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

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

230
tex/thesis.tex
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@ -3668,8 +3668,6 @@ interest using \glspl{mab}.
\subsection{DMSs Temporal Modulation} \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 \glspl{dms} were digested in active T cell cultures via addition of sterile
\product{\gls{colb}}{\sigald}{11088807001} or \product{\gls{colb}}{\sigald}{11088807001} or
\product{\gls{cold}}{\sigald}{11088858001}. Collagenase was dissolved in \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 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}. 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 Adding \glspl{dms} was simpler; the number of \gls{dms} used per area on day 0
area on day 0 was scaled up by 3 on day 4 to match the change from a 96 well was scaled up by 3 on day 4 to match the change from a 96 well plate to a 24
plate to a 24 well plate, effectively producing a constant activation signal. well plate, effectively producing a constant activation signal.
\subsection{Mass Cytometry and Clustering Analysis} \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. cells per group were analyzed on a Fluidigm Helios.
Unbiased cell clusters were obtained using \gls{spade} analysis by pooling three 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 representative \gls{fcs} files and running \gls{spade} with k-means clustering
clustering (k = 100), arcsinh transformation with cofactor 5, density (k = 100), arcsinh transformation with cofactor 5, density calculation
calculation neighborhood size of 5 and local density approximation factor of neighborhood size of 5, local density approximation factor of 1.5, target
1.5, target density of 20000 cells, and outlier density cutoff of density of 20000 cells, and outlier density cutoff of
\SI{1}{\percent}\cite{Qiu2017}. All markers in the \gls{cytof} panel were used \SI{1}{\percent}\cite{Qiu2017}. All markers in the \gls{cytof} panel were used
in the analysis in the analysis
@ -3716,7 +3714,7 @@ analyzing via a \bd{} Accuri flow cytometer.
To block the \gls{il15r}, we supplemented T cell To block the \gls{il15r}, we supplemented T cell
cultures activated with \gls{dms} with either 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 control}{RnD}{AB-108-C} at the indicated timepoints and concentrations. T
cells were grown as otherwise described in \cref{sec:tcellculture} with the 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 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 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 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 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 off by the enzyme which would bias our final readout. The marker histograms in
marker histograms in the \gls{cold} group were similar to that of the buffer the \gls{cold} group were similar to that of the buffer group, while the
group, while the \gls{colb} group visibly lowered CD62L and CD4, indicating \gls{colb} group visibly lowered CD62L and CD4, indicating partial enzymatic
partial enzymatic cleavage (\cref{fig:collagenase_fx}). Based on this result, we cleavage (\cref{fig:collagenase_fx}). Based on this result, we used \gls{cold}
used \gls{cold} moving forward. moving forward.
\begin{figure*}[ht!] \begin{figure*}[ht!]
\begingroup \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 the \textit{removed} group relative to the \textit{no change} group
(\cref{fig:add_rem_growth}). Additionally, the \textit{removed} group seemed to (\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 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 that either the lack activation signal had slowed cell growth or that the cells
the cells were growing fast enough to outpace the media feeding schedule. The were growing fast enough to outpace the media feeding schedule. The viability
viability was the same between all groups, indicating that this negative growth was the same between all groups, indicating that this negative growth rate and
rate and the lower growth rate in the \textit{added} group were likely not due the lower growth rate in the \textit{added} group were likely not due to cell
to cell death (\cref{fig:add_rem_viability}). Interestingly, the \textit{added} death (\cref{fig:add_rem_viability}). Interestingly, the \textit{added} group
group had significantly higher \pth{} cells compared to the \textit{no change} had significantly higher \pth{} cells compared to the \textit{no change} group,
group, and the inverse was true for the \textit{removed} 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 (\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. 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 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 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 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 them via \gls{spade} we saw a strong bifurcation of CD4 and CD8 T cells. When
cells. We also observe that among CD27, CD45RA, and CD45RO (markers commonly looking at CD27, CD45RA, and CD45RO (markers commonly used to identify \gls{tcm}
used to identify \gls{tcm} and \gls{tscm} subtypes) we see clear `metaclusters' and \gls{tscm} subtypes) we saw clear ``metaclusters'' composed of individual
composed of individual \gls{spade} clusters which are high for that marker \gls{spade} clusters which are high for these markers
(\cref{fig:spade_msts,fig:spade_gates}). We then gated each of these (\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 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 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 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 these represent low differentiated cells which should be highly potent as
anti-tumor therapies. anti-tumor therapies.
