ADD discussion to aim 2b
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tex/thesis.tex
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tex/thesis.tex
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@ -40,6 +40,7 @@
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\renewcommand{\glossarysection}[2][]{} % remove glossary title
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\renewcommand{\glossarysection}[2][]{} % remove glossary title
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\makeglossaries
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\makeglossaries
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\newacronym{act}{ACT}{adoptive cell therapies}
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\newacronym{act}{ACT}{adoptive cell therapies}
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\newacronym{qc}{QC}{quality control}
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\newacronym{tcm}{T\textsubscript{cm}}{central memory T cell}
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\newacronym{tcm}{T\textsubscript{cm}}{central memory T cell}
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\newacronym{tscm}{T\textsubscript{scm}}{stem-memory T cell}
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\newacronym{tscm}{T\textsubscript{scm}}{stem-memory T cell}
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\newacronym{car}{CAR}{chimeric antigen receptor}
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\newacronym{car}{CAR}{chimeric antigen receptor}
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@ -1823,32 +1824,6 @@ exhaustion, and apoptosis23. Despite evidence for the importance of CD4 T cells,
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more work is required to determine the precise ratios of CD4 and CD8 T cell
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more work is required to determine the precise ratios of CD4 and CD8 T cell
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subsets to be included in CAR T cell therapy given a disease state.
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subsets to be included in CAR T cell therapy given a disease state.
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% TODO this might be more appropriate for aim 2b where I actually talk about
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% the signaling and why this might matter
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There are several plausible explanations for the observed phenotypic differences
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between beads and DMSs. First, the DMSs are composed of a collagen derivative
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(gelatin); collagen has been shown to costimulate activated T cells via
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\gls{a2b1} and \gls{a2b2}, leading to enhanced proliferation, increased
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IFN$\upgamma$ production, and upregulated CD25 (IL2R$\upalpha$) surface
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expression8,10,11,41,42. Second, there is evidence that providing a larger
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contact area for T cell activation provides greater stimulation16,43; the DMSs
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have a rougher interface than the 5 µm magnetic beads, and thus could facilitate
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these larger contact areas. Third, the DMSs may allow the T cells to cluster
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more densely compared to beads, as evidenced by the large clusters on the
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outside of the DMSs (Figure 1f) as well as the significant fraction of DMSs
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found within their interiors (Supplemental Figure 2a and b). This may alter the
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local cytokine environment and trigger different signaling pathways.
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Particularly, IL15 and IL21 are secreted by T cells and known to drive memory
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phenotype44–46. We noted that the IL15 and IL21 concentration was higher in a
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majority of samples when comparing beads and DMSs across multiple timepoints
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(Supplemental Figure 18) in addition to many other cytokines. IL15 and IL21 are
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added exogenously to T cell cultures to enhance memory frequency,45,47 and our
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data here suggest that the DMSs are better at naturally producing these
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cytokines and limiting this need. Furthermore, IL15 unique signals in a trans
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manner in which IL15 is presented on IL15R to neighboring cells48. The higher
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cell density in the DMS cultures would lead to more of these trans interactions,
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and therefore upregulate the IL15 pathway and lead to more memory T cells.
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% TODO this mentions the DOE which is in the next aim
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% TODO this mentions the DOE which is in the next aim
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When analyzing all our experiments comprehensively using causal inference, we
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When analyzing all our experiments comprehensively using causal inference, we
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found that all three of our responses were significantly increased when
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found that all three of our responses were significantly increased when
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@ -2423,6 +2398,88 @@ gains its advantages via the \gls{il15} pathway.
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\section{discussion}
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\section{discussion}
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This work provides insight for how the \gls{dms} operates and may be optimized
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further. The data showing increased \pthp{} when \glspl{dms} are added and the
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reverse when removed is consistent with other data we produced via \gls{doe}
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showing that higher \gls{dms} concentrations lead to higher \pthp{}
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(\cref{fig:doe_responses_cd4,fig:add_rem_cd4}). The difference in this case is
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that we showed that altering activation signal analogously affects the \pthp{}
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in the dimension of time as well as space. A similar trend was observed with
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memory T cells in this aim. Our previous \gls{doe} data showed that, to a point,
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lower \gls{dms} concentration leads to higher \ptmemp{}
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(\cref{fig:doe_responses_mem}). In this aim, we showed that decreasing
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activation signal temporally by removing \glspl{dms} leads to the same effect in
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the \gls{tcm}, \gls{tscm} and `transitory' \gls{tscm} populations, (all of which
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are included in the \ptmem{} phenotype). Taken together, these imply that
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temporally or spatially altering the \gls{dms} concentration, and thus the
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activation signal, has similar effects.
