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ENH integrate some of the new figures

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Nathan Dwarshuis 2021-07-31 20:46:41 -04:00
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@ -1390,7 +1390,7 @@ model, the \gls{mab} binding reaction should be complete within \SI{15}{\minute}
under the conditions used for our protocol (\cref{fig:dms_mab_per_time}). Note
that our unoptimized coated steps were done in \SI{45}{\minute}, which seemed
reasonable given the slightly larger hydrodynamic radius of \glspl{mab} compared
to \gls{stp} which was shown to react in \SI{30}{\minutes} experimentally. The
to \gls{stp} which was shown to react in \SI{30}{\minute} experimentally. The
results of this model should be experimentally verified.
% TODO find the actual numbers for this
@ -1415,21 +1415,6 @@ far less severe than that of \gls{snb}.
\subsection{DMSs can efficiently expand T cells compared to beads}
% RESULT add other subfigures here
We next sought to determine how our \glspl{dms} could expand T cells compared to
state-of-the-art methods used in industry. All bead expansions were performed as
per the manufacturer’s protocol, with the exception that the starting cell
densities were matched between the beads and carriers to
~\SI{2.5e6}{\cell\per\ml}. Throughout the culture we observed that T cells in
\gls{dms} culture grew in tight clumps on the surface of the \glspl{dms} as well
as inside the pores of the \glspl{dms}
(\cref{fig:dms_cells_phase,fig:dms_cells_fluor}). Furthermore, we observed that
the \glspl{dms} conferred greater expansion compared to traditional beads, and
significantly greater expansion after \SI{12}{\day} of culture {Figure X}. We
also observed no T cell expansion using \glspl{dms} coated with an isotype
control mAb compared to \glspl{dms} coated with \acd{3}/\acd{28} \glspl{mab}
{Figure X}, confirming specificity of the expansion method.
% FIGURE make sure the day on these is correct
\begin{figure*}[ht!]
\begingroup
@ -1450,7 +1435,6 @@ control mAb compared to \glspl{dms} coated with \acd{3}/\acd{28} \glspl{mab}
\label{fig:dms_cells}
\end{figure*}
% RESULT for this figure
\begin{figure*}[ht!]
\begingroup
@ -1470,26 +1454,23 @@ control mAb compared to \glspl{dms} coated with \acd{3}/\acd{28} \glspl{mab}
\label{fig:dms_expansion}
\end{figure*}
% RESULT talk about this table somewhere
\begin{table}[!h] \centering
\caption{Regression for fraction of cells in \gls{dms} at day 14}
\label{tab:inside_regression}
\input{../tables/inside_fraction_regression.tex}
\end{table}
% RESULT state the CI of what are inside the carriers
We also asked how many cells were inside the \glspl{dms} vs. free-floating in
suspension and/or loosely attached to the surface. We qualitatively verified the
presence of cells inside the \glspl{dms} using a \gls{mtt} stain to opaquely
mark cells and enable visualization on a brightfield microscope
(\cref{fig:dms_inside_bf}). After seeding \glspl{dms} at different densities and
expanding for \SI{14}{\day}, we filtered the \glspl{dms} out of the cell
suspension and digested them using dispase to free any cells attached on the
inner surface. We observed that approximately \SI{15}{\percent} of the total
cells after \SI{14}{\day} were on the interior surface of the \glspl{dms}
(\cref{fig:dms_inside_regression}).
%, and this did not significantly change with initial seeding density (Supplemental Table 1).
% DISCUSSION krish seems concerned about this isotype control figure, add some
% discussion saying that IL2 does not spontaneously activate T cells to appease
% him
We next sought to determine how our \glspl{dms} could expand T cells compared to
state-of-the-art methods used in industry. All bead expansions were performed as
per the manufacturer’s protocol, with the exception that the starting cell
densities were matched between the beads and carriers to
~\SI{2.5e6}{\cell\per\ml}. Throughout the culture we observed that T cells in
\gls{dms} culture grew in tight clumps on the surface of the \glspl{dms} as well
as inside the pores of the \glspl{dms}
(\cref{fig:dms_cells_phase,fig:dms_cells_fluor}). Furthermore, we observed that
the \glspl{dms} conferred greater expansion compared to traditional beads, and
significantly greater expansion after \SI{12}{\day} of culture
(\cref{fig:dms_expansion_bead}). We also observed no T cell expansion using
\glspl{dms} coated with an isotype control mAb compared to \glspl{dms} coated
with \acd{3}/\acd{28} \glspl{mab} (\cref{fig:dms_expansion_isotype}), confirming
specificity of the expansion method.
\begin{figure*}[ht!]
