From 3b28dc5612421e7bb3768c3f8f30955368e278b1 Mon Sep 17 00:00:00 2001
From: ndwarshuis <ndwar@yavin4.ch>
Date: Sun, 25 Jul 2021 22:53:14 -0400
Subject: [PATCH] ADD a bunch of discussion stuff

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 tex/thesis.tex | 175 +++++++++++++++++++++++++++++++++++++++++++++++++
 1 file changed, 175 insertions(+)

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@@ -1366,6 +1366,181 @@ the responses.
 
 \section{discussion}
 
+% TODO this is fluffy
+We have developed a T cell expansion system that recapitulates key features of
+the in vivo lymph node microenvironment using DMSs functionalized with
+activating mAbs. This strategy provided superior expansion with higher number of
+naïve/memory and CD4+ T cells compared to state-of-the-art microbead technology
+(Figure 2). Other groups have used biomaterials approaches to mimic the in vivo
+microenvironment13–15,17,34; however, to our knowledge this is the first system
+that specifically drives naïve/memory and CD4+ T cell formation in a scalable,
+potentially bioreactor-compatible manufacturing process.
+
+Memory and naïve T cells have been shown to be important clinically. Compared to
+effectors, they have a higher proliferative capacity and are able to engraft for
+months; thus they are able to provide long-term immunity with smaller
+doses19,35. Indeed, less differentiated T cells have led to greater survival
+both in mouse tumor models and human patients20,36,37. Furthermore, clinical
+response rates have been positively correlated with T cell expansion, implying
+that highly-proliferative naïve and memory T cells are a significant
+contributor18,38. Circulating memory T cells have also been found in complete
+responders who received CAR T cell therapy39.
+
+Similarly, CD4 T cells have been shown to play an important role in CAR T cell
+immunotherapy. It has been shown that CAR T doses with only CD4 or a mix of CD4
+and CD8 T cells confer greater tumor cytotoxicity than only CD8 T cells22,40.
+There are several possible reasons for these observations. First, CD4 T cells
+secrete proinflammatory cytokines upon stimulation which may have a synergistic
+effect on CD8 T cells. Second, CD4 T cells may be less prone to exhaustion and
+may more readily adopt a memory phenotype compared to CD8 T cells22. Third, CD8
+T cells may be more susceptible than CD4 T cells to dual stimulation via the CAR
+and endogenous T Cell Receptor (TCR), which could lead to overstimulation,
+exhaustion, and apoptosis23. Despite evidence for the importance of CD4 T cells,
+more work is required to determine the precise ratios of CD4 and CD8 T cell
+subsets to be included in CAR T cell therapy given a disease state.
+
+% TODO this might be more appropriate for aim 2b where I actually talk about
+% the signaling and why this might matter
+There are several plausible explanations for the observed phenotypic differences
+between beads and DMSs. First, the DMSs are composed of a collagen derivative
+(gelatin); collagen has been shown to costimulate activated T cells via α1β1 and
+α2β1 integrins, leading to enhanced proliferation, increased IFNγ production,
+and upregulated CD25 (IL2Rα) surface expression8,10,11,41,42. Second, there is
+evidence that providing a larger contact area for T cell activation provides
+greater stimulation16,43; the DMSs have a rougher interface than the 5 µm
+magnetic beads, and thus could facilitate these larger contact areas. Third, the
+DMSs may allow the T cells to cluster more densely compared to beads, as
+evidenced by the large clusters on the outside of the DMSs (Figure 1f) as well
+as the significant fraction of DMSs found within their interiors (Supplemental
+Figure 2a and b). This may alter the local cytokine environment and trigger
+different signaling pathways. Particularly, IL15 and IL21 are secreted by T
+cells and known to drive memory phenotype44–46. We noted that the IL15 and IL21
+concentration was higher in a majority of samples when comparing beads and DMSs
+across multiple timepoints (Supplemental Figure 18) in addition to many other
+cytokines. IL15 and IL21 are added exogenously to T cell cultures to enhance
+memory frequency,45,47 and our data here suggest that the DMSs are better at
+naturally producing these cytokines and limiting this need. Furthermore, IL15
+unique signals in a trans manner in which IL15 is presented on IL15R to
+neighboring cells48. The higher cell density in the DMS cultures would lead to
+more of these trans interactions, and therefore upregulate the IL15 pathway and
+lead to more memory T cells.
+
+% TODO this mentions the DOE which is in the next aim
+When analyzing all our experiments comprehensively using causal inference, we
+found that all three of our responses were significantly increased when
+controlling for covariates (Figure 3, Table 2). By extension, this implies that
+not only will DMSs lead to higher fold change overall, but also much higher fold
+change in absolute numbers of memory and CD4+ T cells. Furthermore, we found
+that using a Grex bioreactor is detrimental to fold change and memory percent
+while helping CD4+. Since there are multiple consequences to using a Grex
+compared to tissue-treated plates, we can only speculate as to why this might be
+the case. Firstly, when using a Grex we did not expand the surface area on which
+the cells were growing in a comparable way to that of polystyrene plates. In
+conjunction with our DOE data {Figure X} which shows that high DMS
+concentrations favor CD4+ and don’t favor memory fraction, one possible
+explanation is that the T cells spent longer times in highly activating
+conditions (since the beads and DMSs would have been at higher per-area
+concentrations in the Grex vs polystyrene plates). Furthermore, the simple fact
+that the T cells spent more time at high surface densities could simply mean
+that the T cells didn’t expands as much due to spacial constraints. This would
+all be despite the fact that Grex bioreactors are designed to lead to better T
+cell expansion due to their gas-permeable membranes and higher media-loading
+capacities. If anything, our data suggests we were using the bioreactor
+sub-optimally, and the hypothesized causes for why our T cells did not expand
