From 35ba50b7e6ea4ee6dfc8375eb5fe03746ef28d53 Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Wed, 28 Jul 2021 15:32:35 -0400 Subject: [PATCH] ADD results for mouse 2 experiment --- figures/mouse_timecourse_ivis.svg | 139 +---- figures/mouse_timecourse_qc.svg | 818 +++++++++++++++--------------- tex/thesis.tex | 65 ++- 3 files changed, 490 insertions(+), 532 deletions(-) diff --git a/figures/mouse_timecourse_ivis.svg b/figures/mouse_timecourse_ivis.svg index c3e741b..91b66eb 100644 --- a/figures/mouse_timecourse_ivis.svg +++ b/figures/mouse_timecourse_ivis.svg @@ -293,9 +293,9 @@ borderopacity="1.0" inkscape:pageopacity="1" inkscape:pageshadow="2" - inkscape:zoom="1.4" - inkscape:cx="200.71429" - inkscape:cy="291.07143" + inkscape:zoom="1.979899" + inkscape:cx="217.94041" + inkscape:cy="243.6993" inkscape:document-units="px" inkscape:current-layer="layer1" showgrid="true" @@ -448,7 +448,7 @@ preserveAspectRatio="none" 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@@ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 0.00% - 2.00% - 4.00% - 6.00% - 8.00% - - - - - - - - - - - - - - - - - - - - - - - - - - - - Day 6 - Day 10 - Day 14 - - - - - - - - - - CAR+CD4+ % (of CD3+) - - - - - - - - - - - - - - - - Method - - - - - - - - - - - - - - - - - - - Bead - - - - - - - DMS - - - - - - - - - - - - - - - d. + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 0.0% + 20.0% + 40.0% + 60.0% + + + + + + + + + + + + + + + + + + + + + + + + + + + Day 6 + Day 10 + Day 14 + + + + + + + + + + CD4 Fraction (of CD3+) + + + + + + + + + + + + + + + + Method + + + + + + + + + + + + + + + + + + + Bead + + + + + + + DMS + + + + + + + + + + + + + + + diff --git a/tex/thesis.tex b/tex/thesis.tex index 9921fa9..da9fc98 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -102,6 +102,9 @@ \newacronym{tnfa}{TNF$\upalpha$}{tumor necrosis factor-$\upalpha$} \newacronym{sql}{SQL}{structured query language} \newacronym{fcs}{FCS}{flow cytometry standard} +\newacronym{ivis}{ivis}{in vivo imaging system} +\newacronym{iacuc}{IACUC}{institutional animal care and use committee} + %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % SI units for uber nerds @@ -2232,18 +2235,18 @@ day 0 (-7 day relative to T cell injection), 1e6 firefly luciferase-expressing via tail vein into each mouse. At day 7, saline or CAR T cells at the indicated doses from either bead or DMS-expanded T cell cultures (for 14 days) were injected into each mouse via tail vein. Tumor burden was quantified -longitudinally via IVIS Spectrum In Vivo Imaging System (Perkin Elmer). Briefly, -200ug/mice luciferin at 15 mg/ml in PBS was injected intraperitoneally under -isoflurane anesthesia into each mouse and waited for at least 10 minutes before -imaging. Mice were anesthetized again and imaged using the IVIS. Mice from each +longitudinally via an \gls{ivis} Spectrum (Perkin Elmer). Briefly, 200ug/mice +luciferin at 15 mg/ml in PBS was injected intraperitoneally under isoflurane +anesthesia into each mouse and waited for at least 10 minutes before imaging. +Mice were anesthetized again and imaged using the \gls{ivis}. Mice from each treatment group/dose were anesthetized, injected, and imaged together, and -exposure time of the IVIS was limited to avoid saturation based on the signal -from the saline group. IVIS images were processed by normalizing them to common -minimum and maximum photon counts and total flux was estimated in terms of -photons/second. Endpoint for each mouse was determined by IACUC euthanasia -criteria (hunched back, paralysis, blindness, lethargy, and weight loss). -Mice were euthanized according to these endpoint criteria using carbon dioxide -asphyxiation. +exposure time of the \gls{ivis} was limited to avoid saturation based on the +signal from the saline group. \gls{ivis} images were processed by normalizing +them to common minimum and maximum photon counts and total flux was estimated in +terms of photons/second. Endpoint for each mouse was determined by \gls{iacuc} +euthanasia criteria (hunched back, paralysis, blindness, lethargy, and weight +loss). Mice were euthanized according to these endpoint criteria using carbon +dioxide asphyxiation. \subsection{statistics} @@ -2384,6 +2387,15 @@ case of beads (\cref{fig:mouse_dosing_qc_mem}). \subsection{Beads and DMSs perform similarly at earlier timepoints} +We then asked how T cells harvested using either beads or \gls{dms} performed +when harvested at earlier timepoints {\#}{levine paper with early timepoints}. +We performed the same experiments as described in +\cref{fig:mouse_dosing_overview} with the modification that T cells were only +grown and harvested after \SI{6}{\day}, \SI{10}{\day}, or \SI{14}{\day} of +expansion (\cref{fig:mouse_timecourse_overview}). T cells were frozen after +harvest, and all timepoints were thawed at the same time prior to injection. All +other characteristics of the experiment were the same. + \begin{figure*}[ht!] \begingroup @@ -2397,6 +2409,22 @@ case of beads (\cref{fig:mouse_dosing_qc_mem}). \label{fig:mouse_timecourse_overview} \end{figure*} +% TODO find literature saying that CAR T cells grow slower +As was the case with the first \invivo{} experiment, T cells activated with +\glspl{dms} expanded much more efficiently compared to those expanded with beads +(\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} of T +cells harvested at each timepoint, we noted that the bead group had much higher +\ptcar{} expression at earlier timpoints compared to \gls{dms}, while they +equalized at later timepoints (\cref{fig:mouse_timecourse_qc_car}). In addition, +overall \ptcar{} expression decreased at later timepoints, indicating that +\gls{car} transduced T cells either grow slower or die faster compared to +untransduced cells. The \pthp{} of the harvested T cells was higher overall in +\gls{dms} expanded T cells but decreased with increasing timepoints +(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6 +between bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{} +at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere +(\cref{fig:mouse_timecourse_qc_mem}) {\#}{levine paper mem over time}. + \begin{figure*}[ht!] \begingroup @@ -2420,6 +2448,21 @@ case of beads (\cref{fig:mouse_dosing_qc_mem}). \label{fig:mouse_timecourse_qc} \end{figure*} +We analyzed the tumor burden using \gls{ivis} which showed that mice that +received T cells from any group performed better than those that received only +saline (\cref{fig:mouse_timecourse_ivis}). Note that unlike the previous +experiment, many of the mice survived until day 40 at which point \gls{gvhd} +began to take effect (after euthanizing the mice at day 42, most had small or no +spleen). When comparing bead and \gls{dms} groups, the \gls{dms} T cells still +seemed superior to the bead group, at least initially (note that in this case +they had similar numbers of \ptcar{} cells). At day 6, both \gls{dms} and bead +groups seemed to eradicate the tumor initially, after which it came back after +day 21 for the bead and day 28 for the \gls{dms} group. The day 10 groups +performed somewhere in between, where they increased linearly unlike the day 6 +groups but not as quickly as the day 14 groups. In the case of the \gls{dms} day +10 group, it also appeared like a few mice actually performed better than all +other groups in regard to the final tumor burden. + \begin{figure*}[ht!] \begingroup