When quantifying the number of cells from each experimental group in these 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 phenotypes, the number of lower differentiated cells was much higher in the
higher in the \textit{no change} or \textit{removed} groups compared to the \textit{no change} or \textit{removed} groups compared to the \textit{added}
\textit{added} group (\cref{fig:spade_quant}). Furthermore, the \textit{removed} group (\cref{fig:spade_quant}). Furthermore, the \textit{removed} group had a
group had a much higher fraction of \gls{tscm} cells compared to the \textit{no much higher fraction of \gls{tscm} cells compared to the \textit{no change}
change} group, which had more `transitory \gls{tscm} cells'. The majority of group, which had more ``transitory \gls{tscm} cells.'' The majority of these
these cells were \cdp{8} cells. When analyzing the same data using \gls{tsne}, cells were \cdp{8} cells. When analyzing the same data using \gls{tsne}, we
we observe a higher fraction of CD27 and lower fraction of CD45RO in the the 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 \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} CD27+CD45RO- population, we see there is higher density in the \textit{removed}
group, indicating more of this population (\cref{fig:spade_tsne_stem}). group, indicating more of this population (\cref{fig:spade_tsne_stem}).
Together, these data indicate that removing \glspl{dms} at lower timepoints Together, these data indicate that removing \glspl{dms} at lower timepoints
leads to potentially higher expansion, lower \pthp{}, and higher fraction of leads to higher expansion, lower \pthp{}, and higher fraction of
lower differentiated T cells such as \gls{tscm}, and adding \gls{dms} seems to lower differentiated T cells such as \gls{tscm}, and adding \gls{dms} does the
do the inverse. inverse.
\subsection{Blocking Integrin Does Not Alter Expansion or Phenotype} \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 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 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 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 \gls{dms} group could provide pro-survival and pro-expansion signals to the T
T cells through \gls{a2b1} and \gls{a2b2}, causing them to grow better in the cells through \gls{a2b1} and \gls{a2b2}, causing them to grow better in the
\gls{dms} system. \gls{dms} system.
\begin{figure*}[ht!] \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} 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}. 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 These blocking \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 \gls{a2b2} were known to be expressed\cite{Hemler1990}. The fold expansion was
expansion was identical in all the blocked groups vs the unblocked control group identical between the blocked and unblocked groupds (\cref{fig:inegrin_1_fc}).
(\cref{fig:inegrin_1_fc}). Furthermore, we observed that the \ptmemp{} (total Furthermore, the \ptmemp{} (total and across the CD4/CD8 compartments) was not
and across the CD4/CD8 compartments) was not significantly different between any significantly different between any of the groups
of the groups (\cref{fig:inegrin_1_mem,tab:integrin_1_reg}). We also noted that (\cref{fig:inegrin_1_mem,tab:integrin_1_reg}). Furthermore, \gls{a2b1} and
\gls{a2b1} and \gls{a2b2} were present on the surface of a significant subset of \gls{a2b2} were present on the surface of a significant subset of T cells at day
T cells at day 6, showing that the target we wished to block was present 6, showing that the target we wished to block was present
(\cref{fig:inegrin_1_cd49}). (\cref{fig:inegrin_1_cd49}).
\begin{figure*}[ht!] \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} \input{../tables/integrin_2_reg.tex}
\end{table} \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 \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 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 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 its maximum. Once again, there was no difference between the blocked and
conditions and the unblocked controls in regard to expansion unblocked conditions in regard to expansion (\cref{fig:inegrin_2_fc}).
(\cref{fig:inegrin_2_fc}). Furthermore, none of the \ptmemp{} readouts (total, Furthermore, none of the \ptmemp{} readouts (total, CD4, or CD8) were
CD4, or CD8) were statistically different between groups statistically different between groups
(\cref{fig:inegrin_2_mem,tab:integrin_2_reg}). (\cref{fig:inegrin_2_mem,tab:integrin_2_reg}).
