ENH proofread aim 3

tex/references.bib

@@ -2729,6 +2729,19 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec
   isbn      = {0262111705},
 }
 
+@Article{GomesSilva2017,
+  author    = {Diogo Gomes-Silva and Malini Mukherjee and Madhuwanti Srinivasan and Giedre Krenciute and Olga Dakhova and Yueting Zheng and Joaquim M.S. Cabral and Cliona M. Rooney and Jordan S. Orange and Malcolm K. Brenner and Maksim Mamonkin},
+  journal   = {Cell Reports},
+  title     = {Tonic 4-1BB Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector-Dependent},
+  year      = {2017},
+  month     = {oct},
+  number    = {1},
+  pages     = {17--26},
+  volume    = {21},
+  doi       = {10.1016/j.celrep.2017.09.015},
+  publisher = {Elsevier {BV}},
+}
+
 @Comment{jabref-meta: databaseType:bibtex;}
 
 @Comment{jabref-meta: grouping:

tex/thesis.tex

@@ -178,7 +178,7 @@
 \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{ivis}{IVIS}{in vivo imaging system}
 \newacronym{iacuc}{IACUC}{institutional animal care and use committee}
 \newacronym{hbss}{HBSS}{Hank's buffered saline solution}
 \newacronym{leaf}{LEAF}{low endotoxin, azide-free}
@@ -4047,31 +4047,34 @@ potency\cite{Ghassemi2018}.
 
 \subsection{CD19-CAR T cell generation}
 
-% METHOD describe how T cells were grown for this aim
+\subsection{T cell culture}
+
+T cells were grown as described in \cref{sec:tcellculture}.
 
-% METHOD describe how the luciferase cells were generated (eg the kwong lab)
 
 \subsection{\invivo{} therapeutic efficacy in NSG mice model}
 
+% METHOD describe how the luciferase cells were generated (eg the kwong lab)
 % METHOD use actual product numbers for mice
 All mice in this study were male \gls{nsg} mice from Jackson Laboratories. At
-day 0 (-7 day relative to T cell injection), 1e6 firefly luciferase-expressing
+day 0 (\SI{-7}{\day} relative to T cell injection), 1e6 firefly
-\product{Nalm-6 cells}{ATCC}{CRL-3273} suspended in ice-cold PBS were injected
+luciferase-expressing \product{Nalm-6 cells}{ATCC}{CRL-3273} suspended in
-via tail vein into each mouse. At day 7, saline or CAR T cells at the indicated
+ice-cold \gls{pbs} were injected via tail vein into each mouse. At day 7, saline
-doses from either bead or DMS-expanded T cell cultures (for 14 days) were
+or \gls{car} T cells at the indicated doses from either bead or
-injected into each mouse via tail vein. Tumor burden was quantified
+\gls{dms}-expanded T cell cultures (for \SI{14}{\day}) were injected into each
-longitudinally via an \gls{ivis} Spectrum (Perkin Elmer). Briefly, 200ug/mice
+mouse via tail vein. Tumor burden was quantified longitudinally via an
-luciferin at 15 mg/ml in PBS was injected intraperitoneally under isoflurane
+\gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug} luciferin at
-anesthesia into each mouse and waited for at least 10 minutes before imaging.
+\SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally under isoflurane
-Mice were anesthetized again and imaged using the \gls{ivis}. Mice from each
+anesthesia into each mouse and allowed to circulate for at least
-treatment group/dose were anesthetized, injected, and imaged together, and
+\SI{10}{\minute} before imaging. Mice were anesthetized again and imaged using
-exposure time of the \gls{ivis} was limited to avoid saturation based on the
+the \gls{ivis}. Mice from each treatment group/dose were anesthetized, injected,
-signal from the saline group. \gls{ivis} images were processed by normalizing
+and imaged together, and exposure time of the \gls{ivis} was limited to avoid
-them to common minimum and maximum photon counts and total flux was estimated in
+saturation based on the signal from the saline group. \gls{ivis} images were
-terms of photons/second. Endpoint for each mouse was determined by \gls{iacuc}
+processed by normalizing them to common minimum and maximum photon counts and
-euthanasia criteria (hunched back, paralysis, blindness, lethargy, and weight
+total flux was estimated in terms of photons/second. Endpoint for each mouse was
-loss). Mice were euthanized according to these endpoint criteria using carbon
+determined by \gls{iacuc} euthanasia criteria (hunched back, paralysis,
-dioxide asphyxiation.
+blindness, lethargy, and weight loss). Mice were euthanized according to these
+endpoint criteria using carbon dioxide asphyxiation.
 