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% TODO this sounds like background?
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% There are several plausible explanations for the observed phenotypic differences
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% between beads and DMSs. First, the DMSs are composed of a collagen derivative
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% (gelatin); collagen has been shown to costimulate activated T cells via
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% \gls{a2b1} and \gls{a2b2}, leading to enhanced proliferation, increased
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% \gls{ifng} production, and upregulated CD25 (IL2R$\upalpha$) surface
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% expression8,10,11,41,42.
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While we did not find support for our hypothesis that the \gls{dms} signal
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through the \gls{a2b1} and/or \gls{a2b2} receptors, we can speculate as to why
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either this experiment failed and may be done better in the future, or why these
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receptors may simply be irrelevant for our system.
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On the first point, we did not verify that these \glspl{mab} indeed blocked the
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receptor we were targeting. There has been evidence from other groups that these
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particular clones work at the concentrations we used {\#}. This does not
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necessarily mean that the \glspl{mab} we obtained were functional in blocking
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their intended targets (although they were from a reputable manufacturer, \bl).
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Furthermore, we can safely rule out the possibility that the \glspl{mab} never
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reached their targets, as they were added immediately after the T cells were
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resuspended as required for cell counting, hence their resting clustered state
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was disrupted.
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% TODO define Bite
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On the second point, the collagen domains may not even be relevant to our system
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depending on the nature of the \gls{stp} coating. We intended by design for the
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system to be fully coated or nearly fully-coated with \gls{stp}
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(\cref{fig:stp_coating}). Thus the domains that \gls{a2b1} and \gls{a2b2} may be
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targeting could be sterically hindered by a layer of \gls{stp}, and if not that,
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also a layer of CD3/CD28 \glspl{mab}. The other possibility is that these
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domains are simply denatured to beyond recognition due to the fabrication
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process for the microcarriers we used (which involves a proprietary
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cross-linking step to make the material autoclave-safe). Either of these could
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be tested and verified by staining the \glspl{dms} with a fluorescently-tagged
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\gls{mab} (or something smaller such as a BiTE to reduce the possibility of
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steric hindrance) and verifying binding via confocal microscopy or indirect
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protein quantification as we do for the \gls{qc} of the \gls{dms}. If this test
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came back negative, we would be fairly confident that the \gls{a2b1} and
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\gls{a2b1} domains are either unreachable or unrecognizable.
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% DISCUSSION not sure exactly how to explain this
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We also failed to uphold our hypothesis that the \gls{dms} system gains its
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advantage via \gls{il15} signaling.
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% TODO not sure if this belongs here, although it might make sense to offer
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% alternative explanations of why the DMSs "work" given this negative data
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% Second, there is evidence that providing a larger
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% contact area for T cell activation provides greater stimulation16,43; the DMSs
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% have a rougher interface than the 5 µm magnetic beads, and thus could facilitate
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% these larger contact areas. Third, the DMSs may allow the T cells to cluster
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% more densely compared to beads, as evidenced by the large clusters on the
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% outside of the DMSs (Figure 1f) as well as the significant fraction of DMSs
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% found within their interiors (Supplemental Figure 2a and b). This may alter the
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% local cytokine environment and trigger different signaling pathways.
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% Particularly, IL15 and IL21 are secreted by T cells and known to drive memory
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% phenotype44–46. We noted that the IL15 and IL21 concentration was higher in a
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% majority of samples when comparing beads and DMSs across multiple timepoints
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% (Supplemental Figure 18) in addition to many other cytokines. IL15 and IL21 are
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% added exogenously to T cell cultures to enhance memory frequency,45,47 and our
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% data here suggest that the DMSs are better at naturally producing these
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% cytokines and limiting this need. Furthermore, IL15 unique signals in a trans
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% manner in which IL15 is presented on IL15R to neighboring cells48. The higher
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% cell density in the DMS cultures would lead to more of these trans interactions,
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% and therefore upregulate the IL15 pathway and lead to more memory T cells.
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\chapter{aim 3}\label{aim3}
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\chapter{aim 3}\label{aim3}
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\section{introduction}
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\section{introduction}
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