From 48989b763b787d0fda08ebf5b92176812befd688 Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Wed, 28 Jul 2021 22:13:37 -0400 Subject: [PATCH] ADD results for il15 and integrin blocking sections --- tex/thesis.tex | 74 ++++++++++++++++++++++++++++++++++++++++++++++++++ 1 file changed, 74 insertions(+) diff --git a/tex/thesis.tex b/tex/thesis.tex index de1da9a..1d6a890 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -2279,9 +2279,20 @@ 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. + % TODO perhaps these figs should be combined % TODO actually make the captions for these % TODO add some background into why integrins are important and the proposed mechanism +% TODO add an experimental timeline to these showing when I added the mabs \begin{figure*}[ht!] \begingroup @@ -2297,6 +2308,20 @@ do the inverse. \label{fig:integrin_1} \end{figure*} +% TABLE add regression table showing the results from this analysis + +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}. +These block \glspl{mab} were added at day 6 of culture when \gls{a2b1} and +\gls{a2b2} were known to be expressed {\#}. We found that the fold expansion was +identical in all the blocked groups vs the unblocked control group +(\cref{fig:inegrin_1_fc}). Furthermore, we observed that the \ptmemp{} (total +and across the CD4/CD8 compartments) was not significantly different between any +of the groups (\cref{fig:inegrin_1_mem}). We also noted that \gls{a2b1} and +\gls{a2b2} were present on the surface of a significant subset of T cells at day +6, showing that the target we wished to block was present +(\cref{fig:inegrin_1_cd49}). + \begin{figure*}[ht!] \begingroup @@ -2311,8 +2336,33 @@ do the inverse. \label{fig:integrin_2} \end{figure*} +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 +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, +CD4, or CD8) were statistically different between groups +(\cref{fig:inegrin_2_mem}). + +Taken together, these data suggest that the advantage of the \gls{dms} platform +is not due to signaling through \gls{a2b1} or \gls{a2b2}. + \subsection{blocking IL15 signaling does not alter expansion or phenotype} +% BACKGROUND why is IL15 important? + +% TODO cite the luminex data +\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 +greater extend in \gls{dms} compared to bead cultures. One of our driving +hypotheses in designing the \gls{dms} system was that the higher cell density +would lead to greater local signaling. Since we observed higher \ptmemp{} across +many conditions, we hypothesized that \gls{il15} may be 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. + % TODO actually add captions % TODO add fold change and viability to these % TODO maybe combine these @@ -2332,6 +2382,20 @@ do the inverse. \label{fig:il15_1} \end{figure*} +% TODO how did I determine how much to add? +% TODO how often was this added? +% TODO 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 +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 +between any group (\cref{fig:il15_1_viability}). We also performed flow +cytometry to asses the \ptmemp{} and \pthp{} outputs. Without even gating the +samples, simply lining up their histograms showed no difference between any of +the markers, and by extension showing no difference in phenotype +(\cref{fig:il15_1_mem}). + \begin{figure*}[ht!] \begingroup @@ -2347,6 +2411,16 @@ do the inverse. \label{fig:il15_2} \end{figure*} +We next tried blocking soluble \gls{il15} itself using either a \gls{mab} or an +\gls{igg} isotype control. 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. + +% TODO 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 +gains its advantages via the \gls{il15} pathway. + \section{discussion} \chapter{aim 3}\label{aim3}