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ENH proof aim 2b

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4
tables/integrin_1_reg.tex
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@ -3,13 +3,13 @@
\begin{tabular}{@{\extracolsep{5pt}}lcc}
\\[-1.8ex]\hline
\hline \\[-1.8ex]
\\[-1.8ex] & \ptmemp{} \% & \pthp{} \\
\\[-1.8ex] & \ptmemp{} & \pthp{} \\
\hline \\[-1.8ex]
CD49a & 0.034 & 34.500 \\
CD49b & 0.024 & 397.000 \\
Constant & 0.313$^{***}$ & 1,233.250$^{***}$ \\
\hline \\[-1.8ex]
Observations & 8 & 8 \\
% Observations & 8 & 8 \\
R$^{2}$ & 0.222 & 0.270 \\
Adjusted R$^{2}$ & $-$0.089 & $-$0.022 \\
\hline

2
tables/integrin_2_reg.tex
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@ -9,7 +9,7 @@
CD49b & 0.007 & $-$0.015 \\
Constant & 0.105$^{***}$ & 0.272$^{***}$ \\
\hline \\[-1.8ex]
Observations & 12 & 12 \\
% Observations & 12 & 12 \\
R$^{2}$ & 0.082 & $-$0.153 \\
\hline
\hline \\[-1.8ex]

230
tex/thesis.tex
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@ -3668,8 +3668,6 @@ interest using \glspl{mab}.
\subsection{DMSs Temporal Modulation}
% METHOD The concentration for the surface marker cleavage experiment was much
% higher, if that matters
\glspl{dms} were digested in active T cell cultures via addition of sterile
\product{\gls{colb}}{\sigald}{11088807001} or
\product{\gls{cold}}{\sigald}{11088858001}. Collagenase was dissolved in
@ -3680,9 +3678,9 @@ media normally used to feed the cells during the regular media addition cycle at
day 4. Cultures were then incubated as described in \cref{sec:tcellculture}, and
the \glspl{dms} were verified to have been digested after \SI{24}{\hour}.
Adding \glspl{dms} was relatively much simpler; the number of \gls{dms} used per
area on day 0 was scaled up by 3 on day 4 to match the change from a 96 well
plate to a 24 well plate, effectively producing a constant activation signal.
Adding \glspl{dms} was simpler; the number of \gls{dms} used per area on day 0
was scaled up by 3 on day 4 to match the change from a 96 well plate to a 24
well plate, effectively producing a constant activation signal.
\subsection{Mass Cytometry and Clustering Analysis}
@ -3692,10 +3690,10 @@ used according to the manufacturer’s instructions. \numrange{2e6}{3e6} stained
cells per group were analyzed on a Fluidigm Helios.
Unbiased cell clusters were obtained using \gls{spade} analysis by pooling three
representative \gls{fcs} files and running the \gls{spade} pipeline with k-means
clustering (k = 100), arcsinh transformation with cofactor 5, density
calculation neighborhood size of 5 and local density approximation factor of
1.5, target density of 20000 cells, and outlier density cutoff of
representative \gls{fcs} files and running \gls{spade} with k-means clustering
(k = 100), arcsinh transformation with cofactor 5, density calculation
neighborhood size of 5, local density approximation factor of 1.5, target
density of 20000 cells, and outlier density cutoff of
\SI{1}{\percent}\cite{Qiu2017}. All markers in the \gls{cytof} panel were used
in the analysis
@ -3716,7 +3714,7 @@ analyzing via a \bd{} Accuri flow cytometer.
