these benefits. \section{introduction} \section{methods} \section{results} + +\subsection{adding or removing DMSs alters expansion and phenotype} + +% TODO this figure is tall and skinny like me +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/collagenase.png} + + \endgroup + \caption[Effects Collagenase Treatment on T cells] + {T cells treated with either \gls{colb}, \gls{cold}, or buffer and then + stained for various surface markers and analyzing via flow cytometry.} + \label{fig:collagenase_fx} +\end{figure*} + +% TODO this figure still says "carrier" +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/add_remove_endpoint.png} + \phantomsubcaption\label{fig:add_rem_growth} + \phantomsubcaption\label{fig:add_rem_viability} + \phantomsubcaption\label{fig:add_rem_cd4} + + \endgroup + \caption[Endpoint results from adding/removing \gls{dms} on day 4] + {Changing \gls{dms} concentration on day 4 has profound effects on phenotype + and growth. + \subcap{fig:add_rem_growth}{Longitudinal fold change}, + \subcap{fig:add_rem_viability}{longitudinal viability}, and + \subcap{fig:add_rem_cd4}{day 14 \pthp{}} of T cell cultures with \glspl{dms} + added, removed, or kept the same on day 4. + } + \label{fig:add_rem} +\end{figure*} + +% TODO this needs some better annotations +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/add_remove_spade.png} + \phantomsubcaption\label{fig:spade_msts} + \phantomsubcaption\label{fig:spade_tsne_all} + \phantomsubcaption\label{fig:spade_tsne_stem} + \phantomsubcaption\label{fig:spade_quant} + + \endgroup + \caption[SPADE and tSNE analysis temporally-modified DMS concentration] + {Removing \glspl{dms} leads to a higher fraction of potent stem-memory T + cells compared to both adding and not changing the \gls{dms} concentration + at day 4. + \subcap{fig:spade_msts}{SPADE plots of CD4, CD45RA, CD27, and CD45RO + expression on T cells. All cells from the added, removed, or no change + groups were pooled and clustered at once.} + \subcap{fig:spade_tsne_all}{\gls{tsne} plots of all cells pooled from all + groups.} + \subcap{fig:spade_tsne_stem}{\gls{tsne} plots of T cells from all groups + manually gated on \cdp{8}\cdp{27}\cdp{45RO}.} + \subcap{fig:spade_quant}{T cells from SPADE plots clustered by expression in + (\subref{fig:spade_msts}) quantified against total cell number from each + group.} + } + \label{fig:spade} +\end{figure*} + +\subsection{blocking integrin binding does not alter expansion or phenotype} + +% TODO perhaps these figs should be combined +% TODO actually make the captions for these +% TODO add some background into why integrins are important and the proposed mechanism +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/integrin_1.png} + \phantomsubcaption\label{fig:inegrin_1_fc} + \phantomsubcaption\label{fig:inegrin_1_mem} + \phantomsubcaption\label{fig:inegrin_1_cd49} + + \endgroup + \caption[Integrin blocking I] + {Blocking with integrin does not lead to differences in memory or growth. + } + \label{fig:integrin_1} +\end{figure*} + +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/integrin_2.png} + \phantomsubcaption\label{fig:inegrin_2_fc} + \phantomsubcaption\label{fig:inegrin_2_mem} + + \endgroup + \caption[Integrin blocking II] + {Blocking with integrin does not lead to differences in memory or growth. + } + \label{fig:integrin_2} +\end{figure*} + +\subsection{blocking IL15 signaling does not alter expansion or phenotype} + +% TODO actually add captions +% TODO add fold change and viability to these +% TODO maybe combine these +% TODO add some background into why IL15 is important and the proposed mechanism +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/il15_blockade_1.png} + \phantomsubcaption\label{fig:il15_1_fc} + \phantomsubcaption\label{fig:il15_1_viability} + \phantomsubcaption\label{fig:il15_1_mem} + + \endgroup + \caption[IL15 blocking I] + {Blocking with IL15 does not lead to differences in memory or growth. + } + \label{fig:il15_1} +\end{figure*} + +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/il15_blockade_2.png} + \phantomsubcaption\label{fig:il15_2_fc} + \phantomsubcaption\label{fig:il15_2_viability} + \phantomsubcaption\label{fig:il15_2_mem} + + \endgroup + \caption[IL15 blocking II] + {Blocking with IL15 does not lead to differences in memory or growth. + } + \label{fig:il15_2} +\end{figure*} + \section{discussion} \chapter{aim 3}\label{aim3}