+could be verified with additional experiments varying the starting cell density
+and/or using larger bioreactors.
+
+A key question in the space of cell manufacturing is that of donor variability.
+To state this precisely, this is a second order interaction effect that
+represents the change in effect of treatment (eg bead vs DMS) given the donor.
+While our meta-analysis was relatively large compared to many published
+experiments usually seen for technologies at this developmental stage, we have a
+limited ability in answering this question. We can control for donor as a
+covariate, and indeed our models show that many of the donor characteristics are
+strongly associated with each response on average, but these are first order
+effects and represent the association of age, gender, demographic, etc given
+everything else in the model is held constant. Second order interactions require
+that our treatments be relatively balanced and random across each donor, which
+is a dubious assumption for our dataset. However, this can easily be solved by
+performing more experiments with these restrictions in mind, which will be a
+subject of our future work.
+
+Furthermore, this dataset offers an interesting insight toward novel hypothesis
+that might be further investigated. One limitation of our dataset is that we
+were unable to investigate the effects of time using a method such as
+autoregression, and instead relied on aggregate measures such as the total
+amount of a reagent added over the course of the expansion. Further studies
+should be performed to investigate the temporal relationship between phenotype,
+cytokine concentrations, feed rates, and other measurements which may perturb
+cell cultures, as this will be the foundation of modern process control
+necessary to have a fully-automated manufacturing system.
+
+In addition to larger numbers of potent T cells, other advantages of our DMS
+approach are that the DMSs are large enough to be filtered (approximately 300
+µm) using standard 40 µm cell filters or similar. If the remaining cells inside
+that DMSs are also desired, digestion with dispase or collagenase may be used.
+Collagenase D may be selective enough to dissolve the DMSs yet preserve surface
+markers which may be important to measure as critical quality attributes CQAs
+{Figure X}. Furthermore, our system should be compatible with
+large-scale static culture systems such as the G-Rex bioreactor or perfusion
+culture systems, which have been previously shown to work well for T cell
+expansion12,50,51. The microcarriers used to create the DMSs also have a
+regulatory history in human cell therapies that will aid in clinical
+translation.; they are already a component in an approved retinal pigment
+epithelial cell product for Parkinson’s patients, and are widely available in 30
+countries26.
+
+It is important to note that all T cell cultures in this study were performed up
+to 14 days. Others have demonstrated that potent memory T cells may be obtained
+simply by culturing T cells as little as 5 days using traditional beads30. It is
+unknown if the naïve/memory phenotype of our DMS system could be further
+improved by reducing the culture time, but we can hypothesize that similar
+results would be observed given the lower number of doublings in a 5 day
+culture. We should also note that we investigated one subtype (\ptmem{}) in
+this study. Future work will focus on other memory subtypes such as tissue
+resident memory and stem memory T cells, as well as the impact of using the DMS
+system on the generation of these subtypes.
+
+% TODO this sounds sketchy
+Another advantage is that the DMS system appears to induce a faster growth rate
+of T cells given the same IL2 concentration compared to beads (Supplemental
+Figure 8) along with retaining naïve and memory phenotype. This has benefits in
+multiple contexts. Firstly, some patients have small starting T cell populations
+(such as infants or those who are severely lymphodepleted), and thus require
+more population doublings to reach a usable dose. Our data suggests the time to
+reach this dose would be reduced, easing scheduling a reducing cost. Secondly,
+the allogeneic T cell model would greatly benefit from a system that could
+create large numbers of T cells with naïve and memory phenotype. In contrast to
+the autologous model which is currently used for Kymriah and Yescarta,
+allogeneic T cell therapy would reduce cost by spreading manufacturing expenses
+across many doses for multiple patients52. Since it is economically advantageous
+to grow as many T cells as possible in one batch in the allogeneic model
+(reduced start up and harvesting costs, fewer required cell donations), the DMSs
+offer an advantage over current technology.
+
+% TODO this is already stated in the innovation section
+It should be noted that while we demonstrate a method providing superior
+performance compared to bead-based expansion, the cell manufacturing field would
+tremendously benefit from simply having an alternative to state-of-the-art
+methods. The patents for bead-based expansion are owned by few companies and
+licensed accordingly; having an alternative would provide more competition in
+the market, reducing costs and improving access for academic researchers and
+manufacturing companies.
+
+% TODO this isn't relevent to this aim but should be said somewhere
+Finally, while we have demonstrated the DMS system in the context of CAR T
+cells, this method can theoretically be applied to any T cell immunotherapy
+which responds to anti-CD3/CD28 mAb and cytokine stimulation. These include
+tumor infiltrating lymphocytes (TILs), virus-specific T cells (VSTs), T cells
+engineered to express γδTCR (TEGs), γδ T cells, T cells with transduced-TCR, and
+CAR-TCR T cells53–58. Similar to CD19-CARs used in liquid tumors, these T cell
+immunotherapies would similarly benefit from the increased proliferative
+capacity, metabolic fitness, migration, and engraftment potential characteristic
+of naïve and memory phenotypes59–61. Indeed, since these T cell immunotherapies
+are activated and expanded with either soluble mAbs or bead-immobilized mAbs,
+our system will likely serve as a drop-in substitution to provide these
+benefits.
+
 \chapter{aim 2}\label{aim2}
 
 \section{introduction}