To block the \gls{il15r}, we supplemented T cell
cultures activated with \gls{dms} with either
\product{\anti{\gls{il15r}}}{Rnd}{AF247} or \product{\gls{igg} isotype
\product{\anti{\gls{il15r}}}{RnD}{AF247} or \product{\gls{igg} isotype
control}{RnD}{AB-108-C} at the indicated timepoints and concentrations. T
cells were grown as otherwise described in \cref{sec:tcellculture} with the
exception that volumes were split by $\frac{1}{3}$ to keep the culture volume
@ -3739,11 +3737,11 @@ porcine-derived collagen, this enzyme should target the \gls{dms} while sparing
the cells along with any markers we wish to analyze. We tested this specific
hypothesis using either \gls{colb}, \gls{cold} or \gls{hbss}, and stained the
cells using a typical marker panel to assess if any of the markers were cleaved
off by the enzyme which would bias our final readout. We observed that the
marker histograms in the \gls{cold} group were similar to that of the buffer
group, while the \gls{colb} group visibly lowered CD62L and CD4, indicating
partial enzymatic cleavage (\cref{fig:collagenase_fx}). Based on this result, we
used \gls{cold} moving forward.
off by the enzyme which would bias our final readout. The marker histograms in
the \gls{cold} group were similar to that of the buffer group, while the
\gls{colb} group visibly lowered CD62L and CD4, indicating partial enzymatic
cleavage (\cref{fig:collagenase_fx}). Based on this result, we used \gls{cold}
moving forward.
\begin{figure*}[ht!]
\begingroup
@ -3763,13 +3761,13 @@ inhibited in the \textit{added} group while the cells seemed to grow faster in
the \textit{removed} group relative to the \textit{no change} group
(\cref{fig:add_rem_growth}). Additionally, the \textit{removed} group seemed to
have a negative growth rate in the final \SI{4}{\day} of culture, indicating
that either the lack activation signal had slowed the cell growth down or that
the cells were growing fast enough to outpace the media feeding schedule. The
viability was the same between all groups, indicating that this negative growth
rate and the lower growth rate in the \textit{added} group were likely not due
to cell death (\cref{fig:add_rem_viability}). Interestingly, the \textit{added}
group had significantly higher \pth{} cells compared to the \textit{no change}
group, and the inverse was true for the \textit{removed} group
that either the lack activation signal had slowed cell growth or that the cells
were growing fast enough to outpace the media feeding schedule. The viability
was the same between all groups, indicating that this negative growth rate and
the lower growth rate in the \textit{added} group were likely not due to cell
death (\cref{fig:add_rem_viability}). Interestingly, the \textit{added} group
had significantly higher \pth{} cells compared to the \textit{no change} group,
and the inverse was true for the \textit{removed} group
(\cref{fig:add_rem_cd4}). These results show that the growth rate and phenotype
are fundamentally altered by changing the number of \glspl{dms} temporally.
@ -3838,33 +3836,33 @@ We next asked what the effect of removing the \glspl{dms} would have on other
phenotypes, specifically \gls{tcm} and \gls{tscm} cells. To this end we stained
cells using a 34-marker mass cytometry panel and analyzed them using a Fluidigm
Helios. After pooling the \gls{fcs} file events from each group and analyzing
them via \gls{spade} we see that there is a strong bifurcation of CD4 and CD8 T
cells. We also observe that among CD27, CD45RA, and CD45RO (markers commonly
used to identify \gls{tcm} and \gls{tscm} subtypes) we see clear `metaclusters'
composed of individual \gls{spade} clusters which are high for that marker
them via \gls{spade} we saw a strong bifurcation of CD4 and CD8 T cells. When
looking at CD27, CD45RA, and CD45RO (markers commonly used to identify \gls{tcm}
and \gls{tscm} subtypes) we saw clear ``metaclusters'' composed of individual
\gls{spade} clusters which are high for these markers
(\cref{fig:spade_msts,fig:spade_gates}). We then gated each of these
metaclusters according to their marker levels and assigned them to one of three
phenotypes for both the CD4 and CD8 compartments: \gls{tcm} (high CD45RO, low
CD45RA, high CD27), \gls{tscm} (low CD45RO, high CD45RA, high CD27), and
`transitory' \gls{tscm} cells (mid CD45RO, mid CD45RA, high CD27). Together
``transitory'' \gls{tscm} cells (mid CD45RO, mid CD45RA, high CD27). Together
these represent low differentiated cells which should be highly potent as
anti-tumor therapies.