\begingroup
@ -1509,11 +1490,34 @@ cells after \SI{14}{\day} were on the interior surface of the \glspl{dms}
CellEvent dye.}
\subcap{fig:apoptosis_bcl2}{Quantification of BCL-2 expression using
\gls{elisa}. All statistical tests shown are two-tailed homoschodastic
t-tests.}
t-tests. All cells were harvested at day 8.}
}
\label{fig:dms_flowchart}
\label{fig:dms_apoptosis}
\end{figure*}
Given that the \gls{dms} system seemed to expand T cells more effectively, we
asked if this difference was due to a reduction in apoptosis or an increase in
proliferation rate (or both). We assessed the apoptotic state of T cells grown
using either bead or \gls{dms} harvested on day 8 using \gls{pi} and \gls{anv}.
\gls{anv} is a marker which stains phospholipid phosphatidylserine, which is
usually present only on the cytoplasmic surface of the cell membrane, but flips
to the outside when the cell becomes apoptotic. \gls{pi} stains the nucleus of
the cell, but only penetrates necrotic cells which have a perforated cell
membrane. When staining for these two markers and assessing via flow cytometry,
we observe that the \gls{dms}-expanded T cells have lower frequencies of
apoptotic and necrotic cells (\cref{fig:apoptosis_annV}). Furthermore, we
stained our cultures with CellEvent dye, which is an indicator of \gls{cas37},
which is activated in apoptotic cells {\#}{cas37 activation}. In line with the
\gls{pi}/\gls{anv} results, we observed that the \gls{dms} T cells had lower
frequency of \gls{cas37} expression, indicating less apoptosis for our method
(\cref{fig:apoptosis_cas}). Finally, we lysed our cells and stained for
\gls{bcl2}, which is also upregulated in apoptosis. In this case, some (but not
all) of the bead-expanded cultures showed higher \gls{bcl2} expression, which
could indicate more apoptosis in those groups (\cref{fig:apoptosis_bcl2}). None
of the \gls{dms} cultures showed similar heightened expression. Taken together,
these data suggest that the \gls{dms} platform at least in part achieves higher
expansion by lowering apoptosis of the cells in culture.
% FIGURE double check the timing of this experiment (it might not be day 14)
\begin{figure*}[ht!]
\begingroup
@ -1535,31 +1539,29 @@ cells after \SI{14}{\day} were on the interior surface of the \glspl{dms}
\label{fig:dms_inside}
\end{figure*}
\begin{table}[!h] \centering
\caption{Regression for fraction of cells in \gls{dms} at day 14}
\label{tab:inside_regression}
\input{../tables/inside_fraction_regression.tex}
\end{table}
% RESULT state the CI of what are inside the carriers
We also asked how many cells were inside the \glspl{dms} vs. free-floating in
suspension and/or loosely attached to the surface. We qualitatively verified the
presence of cells inside the \glspl{dms} using a \gls{mtt} stain to opaquely
mark cells and enable visualization on a brightfield microscope
(\cref{fig:dms_inside_bf}). After seeding \glspl{dms} at different densities and
expanding for \SI{14}{\day}, we filtered the \glspl{dms} out of the cell
suspension and digested them using dispase to free any cells attached on the
inner surface. We observed that approximately \SI{15}{\percent} of the total
cells after \SI{14}{\day} were on the interior surface of the \glspl{dms}
(\cref{fig:dms_inside_regression,tab:inside_regression}). Performing linear
regression on this data revealed that the percentage of T cells inside the
\glspl{dms} does not depend on the initial starting cell density (at least when
harvested after \SI{14}{\day}) (\cref{tab:inside_regression}).