Taken together, these data suggest that the advantage of the \gls{dms} platform 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} \subsection{Blocking IL15 Does Not Alter Expansion or Phenotype}
\gls{il15} is a cytokine responsible for memory T cell survival and maintenance. \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 Furthermore, previous experiments showed that it is secreted to a much greater
greater extend in \gls{dms} compared to bead cultures (\cref{fig:doe_luminex}). extend in \gls{dms} compared to bead cultures (\cref{fig:doe_luminex}). One of
One of our driving hypotheses in designing the \gls{dms} system was that the our driving hypotheses in designing the \gls{dms} system was that the higher
higher cell density would lead to greater local signaling. Since we observed cell density would lead to greater local signaling. Since we observed higher
higher \ptmemp{} across many conditions, we hypothesized that \gls{il15} may be \ptmemp{} across many conditions, we hypothesized that \gls{il15} may be
responsible for this, and further that the unique \textit{cis/trans} activity of 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 \gls{il15} may be more active in the \gls{dms} system due to higher cell
density. density.
@ -3980,7 +3978,7 @@ density.
\endgroup \endgroup
\caption[IL15 Blocking I] \caption[IL15 Blocking I]
{Blocking IL15Ra does not lead to differences in memory or growth. {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 Longitudinal measurements of
\subcap{fig:il15_1_fc}{fold change} and \subcap{fig:il15_1_fc}{fold change} and
\subcap{fig:il15_1_viability}{viability} for blocked and unblocked \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 We first tested this hypothesis by blocking \gls{il15r} with either a specific
\gls{mab} or an \gls{igg} isotype control at \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 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) 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 (\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 \endgroup
\caption[IL15 Blocking II] \caption[IL15 Blocking II]
{Blocking soluble IL15 does not lead to differences in memory or growth. {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 Longitudinal measurements of
\subcap{fig:il15_2_fc}{fold change} and \subcap{fig:il15_2_fc}{fold change} and
\subcap{fig:il15_2_viability}{viability} for blocked and unblocked \subcap{fig:il15_2_viability}{viability} for blocked and unblocked
conditions expanded with \glspl{dms}. conditions expanded with \glspl{dms}.
\subcap{fig:il15_2_mem}{Flow cytometry markers for \gls{dms}-expanded T \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} \label{fig:il15_2}
\end{figure*} \end{figure*}
We next tried blocking soluble \gls{il15} itself using either a \gls{mab} or an 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 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 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 viability, or marker histograms between any of these markers, showing that
blocking \gls{il15} led to no difference in growth or phenotype. 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 In summary, this data did not support the hypothesis that the \gls{dms} platform
gains its advantages via the \gls{il15} pathway. gains its advantages via the \gls{il15} pathway.
\section{Discussion} \section{Discussion}
This work provides insight for how the \gls{dms} operates and may be optimized This work provides insight for how the \gls{dms} platform operates and how it
further. The data showing increased \pthp{} when \glspl{dms} are added and the may be optimized further. The data showing increased \pthp{} when \glspl{dms}
reverse when removed is consistent with other data we produced via \gls{doe} are added and the reverse when removed is consistent with other data we produced
showing that higher \gls{dms} concentrations lead to higher \pthp{} via \gls{doe} showing that higher \gls{dms} concentrations lead to higher
(\cref{fig:doe_responses_cd4,fig:add_rem_cd4}). The difference in this case is \pthp{} (\cref{fig:doe_responses_cd4,fig:add_rem_cd4}). The difference in this
that we showed that altering activation signal analogously affects the \pthp{} case is that altering activation signal analogously affects the \pthp{} in the
in the dimension of time as well as space. A similar trend was observed with dimension of time as well as space. A similar trend was observed with memory T
memory T cells in this aim. Our previous \gls{doe} data showed that, to a point, cells in this aim. Our previous \gls{doe} data showed that, to a point, lower
lower \gls{dms} concentration leads to higher \ptmemp{} \gls{dms} concentration leads to higher \ptmemp{}
(\cref{fig:doe_responses_mem}). In this aim, we showed that decreasing (\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 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 the \gls{tcm}, \gls{tscm} and ``transitory'' \gls{tscm} populations, (all of
are included in the \ptmem{} phenotype). Taken together, these imply that which are included in the \ptmem{} phenotype). Taken together, these imply that
temporally or spatially altering the \gls{dms} concentration, and thus the temporally or spatially decreasing the \gls{dms} concentration, and thus the
activation signal, has similar effects. activation signal, increases memory and lowers CD4+ fractions.