 \subsection{statistics}
 
@@ -4081,67 +4084,6 @@ between survival groups.
 
 \section{results}
 
-We asked if the higher memory/naive phenotype and more balanced CD4/CD8 ratio of
-our \gls{dms}-expanded CAR T cells would lead to better anti-tumor potency in
-vivo compared to bead-expanded CAR T cells. We also asked if this superior
-anti-tumor potency would hold true at lower doses of CAR expressing T cells in
-the DMS group vs the bead group. To test this, we used a human xenograft model
-of B cell \gls{all} by intravenously injecting \gls{nsg} mice with \num{1e6}
-Nalm-6 tumor cells expression firefly luciferase\cite{Fraietta2018}. After
-\SI{7}{\day} of tumor cell growth (\cref{fig:mouse_dosing_overview}), we
-intravenously injected saline or three doses (high, medium, and low) of
-\gls{dms} T cells from either bead or DMS cultures expanded for \SI{14}{\day}.
-We quantified total \gls{dms} expressing T cell percentage for bead and
-\gls{dms} groups using the \gls{ptnl} assay (\cref{tab:mouse_dosing_results}).
-
-Before injecting the T cells into the mice, we quantified their phenotype and
-growth. We observed that for this expansion, the bead and \gls{dms} T cells
-produced similar numbers of \ptmem{} T cells, and the beads even had a higher
-fraction of CD45RA, which is present on lower-differentiated naive and
-stem-memory T cells (\cref{fig:mouse_dosing_qc_mem}). However, the \pthp{} of
-the final product was higher in \gls{dms} (\cref{fig:mouse_dosing_qc_cd4}). The
-\gls{dms} T cells also expanded more robustly than the beads
-(\cref{fig:mouse_dosing_qc_growth}).
-
-In the Nalm-6/\gls{nsg} xenograft model, we observed lower tumor burden and
-significantly longer survival of bead and \gls{dms}-treated mice at all doses
-compared to the saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at
-each dose we observed that the \gls{dms}-treated mice had much lower tumor
-burden and significantly higher survival than their bead-treated counterparts
-(\cref{fig:mouse_dosing_ivis_survival}). When factoring the percentage T cells
-in each dose that expressed the \gls{car}, we note that survival of the low
-\gls{dms} dose (which had similar total \gls{car} T cells compared to the bead
-medium dose and less than the bead high dose) is significantly higher than that
-of both the bead medium dose and the bead high dose
-(\cref{fig:mouse_dosing_ivis_survival_comp}). Overall, the Kaplan-Meier survival
-of Nalm-6 tumor bearing \gls{nsg} mice shown in the
-\cref{fig:mouse_dosing_ivis_survival} was up to day 40 as reported
-elsewhere\cite{Fraietta2018}. However, we also included a Kaplan-Meier figure up
-to day 46 (\cref{fig:mouse_dosing_ivis_survival_full}) where most of the mice
-euthanized from day 40 through day 46 from \gls{dms} groups showed no or very
-small fragment of spleen which was due to \gls{gvhd} responses. Similar
-\gls{gvhd} responses were reported earlier in \gls{nsg} mice where the mice
-injected with human \gls{pbmc} exhibited acute \gls{gvhd} between
-\SIrange{40}{50}{\day} post intravenous injection\cite{Ali2012}. Notably, both
-survival analyses (up to day 40 in \cref{fig:mouse_dosing_ivis_survival} and up
-to day 46 in \cref{fig:mouse_dosing_ivis_survival_full}) confirmed that
-\gls{dms}-expanded groups outperformed bead-expanded groups in terms of
-prolonging survival of Nalm-6 tumor challenged \gls{nsg} mice.
-
-Together, these data suggested that \glspl{dms} produce T cells that are not
-only more potent that bead-expanded T cells (even when accounting for
-differences in \gls{car} expression) but also showed that \gls{dms} expanded T
-cells are effective at lower doses. Given the quality control data of the T
-cells prior to injecting into the mice, it seems that this advantage is either
-due to the higher \pthp{} or the overall fitness of the T cells given the higher
-expansion in the case of \gls{dms}
-(\cref{fig:mouse_dosing_qc_cd4,fig:mouse_dosing_qc_growth}). It was likely not
-due to the memory phenotype given that it was actually slightly higher in the
-case of beads (\cref{fig:mouse_dosing_qc_mem}).
-
-\subsection{DMS-expanded T cells show greater anti-tumor activity \invivo{}
-  compared to beads}
-
 \begin{figure*}[ht!]
   \begingroup
 