\subsection{DMSs lead to greater expansion and memory and CD4+ phenotypes}
After observing differences in expansion, we further hypothesized that the
\gls{dms} cultures could lead to a different T cell phenotype. In particular, we
were interested in the formation of naïve and memory T cells, as these represent
a subset with higher replicative potential and therefore improved clinical
prognosis\cite{Gattinoni2011, Wang2018}. We measured naïve and memory T cell
frequency staining for CCR7 and CD62L (both of which are present on lower
differentiated T cells such as naïve, central memory, and stem memory
cells\cite{Gattinoni2012}). Using three donors, we noted again \glspl{dms}
produced more T cells over a \SI{14}{\day} expansion than beads, with
significant differences in number appearing as early after \SI{5}{\day}
(\cref{fig:dms_exp_fold_change}). Furthermore, we noted that \glspl{dms}
produced more memory/naïve cells after \SI{14}{\day} when compared to beads for
all donors (\cref{fig:dms_exp_mem,fig:dms_exp_cd4}) showing that the \gls{dms}
platform is able to selectively expand potent, early differentiation T cells.
Of additional interest was the preservation of the CD4+ compartment. In healthy
donor samples (such as those used here), the typical CD4:CD8 ratio is 2:1. We
noted that \glspl{dms} produced more CD4+ T cells than bead cultures as well as
naïve/memory, showing that the \gls{dms} platform can selectively expand CD4 T
cells to a greater degree than beads (Figure 2c). The trends held true when
observing the CD4+ and CD8+ fractions of the naïve/memory subset (CD62L+CCR7+)
(\cref{fig:dms_exp_mem4,fig:dms_exp_mem8}).
\begin{figure*}[ht!]
\begingroup
@ -1587,7 +1589,28 @@ observing the CD4+ and CD8+ fractions of the naïve/memory subset (CD62L+CCR7+)
\label{fig:dms_exp}
\end{figure*}
% RESULT add a paragraph for this figure
After observing differences in expansion, we further hypothesized that the
\gls{dms} cultures could lead to a different T cell phenotype. In particular, we
were interested in the formation of naïve and memory T cells, as these represent
a subset with higher replicative potential and therefore improved clinical
prognosis\cite{Gattinoni2011, Wang2018}. We measured naïve and memory T cell
frequency staining for CCR7 and CD62L (both of which are present on lower
differentiated T cells such as naïve, central memory, and stem memory
cells\cite{Gattinoni2012}). Using three donors, we noted again \glspl{dms}
produced more T cells over a \SI{14}{\day} expansion than beads, with
significant differences in number appearing as early after \SI{5}{\day}
(\cref{fig:dms_exp_fold_change}). Furthermore, we noted that \glspl{dms}
produced more memory/naïve cells after \SI{14}{\day} when compared to beads for
all donors (\cref{fig:dms_exp_mem,fig:dms_exp_cd4}) showing that the \gls{dms}
platform is able to selectively expand potent, early differentiation T cells.
Of additional interest was the preservation of the CD4+ compartment. In healthy
donor samples (such as those used here), the typical CD4:CD8 ratio is 2:1. We
noted that \glspl{dms} produced more CD4+ T cells than bead cultures as well as
naïve/memory, showing that the \gls{dms} platform can selectively expand CD4 T
cells to a greater degree than beads (Figure 2c). The trends held true when
observing the CD4+ and CD8+ fractions of the naïve/memory subset (\ptmem{})
(\cref{fig:dms_exp_mem4,fig:dms_exp_mem8}).
% FIGURE this figure has weird proportions
\begin{figure*}[ht!]
@ -1608,6 +1631,25 @@ observing the CD4+ and CD8+ fractions of the naïve/memory subset (CD62L+CCR7+)
\label{fig:dms_phenotype}
\end{figure*}
We also observed that, at least with the donors and conditions tested in these
experiments\footnote{these results were not always consistent, see the
metaanalysis at the end of this aim for an in-depth quantification of this
observation} that the fraction of \ptmem{} and \pth{} T cells was higher in
the \gls{dms} groups compared to the bead groups (\cref{fig:dms_phenotype}).
