ENH proofread aim2b

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Nathan Dwarshuis 2021-08-04 18:59:54 -04:00
parent 4789723e72
commit f05bbc3d01
1 changed files with 58 additions and 32 deletions

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@ -3401,8 +3401,8 @@ available data, mostly small \gls{doe} datasets. \gls{sr} has the necessary
capabilities to resolve the issues of process effects modeling and has been capabilities to resolve the issues of process effects modeling and has been
applied across multiple industries\cite{Kordona}. \gls{sr} discovers applied across multiple industries\cite{Kordona}. \gls{sr} discovers
mathematical expressions that fit a given sample and differs from conventional mathematical expressions that fit a given sample and differs from conventional
regression techniques in that a model structure is not defined a regression techniques in that a model structure is not defined \textit{a
priori\cite{Koza1992}. Hence, a key advantage of this methodology is that priori}\cite{Koza1992}. Hence, a key advantage of this methodology is that
transparent, human-interpretable models can be generated from small and large transparent, human-interpretable models can be generated from small and large
datasets with no prior assumptions\cite{Kotancheka}. datasets with no prior assumptions\cite{Kotancheka}.
@ -3554,7 +3554,7 @@ 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 \gls{dms} was relatively much simpler; the number of \gls{dms} used per 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 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. plate to a 24 well plate, effectively producing a constant activation signal.
@ -3570,7 +3570,8 @@ representative \gls{fcs} files and running the \gls{spade} pipeline with k-means
clustering (k = 100), arcsinh transformation with cofactor 5, density clustering (k = 100), arcsinh transformation with cofactor 5, density
calculation neighborhood size of 5 and local density approximation factor of calculation neighborhood size of 5 and local density approximation factor of
1.5, target density of 20000 cells, and outlier density cutoff of 1.5, target density of 20000 cells, and outlier density cutoff of
\SI{1}{\percent}. All markers in the \gls{cytof} panel were used in the analysis \SI{1}{\percent}\cite{Qiu2017}. All markers in the \gls{cytof} panel were used
in the analysis
\subsection{integrin blocking experiments} \subsection{integrin blocking experiments}
@ -3583,7 +3584,7 @@ concentrations and timepoints. T cells were grown as described in
\gls{a2b1} and \gls{a2b2} were verified to be present on active T cell cultures \gls{a2b1} and \gls{a2b2} were verified to be present on active T cell cultures
by staining with \product{\anti{\gls{a2b1}}-\gls{apc}}{\bl}{328313} and by staining with \product{\anti{\gls{a2b1}}-\gls{apc}}{\bl}{328313} and
\product{\anti{\gls{a2b2}}-\gls{fitc}}{\bl}{359305} on day 6 of culture and \product{\anti{\gls{a2b2}}-\gls{fitc}}{\bl}{359305} on day 6 of culture and
analyzing via a BD Accuri flow cytometer. analyzing via a \bd{} Accuri flow cytometer.
\subsection{IL15 blocking experiments} \subsection{IL15 blocking experiments}
@ -3741,15 +3742,13 @@ do the inverse.
\subsection{blocking integrin binding does not alter expansion or phenotype} \subsection{blocking integrin binding does not alter expansion or phenotype}
% BACKGROUND add background into why integrins are important 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
One of the reasons the \gls{dms} platform might be performing better than the beads are simply \gls{mab} attached to a polymer resin coated onto an iron oxide
beads is the fact that they are composed of gelatin, which is a collagen core, and thus have no analogue for collagen. Collagen domains present on the
derivative. The beads are simply \gls{mab} attached to a polymer resin coated \gls{dms} group could be creating pro-survival and pro-expansion signals to the
onto an iron oxide core, and thus have no analogue for collagen. Collagen T cells through \gls{a2b1} and \gls{a2b2}, causing them to grow better in the
domains present on the \gls{dms} group could be creating pro-survival and \gls{dms} system.
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!] \begin{figure*}[ht!]
