ADD results for mouse 2 experiment

This commit is contained in:
Nathan Dwarshuis 2021-07-28 15:32:35 -04:00
parent 21eab442ee
commit 35ba50b7e6
3 changed files with 490 additions and 532 deletions

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@ -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