When quantifying the number of cells from each experimental group in these
phenotypes, we clearly see that the number of lower differentiated cells is much
higher in the \textit{no change} or \textit{removed} groups compared to the
\textit{added} group (\cref{fig:spade_quant}). Furthermore, the \textit{removed}
group had a much higher fraction of \gls{tscm} cells compared to the \textit{no
change} group, which had more `transitory \gls{tscm} cells'. The majority of
these cells were \cdp{8} cells. When analyzing the same data using \gls{tsne},
we observe a higher fraction of CD27 and lower fraction of CD45RO in the the
phenotypes, the number of lower differentiated cells was much higher in the
\textit{no change} or \textit{removed} groups compared to the \textit{added}
group (\cref{fig:spade_quant}). Furthermore, the \textit{removed} group had a
much higher fraction of \gls{tscm} cells compared to the \textit{no change}
group, which had more ``transitory \gls{tscm} cells.'' The majority of these
cells were \cdp{8} cells. When analyzing the same data using \gls{tsne}, we
observed a higher fraction of CD27 and lower fraction of CD45RO in the
\textit{removed} group (\cref{fig:spade_tsne_all}). When manually gating on the
CD27+CD45RO- population, we see there is higher density in the \textit{removed}
group, indicating more of this population (\cref{fig:spade_tsne_stem}).
Together, these data indicate that removing \glspl{dms} at lower timepoints
leads to potentially higher expansion, lower \pthp{}, and higher fraction of
lower differentiated T cells such as \gls{tscm}, and adding \gls{dms} seems to
do the inverse.
leads to higher expansion, lower \pthp{}, and higher fraction of
lower differentiated T cells such as \gls{tscm}, and adding \gls{dms} does the
inverse.
\subsection{Blocking Integrin Does Not Alter Expansion or Phenotype}
@ -3872,8 +3870,8 @@ One of the reasons the \gls{dms} platform might perform better than the beads is
the fact that they are composed of gelatin, which is a collagen derivative. The
beads are simply \gls{mab} attached to a polymer resin coated onto an iron oxide
core, and thus have no analogue for collagen. Collagen domains present on the
\gls{dms} group could be creating pro-survival and pro-expansion signals to the
T cells through \gls{a2b1} and \gls{a2b2}, causing them to grow better in the
\gls{dms} group could provide pro-survival and pro-expansion signals to the T
cells through \gls{a2b1} and \gls{a2b2}, causing them to grow better in the
\gls{dms} system.
\begin{figure*}[ht!]
@ -3907,14 +3905,14 @@ T cells through \gls{a2b1} and \gls{a2b2}, causing them to grow better in the
We tested this hypothesis by adding blocking \glspl{mab} against \gls{a2b1}
and/or \gls{a2b2} to running T cell cultures activated using the \glspl{dms}.
These block \glspl{mab} were added at day 6 of culture when \gls{a2b1} and
\gls{a2b2} were known to be expressed\cite{Hemler1990}. We found that the fold
expansion was 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,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
These blocking \glspl{mab} were added at day 6 of culture when \gls{a2b1} and
\gls{a2b2} were known to be expressed\cite{Hemler1990}. The fold expansion was
identical between the blocked and unblocked groupds (\cref{fig:inegrin_1_fc}).
Furthermore, the \ptmemp{} (total and across the CD4/CD8 compartments) was not
significantly different between any of the groups
(\cref{fig:inegrin_1_mem,tab:integrin_1_reg}). Furthermore, \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!]