@@ -4160,6 +4102,9 @@ case of beads (\cref{fig:mouse_dosing_qc_mem}).
   \input{../tables/mouse_dose_car.tex}
 \end{table}
 
+\subsection{DMS-expanded T cells show greater anti-tumor activity \invivo{}
+  compared to beads}
+
 % FIGURE put growth first in this figure
 \begin{figure*}[ht!]
   \begingroup
@@ -4210,6 +4155,64 @@ case of beads (\cref{fig:mouse_dosing_qc_mem}).
   \label{fig:mouse_dosing_ivis}
 \end{figure*}
 
+We asked if the higher memory/naive phenotype and more balanced CD4/CD8 ratio of
+our \gls{dms}-expanded \gls{car} T cells would lead to better anti-tumor potency
+in vivo compared to bead-expanded \gls{car} T cells. We also asked if this
+superior anti-tumor potency would hold true at lower doses of \gls{car}
+expressing T cells in the DMS group vs the bead group. To test this, we used a
+human xenograft model of B cell \gls{all} by intravenously injecting \gls{nsg}
+mice with \num{1e6} Nalm-6 tumor cells expression firefly
+luciferase\cite{Fraietta2018}. After \SI{7}{\day} of tumor cell growth
+(\cref{fig:mouse_dosing_overview}), we intravenously injected saline or three
+doses (high, medium, and low) of \gls{car} T cells from either bead or \gls{dms}
+cultures expanded for \SI{14}{\day}. We quantified \ptcarp{} bead and \gls{dms}
+groups using the \gls{ptnl} assay (\cref{tab:mouse_dosing_results}).
+
+Before injecting the T cells into the mice, we quantified their phenotype and
+growth. We observed that for this expansion, the bead and \gls{dms} T cells
+produced similar numbers of \ptmem{} T cells, and the beads even had a higher
+fraction of CD45RA, which is present on lower-differentiated \glspl{tn} and
+\glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the \pthp{} of
+the final product was higher in \gls{dms} (\cref{fig:mouse_dosing_qc_cd4}). The
+\gls{dms} T cells also expanded more robustly than the beads
+(\cref{fig:mouse_dosing_qc_growth}).
+
+In the Nalm-6/\gls{nsg} xenograft model, we observed lower tumor burden and
+significantly longer survival of bead and \gls{dms}-treated mice at all doses
+compared to the saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at
+each dose we observed that the \gls{dms}-treated mice had much lower tumor
+burden and significantly higher survival than their bead-treated counterparts
+(\cref{fig:mouse_dosing_ivis_survival}). When factoring the percentage T cells
+in each dose that expressed the \gls{car}, we note that survival of the low
+\gls{dms} dose (which had similar total \gls{car} T cells compared to the bead
+medium dose and less than the bead high dose) is significantly higher than that
+of both the bead medium dose and the bead high dose
+(\cref{fig:mouse_dosing_ivis_survival_comp}). Overall, the Kaplan-Meier survival
+of Nalm-6 tumor bearing \gls{nsg} mice shown in the
+\cref{fig:mouse_dosing_ivis_survival} was up to day 40 as reported
+elsewhere\cite{Fraietta2018}. However, we also included a Kaplan-Meier figure up
+to day 46 (\cref{fig:mouse_dosing_ivis_survival_full}) where most of the mice
+euthanized from day 40 through day 46 from \gls{dms} groups showed no or very
+small fragment of spleen which was due to \gls{gvhd} responses. Similar
+\gls{gvhd} responses were reported earlier in \gls{nsg} mice where the mice
+injected with human \gls{pbmc} exhibited acute \gls{gvhd} between
+\SIrange{40}{50}{\day} post intravenous injection\cite{Ali2012}. Notably, both
