From f05bbc3d01dbed4608500e5a3f1271735c7a2203 Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Wed, 4 Aug 2021 18:59:54 -0400 Subject: [PATCH] ENH proofread aim2b --- tex/thesis.tex | 90 ++++++++++++++++++++++++++++++++------------------ 1 file changed, 58 insertions(+), 32 deletions(-) diff --git a/tex/thesis.tex b/tex/thesis.tex index 7b9de20..f722bd2 100644 --- a/tex/thesis.tex +++ b/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