@ -3943,14 +3941,14 @@ T cells at day 6, showing that the target we wished to block was present
\input{../tables/integrin_2_reg.tex}
\end{table}
Since this last experiment gave a negative result, we decided to block
Since this initial experiment gave a negative result, we decided to block
\gls{a2b1} and \gls{a2b2} harder by adding \glspl{mab} at more timepoints
between day 0 and day 6, hypothesizing that the majority of the signaling would
be during the period of culture where the \gls{dms} surface concentration was at
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
its maximum. Once again, there was no difference between the blocked and
unblocked conditions 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,tab:integrin_2_reg}).
Taken together, these data suggest that the advantage of the \gls{dms} platform
@ -3959,11 +3957,11 @@ is not due to signaling through \gls{a2b1} or \gls{a2b2}.
\subsection{Blocking IL15 Does Not Alter Expansion or Phenotype}
\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 (\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
Furthermore, previous experiments showed that it is secreted to a much 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.
@ -3980,7 +3978,7 @@ density.
\endgroup
\caption[IL15 Blocking I]
{Blocking IL15Ra does not lead to differences in memory or growth.
\subcap{fig:il15_1_overview}{Experimental overview}
\subcap{fig:il15_1_overview}{Experimental overview}.
Longitudinal measurements of
\subcap{fig:il15_1_fc}{fold change} and
\subcap{fig:il15_1_viability}{viability} for blocked and unblocked
@ -3993,7 +3991,7 @@ density.
We first tested this hypothesis by blocking \gls{il15r} with either a specific
\gls{mab} or an \gls{igg} isotype control at
\SI{5}{\ug\per\ml}\cite{MirandaCarus2005}. We observed no difference in the
\SI{5}{\ug\per\ml}\cite{MirandaCarus2005}. There was no difference in the
expansion rate of blocked or unblocked cells (this experiment also had
bead-based groups but they did not expand well and thus were not included)
(\cref{fig:il15_1_fc}). Furthermore, there were no differences in viability
@ -4015,89 +4013,79 @@ the markers, and by extension showing no difference in phenotype
\endgroup
\caption[IL15 Blocking II]
{Blocking soluble IL15 does not lead to differences in memory or growth.
\subcap{fig:il15_2_overview}{Experimental overview}
\subcap{fig:il15_2_overview}{Experimental overview}.
Longitudinal measurements of
\subcap{fig:il15_2_fc}{fold change} and
\subcap{fig:il15_2_viability}{viability} for blocked and unblocked
conditions expanded with \glspl{dms}.
\subcap{fig:il15_2_mem}{Flow cytometry markers for \gls{dms}-expanded T
cells at day 14 for blocked and unblocked groups.}.
cells at day 14 for blocked and unblocked groups.}
}
\label{fig:il15_2}
\end{figure*}
We next tried blocking soluble \gls{il15} itself using either a \gls{mab} or an
\gls{igg} isotype control. \anti{\gls{il15}} or \gls{igg} isotype control was
\gls{igg} isotype control. Anti-\gls{il15} or \gls{igg} isotype control was
added at \SI{5}{\ug\per\ml}, which according to \cref{fig:doe_luminex} was in
excess of the \gls{il15} concentration seen in past experiments by over
\num{20000} times. Similarly, we observed no difference between fold change,
\num{20000} times. Similarly, there was no difference between fold change,
viability, or marker histograms between any of these markers, showing that
blocking \gls{il15} led to no difference in growth or phenotype.
% RESULT this can probably be worded more specifically in terms of the cis/trans
% action of IL15
In summary, this data did not support the hypothesis that the \gls{dms} platform
gains its advantages via the \gls{il15} pathway.