+survival analyses (up to day 40 in \cref{fig:mouse_dosing_ivis_survival} and up
+to day 46 in \cref{fig:mouse_dosing_ivis_survival_full}) confirmed that
+\gls{dms}-expanded groups outperformed bead-expanded groups in terms of
+prolonging survival of Nalm-6 tumor challenged \gls{nsg} mice.
+
+Together, these data suggested that \glspl{dms} produce T cells that are not
+only more potent that bead-expanded T cells (even when accounting for
+differences in \gls{car} expression) but also showed that \gls{dms} expanded T
+cells are effective at lower doses. Given the quality control data of the T
+cells prior to injecting into the mice, it seems that this advantage is either
+due to the higher \pthp{} or the overall fitness of the T cells given the higher
+expansion in the case of \gls{dms}
+(\cref{fig:mouse_dosing_qc_cd4,fig:mouse_dosing_qc_growth}). It was likely not
+due to the memory phenotype given that it was actually slightly higher in the
+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
@@ -4236,7 +4239,6 @@ same.
   \label{fig:mouse_timecourse_overview}
 \end{figure*}
 
-% RESULT 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
@@ -4244,7 +4246,7 @@ 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
+\gls{car} transduced T cells either grow slower or died 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
@@ -4332,20 +4334,21 @@ other groups in regard to the final tumor burden.
   \label{fig:mouse_summary}
 \end{figure*}
 
-The total number of T cells for each \invivo{} experiment are shown in
+The total number of T cells injected for each \invivo{} experiment are shown in
 \cref{fig:mouse_summary}.
 
-When we tested bead and DMS expanded \gls{car} T cells, we found that the
+When we tested bead and \gls{dms} expanded \gls{car} T cells, we found that the
-\gls{dms} expanded CAR-T cells outperformed bead groups in prolonging survival
+\gls{dms} expanded \gls{car} T cells outperformed bead groups in prolonging
-of Nalm-6 tumor challenged (intravenously injected) \gls{nsg} mice. DMS expanded
+survival of Nalm-6 tumor challenged (intravenously injected) \gls{nsg} mice.
-CAR-T cells were very effective in clearing tumor cells as early as 7 days post
+\gls{dms} expanded CAR-T cells were very effective in clearing tumor cells as
-CAR-T injection even at low total T cell dose compared to the bead groups where
+early as \SI{7}{\day} post \gls{car} T injection even at low total T cell dose
-tumor burden was higher than DMS groups across all the total T cell doses tested
+compared to the bead groups where tumor burden was higher than \gls{dms} groups
-here. More interestingly, when only CAR-expressing T cell doses between bead and
+across all the total T cell doses tested here. More interestingly, when only
-DMS groups were compared, DMS group had significantly higher survival effects
+\gls{car}-expressing T cell doses between bead and \gls{dms} groups were
-over similar or higher CAR expression T cell doses from bead group. All these
+compared, \gls{dms} group had significantly higher survival effects over similar
-results suggest that the T cells in DMS groups (compared to bead group) resulted
+or higher CAR expression T cell doses from bead group. All these results suggest
-in highly effective CAR-T cells that can efficiently kill tumor cells.
+that the T cells in \gls{dms} groups (compared to bead group) resulted in highly
+effective \gls{car} T cells that can efficiently kill tumor cells.
 