\begingroup \begingroup
@ -3818,10 +3817,10 @@ 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 hit \gls{a2b1} Since this last experiment gave a negative result, we decided to block
and \gls{a2b2} harder by adding blocking \glspl{mab} at more timepoints between \gls{a2b1} and \gls{a2b2} harder by adding \glspl{mab} at more timepoints
day 0 and day 6, hypothesizing that the majority of the signaling would be between day 0 and day 6, hypothesizing that the majority of the signaling would
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, we observed no difference between any of the blocked
conditions and the unblocked controls in regard to expansion conditions and the unblocked controls in regard to expansion
(\cref{fig:inegrin_2_fc}). Furthermore, none of the \ptmemp{} readouts (total, (\cref{fig:inegrin_2_fc}). Furthermore, none of the \ptmemp{} readouts (total,
@ -3843,6 +3842,7 @@ 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.
% FIGURE this should say ug not mg
\begin{figure*}[ht!] \begin{figure*}[ht!]
\begingroup \begingroup
@ -3866,10 +3866,10 @@ density.
\label{fig:il15_1} \label{fig:il15_1}
\end{figure*} \end{figure*}
% RESULT how did I determine how much to add?
% FIGURE just gate these as normal because this looks sketchy % FIGURE just gate these as normal because this looks sketchy
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. We observed no difference in the \gls{mab} or an \gls{igg} isotype control at
\SI{5}{\ug\per\ml}\cite{MirandaCarus2005}. We observed 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
@ -3906,10 +3906,10 @@ the markers, and by extension showing no difference in phenotype
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 20000X. excess of the \gls{il15} concentration seen in past experiments by over
Similarly, we observed no difference between fold change, viability, or marker \num{20000} times. Similarly, we observed no difference between fold change,
histograms between any of these markers, showing that blocking \gls{il15} led to viability, or marker histograms between any of these markers, showing that
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 % RESULT this can probably be worded more specifically in terms of the cis/trans
% action of IL15 % action of IL15
@ -3957,7 +3957,6 @@ manufacturer, \bl). Furthermore, we can safely rule out the possibility that the
the T cells were resuspended as required for cell counting, hence their resting the T cells were resuspended as required for cell counting, hence their resting
clustered state was disrupted. clustered state was disrupted.
% TODO define Bite
On the second point, the collagen domains may not even be relevant to our system 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 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} system to be fully coated or nearly fully-coated with \gls{stp}
@ -3968,15 +3967,42 @@ domains are simply denatured to beyond recognition due to the fabrication
process for the microcarriers we used (which involves a proprietary process for the microcarriers we used (which involves a proprietary
cross-linking step to make the material autoclave-safe). Either of these could 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 be tested and verified by staining the \glspl{dms} with a fluorescently-tagged
\gls{mab} (or something smaller such as a BiTE to reduce the possibility of \gls{mab} and verifying binding via confocal microscopy or indirect protein
steric hindrance) and verifying binding via confocal microscopy or indirect quantification as we do for the \gls{qc} of the \gls{dms}. If this test came
protein quantification as we do for the \gls{qc} of the \gls{dms}. If this test back negative, we would be fairly confident that the \gls{a2b1} and \gls{a2b1}
came back negative, we would be fairly confident that the \gls{a2b1} and domains are either unreachable or unrecognizable. Even if it turned out that
\gls{a2b1} domains are either unreachable or unrecognizable. 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}.
% DISCUSSION not sure exactly how to explain this
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. advantage via \gls{il15} signaling. There could be multiple reasons for why
blocking either \il{15} itself or its receptor would not influence the response
at all. First, it could be that \il{15} is not important in our system, which is
not likely given the importance of \il{15} in T cells expansion and particularly
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
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
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.
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
complex to the surface of the \gls{dms} might produce interesting and positive
results on expansion and memory phenotype. Essentially this would turn the
\glspl{dms} into stromal cells that present \il{15}, as seen to be important in
the early work with \il{15} in mice\cite{Lodolce1998}.
% DISCUSSION not sure if this belongs here, although it might make sense to offer % DISCUSSION not sure if this belongs here, although it might make sense to offer
% alternative explanations of why the DMSs "work" given this negative data % alternative explanations of why the DMSs "work" given this negative data