% BACKGROUND this sounds like background? While we did not find support for our hypothesis that \glspl{dms} signal via the
% There are several plausible explanations for the observed phenotypic differences \gls{a2b1} and/or \gls{a2b2} receptors, we can speculate that either the
% between beads and DMSs. First, the DMSs are composed of a collagen derivative experiment failed to block the targeted pathways or that this mechanism is
% (gelatin); collagen has been shown to costimulate activated T cells via simply not relevant for our system.
% \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 the \gls{dms} signal On the first point, we did not verify that these \glspl{mab} actually blocked
through the \gls{a2b1} and/or \gls{a2b2} receptors, we can speculate as to why their target receptors (although they were from a reputable manufacturer, \bl).
either this experiment failed and may be done better in the future, or why these However, other groups have shown that these particular clones work at the
receptors may simply be irrelevant for our system. 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 On the second point, collagen domains may not even be relevant to our system
receptor we were targeting. There has been evidence from other groups that these depending on the extent of \gls{stp} coating. We intended by design for the
particular clones work at the concentrations we used\cite{MirandaCarus2005}. system to be fully coated with \gls{stp} (\cref{fig:stp_coating}). Thus the
This does not necessarily mean that the \glspl{mab} we obtained were functional domains that \gls{a2b1} and \gls{a2b2} may be targeting could be sterically
in blocking their intended targets (although they were from a reputable hindered by a layer of \gls{stp}, and if not that, also a layer of CD3/CD28
manufacturer, \bl). Furthermore, we can safely rule out the possibility that the \glspl{mab}. The other possibility is that these domains are simply denatured to
\glspl{mab} never reached their targets, as they were added immediately after beyond recognition due to the fabrication process for the microcarriers (which
the T cells were resuspended as required for cell counting, hence their resting involves a proprietary cross-linking step to make the material autoclave-safe).
clustered state was disrupted. 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
On the second point, the collagen domains may not even be relevant to our system indirect protein quantification as we do for the \gls{qc} of the \gls{dms}. If
depending on the nature of the \gls{stp} coating. We intended by design for the this test came back negative, we would be fairly confident that the \gls{a2b1}
system to be fully coated or nearly fully-coated with \gls{stp} and \gls{a2b1} domains are either unreachable or unrecognizable. Even if it
(\cref{fig:stp_coating}). Thus the domains that \gls{a2b1} and \gls{a2b2} may be turned out that collagen binding domains are non-existent in the \gls{dms}
targeting could be sterically hindered by a layer of \gls{stp}, and if not that, system, previous studies have shown that these domains can enhance proliferation
also a layer of CD3/CD28 \glspl{mab}. The other possibility is that these and survival, and thus adding them along with with the \glspl{mab} could enhance
domains are simply denatured to beyond recognition due to the fabrication T cell expansion\cite{Aoudjit2000, Gendron2003, Boisvert2007}.
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}.
We also failed to uphold our hypothesis that the \gls{dms} system gains its 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 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, 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 which also seems unlikely given that this clone has been observed to inhibit
proliferation in the past (although like the integrin blocking experiments we 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 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 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 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 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 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 \gls{mab} was already very expensive in the concentrations employed for our
experiment). Fourth, the blocking the soluble protein may not have worked experiment). Fourth, blocking the soluble protein may not have worked because
because the \il{15} may have been secreted and immediately captured via \il{15} may have been secreted and immediately captured via \il{15R$\upalpha$}
\il{15R$\upalpha$} either by the cell that secreted it or by a neighboring cell. 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 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 differentiates the \glspl{dms} from the beads, adding \il{15} or its receptor