ENH proofread aim2b
This commit is contained in:
parent
4789723e72
commit
f05bbc3d01
|
@ -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
|
||||
applied across multiple industries\cite{Kordona}. \gls{sr} discovers
|
||||
mathematical expressions that fit a given sample and differs from conventional
|
||||
regression techniques in that a model structure is not defined a
|
||||
priori\cite{Koza1992}. Hence, a key advantage of this methodology is that
|
||||
regression techniques in that a model structure is not defined \textit{a
|
||||
priori}\cite{Koza1992}. Hence, a key advantage of this methodology is that
|
||||
transparent, human-interpretable models can be generated from small and large
|
||||
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
|
||||
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
|
||||
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
|
||||
calculation neighborhood size of 5 and local density approximation factor 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}
|
||||
|
||||
|
@ -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
|
||||
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
|
||||
analyzing via a BD Accuri flow cytometer.
|
||||
analyzing via a \bd{} Accuri flow cytometer.
|
||||
|
||||
\subsection{IL15 blocking experiments}
|
||||
|
||||
|
@ -3741,15 +3742,13 @@ do the inverse.
|
|||
|
||||
\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 be performing 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} system.
|
||||
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} system.
|
||||
|
||||
\begin{figure*}[ht!]
|
||||
\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}
|
||||
\end{table}
|
||||
|
||||
Since this last experiment gave a negative result, we decided to hit \gls{a2b1}
|
||||
and \gls{a2b2} harder by adding blocking \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
|
||||
Since this last 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,
|
||||
|
@ -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
|
||||
density.
|
||||
|
||||
% FIGURE this should say ug not mg
|
||||
\begin{figure*}[ht!]
|
||||
\begingroup
|
||||
|
||||
|
@ -3866,10 +3866,10 @@ density.
|
|||
\label{fig:il15_1}
|
||||
\end{figure*}
|
||||
|
||||
% RESULT how did I determine how much to add?
|
||||
% FIGURE just gate these as normal because this looks sketchy
|
||||
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
|
||||
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
|
||||
|
@ -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
|
||||
\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 20000X.
|
||||
Similarly, we observed 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.
|
||||
excess of the \gls{il15} concentration seen in past experiments by over
|
||||
\num{20000} times. Similarly, we observed 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
|
||||
|
@ -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
|
||||
clustered state was disrupted.
|
||||
|
||||
% TODO define Bite
|
||||
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}
|
||||
|
@ -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
|
||||
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} (or something smaller such as a BiTE to reduce the possibility of
|
||||
steric hindrance) 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.
|
||||
\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}.
|
||||
|
||||
% DISCUSSION not sure exactly how to explain this
|
||||
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
|
||||
% alternative explanations of why the DMSs "work" given this negative data
|
||||
|
|
Loading…
Reference in New Issue