\section{Discussion}
This work provides insight for how the \gls{dms} operates and may be optimized
further. The data showing increased \pthp{} when \glspl{dms} are added and the
reverse when removed is consistent with other data we produced via \gls{doe}
showing that higher \gls{dms} concentrations lead to higher \pthp{}
(\cref{fig:doe_responses_cd4,fig:add_rem_cd4}). The difference in this case is
that we showed that altering activation signal analogously affects the \pthp{}
in the dimension of time as well as space. A similar trend was observed with
memory T cells in this aim. Our previous \gls{doe} data showed that, to a point,
lower \gls{dms} concentration leads to higher \ptmemp{}
This work provides insight for how the \gls{dms} platform operates and how it
may be optimized further. The data showing increased \pthp{} when \glspl{dms}
are added and the reverse when removed is consistent with other data we produced
via \gls{doe} showing that higher \gls{dms} concentrations lead to higher
\pthp{} (\cref{fig:doe_responses_cd4,fig:add_rem_cd4}). The difference in this
case is that altering activation signal analogously affects the \pthp{} in the
dimension of time as well as space. A similar trend was observed with memory T
cells in this aim. Our previous \gls{doe} data showed that, to a point, lower
\gls{dms} concentration leads to higher \ptmemp{}
(\cref{fig:doe_responses_mem}). In this aim, we showed that decreasing
activation signal temporally by removing \glspl{dms} leads to the same effect in
the \gls{tcm}, \gls{tscm} and `transitory' \gls{tscm} populations, (all of which
are included in the \ptmem{} phenotype). Taken together, these imply that
temporally or spatially altering the \gls{dms} concentration, and thus the
activation signal, has similar effects.
the \gls{tcm}, \gls{tscm} and ``transitory'' \gls{tscm} populations, (all of
which are included in the \ptmem{} phenotype). Taken together, these imply that
temporally or spatially decreasing the \gls{dms} concentration, and thus the
activation signal, increases memory and lowers CD4+ fractions.
% BACKGROUND this sounds like background?
% There are several plausible explanations for the observed phenotypic differences
% between beads and DMSs. First, the DMSs are composed of a collagen derivative
% (gelatin); collagen has been shown to costimulate activated T cells via
% \gls{a2b1} and \gls{a2b2}, leading to enhanced proliferation, increased
% \gls{ifng} production, and upregulated CD25 (IL2R$\upalpha$) surface
% expression8,10,11,41,42.
While we did not find support for our hypothesis that \glspl{dms} signal via the
\gls{a2b1} and/or \gls{a2b2} receptors, we can speculate that either the
experiment failed to block the targeted pathways or that this mechanism is
simply not relevant for our system.
While we did not find support for our hypothesis that the \gls{dms} signal
through the \gls{a2b1} and/or \gls{a2b2} receptors, we can speculate as to why
either this experiment failed and may be done better in the future, or why these
receptors may simply be irrelevant for our system.
On the first point, we did not verify that these \glspl{mab} actually blocked
their target receptors (although they were from a reputable manufacturer, \bl).
However, other groups have shown that these particular clones work at the
concentrations we used\cite{MirandaCarus2005}. Furthermore, we can safely rule
out the possibility that the \glspl{mab} never reached their targets, as they
were added immediately after the T cells were resuspended as required for cell
counting, hence their resting clustered state was disrupted. Therefore, the most
likely failure mode was that the \glspl{mab} we obtained were somehow defective
in their intended purpose, which we could experimentally verify using adhesion
assays.
On the first point, we did not verify that these \glspl{mab} indeed blocked the
receptor we were targeting. There has been evidence from other groups that these
particular clones work at the concentrations we used\cite{MirandaCarus2005}.
This does not necessarily mean that the \glspl{mab} we obtained were functional
in blocking their intended targets (although they were from a reputable
manufacturer, \bl). Furthermore, we can safely rule out the possibility that the
\glspl{mab} never reached their targets, as they were added immediately after
the T cells were resuspended as required for cell counting, hence their resting
clustered state was disrupted.