 When comparing the total number of T cells of different phenotypes, we observed
 that when comparing low-dose \gls{dms} group to the mid- bead groups (which had
@@ -4379,17 +4382,20 @@ experiment relative to the first (\cref{fig:mouse_summary}). Only the day 6
 group had \gls{car} T cell numbers comparable to the weakest dose of bead cells
 given in the first experiment, and these T cells were harvested at earlier
 timepoints than the first mouse experiment and thus may not be safely
-comparable. The lower overall \gls{car} doses may explain why at best, the tumor
+comparable. Furthermore, the \ptcarp{} decreased over time, which suggested that
-seemed to be in remission only temporarily. Even so, the \gls{dms} group seemed
+the transduced T cells grew slower. This has been observed elsewhere and could
-to perform better at day 6 as it held off the tumor longer, and also slowed the
+be due to tonic signaling\cite{GomesSilva2017}. The lower overall \gls{car}
-tumor progression relative to the bead group at day 14
+doses may explain why at best, the tumor seemed to be in remission only
-(\cref{fig:mouse_timecourse_ivis_plots}).
+temporarily. Even so, the \gls{dms} group seemed to perform better at day 6 as
+it held off the tumor longer, and also slowed the tumor progression relative to
+the bead group at day 14 (\cref{fig:mouse_timecourse_ivis_plots}).
 
 Taken together, these data suggest that the \gls{dms} platform produces T cells
 that have an advantage \invivo{} over beads. While we may not know the exact
 mechanism, our data suggests that the responses are unsurprisingly influenced by
 the \ptcarp{} of the final product. Followup experiments would need to be
-performed to determine the precise phenotype responsible for these responses.
+performed to determine the precise phenotype responsible for these responses in
+our hands.
 
 \chapter{conclusions and future work}\label{conclusions}
 
@@ -4474,8 +4480,6 @@ integrin interactions with the collagen surface. In the case of \gls{il15} more
 experiments likely need to be done in order to plausibly rule out this mechanism
 and/or determine if it is involved at all.
 
-% TODO make this tighter and cite paper showing that this makes at least some
-% sense
 In \cref{aim3} we determined that the \glspl{dms} expand T cells that also
 performed better than beads \invivo{}. In the first experiment we performed, the
 results were very clearly in favor of the \glspl{dms}. In the second experiment,
@@ -4483,11 +4487,11 @@ even the \gls{dms} group failed to fully control the tumor burden, but this is
 not surprising given the low \ptcarp{} across all groups. Also, despite this,
 the \gls{dms} group appeared to control the tumor better on average for early,
 mid, and late T cell harvesting timepoints. It was not clear if this effect was
-due to increased \cdp{} or overall increased fitness of the \gls{dms}-expanded T
+due to increased \pthp{}, \ptmemp{}, or fitness of the \gls{dms}-expanded T
-cells given their higher expansion rate. The \ptmemp{} did not seem to be a
+cells given their higher expansion rate. More data is needed to establish which
-factor given that it was nearly the same in the first experiment between
+phenotype is responsible for the results we observed, as we did not include the
-\gls{dms} and bead groups despite the clear advantage seen in the \gls{dms}
+\gls{car} in the same panel as the other phenotype surface markers, making it
-group.
+difficult to reliably say the identity of the \ptcar{} cells.
 
 Finally, while we have demonstrated the \gls{dms} system in the context of
 \gls{car} T cells, this method can theoretically be applied to any T cell