ENH proofread aim2b

tex/thesis.tex

@@ -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