On the second point, the collagen domains may not even be relevant to our system
depending on the nature of the \gls{stp} coating. We intended by design for the
system to be fully coated or nearly fully-coated with \gls{stp}
(\cref{fig:stp_coating}). Thus the domains that \gls{a2b1} and \gls{a2b2} may be
targeting could be sterically hindered by a layer of \gls{stp}, and if not that,
also a layer of CD3/CD28 \glspl{mab}. The other possibility is that these
domains are simply denatured to beyond recognition due to the fabrication
process for the microcarriers we used (which involves a proprietary
cross-linking step to make the material autoclave-safe). Either of these could
be tested and verified by staining the \glspl{dms} with a fluorescently-tagged
\gls{mab} and verifying binding via confocal microscopy or indirect protein
quantification as we do for the \gls{qc} of the \gls{dms}. If this test came
back negative, we would be fairly confident that the \gls{a2b1} and \gls{a2b1}
domains are either unreachable or unrecognizable. Even if it turned out that
collagen binding domains are irrelevant in the \gls{dms} system, previous
studies show that these domains can enhance proliferation and survival, and thus
adding them along with with the \glspl{mab} could enhance T cell
expansion\cite{Aoudjit2000, Gendron2003, Boisvert2007}.
On the second point, collagen domains may not even be relevant to our system
depending on the extent of \gls{stp} coating. We intended by design for the
system to be fully coated with \gls{stp} (\cref{fig:stp_coating}). Thus the
domains that \gls{a2b1} and \gls{a2b2} may be targeting could be sterically
hindered by a layer of \gls{stp}, and if not that, also a layer of CD3/CD28
\glspl{mab}. The other possibility is that these domains are simply denatured to
beyond recognition due to the fabrication process for the microcarriers (which
involves a proprietary cross-linking step to make the material autoclave-safe).
Either of these could be tested and verified by staining the \glspl{dms} with a
fluorescently-tagged \gls{mab} and verifying binding via confocal microscopy or
indirect protein quantification as we do for the \gls{qc} of the \gls{dms}. If
this test came back negative, we would be fairly confident that the \gls{a2b1}
and \gls{a2b1} domains are either unreachable or unrecognizable. Even if it
turned out that collagen binding domains are non-existent in the \gls{dms}
system, previous studies have shown that these domains can enhance proliferation
and survival, and thus adding them along with with the \glspl{mab} could enhance
T cell expansion\cite{Aoudjit2000, Gendron2003, Boisvert2007}.
We also failed to uphold our hypothesis that the \gls{dms} system gains its
advantage via \gls{il15} signaling. There could be multiple reasons for why
@ -4108,7 +4096,7 @@ memory phenotypes\cite{Lodolce1998,Kennedy2000}. Second, in the case of the
receptor it could be that that \glspl{mab} we purchased did not actually block,
which also seems unlikely given that this clone has been observed to inhibit
proliferation in the past (although like the integrin blocking experiments we
did not verify that it blocked ourselves), albeit of resting T
did not verify for ourselves that it blocked), albeit of resting T
cells\cite{MirandaCarus2005}. Third, it could be that turnover of the receptor
was so high that there were not enough \glspl{mab} to block (the key difference
between our experiment and that of \cite{MirandaCarus2005} was that they used
@ -4116,9 +4104,9 @@ resting T cells, which are not expressing protein to nearly as high of a
degree). The way to test this would be to simply titrate increasing
concentrations of \gls{mab} (which we did not do in our case because the
\gls{mab} was already very expensive in the concentrations employed for our
experiment). Fourth, the blocking the soluble protein may not have worked
because the \il{15} may have been secreted and immediately captured via
\il{15R$\upalpha$} either by the cell that secreted it or by a neighboring cell.
experiment). Fourth, blocking the soluble protein may not have worked because
\il{15} may have been secreted and immediately captured via \il{15R$\upalpha$}
either by the cell that secreted it or by a neighboring cell.
Regardless of whether or not \il{15} is important for the overall mechanism that
differentiates the \glspl{dms} from the beads, adding \il{15} or its receptor