This result was seen for multiple donors. We should not that in the case of
\pthp{}, the donors we used had an initial \pthp{} that was much higher (healthy
donors generally have a CD4:CD8 ratio of 2:1), so the proper interpretation of
this is that the \pthp{} decreases less over the culture period with the
\gls{dms} platform as opposed to the beads (or alternatively, the \gls{dms} has
less preferential expansion for CD8 T cells). We cannot say the same about
the \ptmemp{} since we did not have the initial data for this phenotype;
however (although it should be the vast majority of cells given that
cryopreserved T cells from a healthy donor should generally be composed of
circulated memory and naive T cells). Taken together, these data indicate the
\gls{dms} platform has the capacity to expand higher numbers and percentages of
highly potent \ptmem{} and \pth{} T cells compared to state-of-the-art bead
technology.
\subsection*{DMSs can be used to produce functional CAR T cells}
After optimizing for naïve/memory and CD4 yield, we sought to determine if the
@ -3077,8 +3119,6 @@ them to grow better in the \gls{dms} system.
\label{fig:integrin_1}
\end{figure*}
% RESULT alude to these tables
\begin{table}[!h] \centering
\caption{Linear regression for day 14 phenotype shown in \cref{fig:integrin_1}}
\label{tab:integrin_1_reg}
@ -3092,9 +3132,9 @@ These block \glspl{mab} were added at day 6 of culture when \gls{a2b1} and
identical in all the blocked groups vs the unblocked control group
(\cref{fig:inegrin_1_fc}). Furthermore, we observed that the \ptmemp{} (total
and across the CD4/CD8 compartments) was not significantly different between any
of the groups (\cref{fig:inegrin_1_mem}). We also noted that \gls{a2b1} and
\gls{a2b2} were present on the surface of a significant subset of T cells at day
6, showing that the target we wished to block was present
of the groups (\cref{fig:inegrin_1_mem,tab:integrin_1_reg}). We also noted that
\gls{a2b1} and \gls{a2b2} were present on the surface of a significant subset of
T cells at day 6, showing that the target we wished to block was present
(\cref{fig:inegrin_1_cd49}).
\begin{figure*}[ht!]
@ -3131,22 +3171,22 @@ its maximum. Once again, we observed no difference between any of the blocked
conditions and the unblocked controls in regard to expansion
(\cref{fig:inegrin_2_fc}). Furthermore, none of the \ptmemp{} readouts (total,
CD4, or CD8) were statistically different between groups
(\cref{fig:inegrin_2_mem}).
(\cref{fig:inegrin_2_mem,tab:integrin_2_reg}).
Taken together, these data suggest that the advantage of the \gls{dms} platform
is not due to signaling through \gls{a2b1} or \gls{a2b2}.
\subsection{blocking IL15 signaling does not alter expansion or phenotype}
% RESULT cite the luminex data
\gls{il15} is a cytokine responsible for memory T cell survival and maintenance.
Furthermore, we observed in other experiments that it is secreted to a much
greater extend in \gls{dms} compared to bead cultures. One of our driving
hypotheses in designing the \gls{dms} system was that the higher cell density
would lead to greater local signaling. Since we observed higher \ptmemp{} across
many conditions, we hypothesized that \gls{il15} may be responsible for this,
and further that the unique \textit{cis/trans} activity of \gls{il15} may be
more active in the \gls{dms} system due to higher cell density.
greater extend in \gls{dms} compared to bead cultures (\cref{fig:doe_luminex}).
One of our driving hypotheses in designing the \gls{dms} system was that the
higher cell density would lead to greater local signaling. Since we observed
higher \ptmemp{} across many conditions, we hypothesized that \gls{il15} may be
responsible for this, and further that the unique \textit{cis/trans} activity of
\gls{il15} may be more active in the \gls{dms} system due to higher cell
density.
\begin{figure*}[ht!]
\begingroup