From c1ff03d5f72d8ae75ab64bf09d434d983ace117e Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Tue, 3 Aug 2021 13:27:50 -0400 Subject: [PATCH] ENH tighten up the mouse discussion --- figures/mouse_summary.svg | 4412 +++++++++++++++++++++++++------------ tex/references.bib | 39 + tex/thesis.tex | 67 +- 3 files changed, 3024 insertions(+), 1494 deletions(-) diff --git a/figures/mouse_summary.svg b/figures/mouse_summary.svg index 61d242d..91368e1 100644 --- a/figures/mouse_summary.svg +++ b/figures/mouse_summary.svg @@ -242,6 +242,888 @@ d="m 266.75,119.98 h 108.78 v 16.27 H 266.75 Z" id="path12662" /> + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + @@ -287,1463 +1169,6 @@ inkscape:label="Layer 1" inkscape:groupmode="layer" id="layer1"> - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CAR+ - - - - - - - - - - - - - - - - CD4+ - - - - - - - - - - - - - - - - CD62L+CCR7+ - - - - - - - - - - - - - - - - - - - - - - - - - - - - Bead - DMS - - - - - - - - - - - - - - - - - Bead - DMS - - - - - - - - - - - - - - - - - Bead - DMS - - - - - - - - - - - - - - - - 0 - 400000 - 800000 - 1200000 - - - - - - - - - - - - - - - - - - - - - - - Number of cells Injected - - - - - - - - - - - - - - - - Dose - - - - - - - - - - - - - - - - - - - - - - - - - Low - - - - - - - Med - - - - - - - High - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CAR+ - - - - - - - - - - - - - - - - CD4+ - - - - - - - - - - - - - - - - CD62L+CCR7+ - - - - - - - - - - - - - - - - - - - - - - - - - - - - Bead - DMS - - - - - - - - - - - - - - - - - Bead - DMS - - - - - - - - - - - - - - - - - Bead - DMS - - - - - - - - - - - - - - - - 0 - 400000 - 800000 - 1200000 - - - - - - - - - - - - - - - - - - - - - - - Number of cells Injected - - - - - - - - - - - - - - - - Harvest Day - - - - - - - - - - - - - - - - - - - - - - - - - 6 - - - - - - - 10 - - - - - - - 14 - - - - - - - - - - - - - - - b. + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + CAR+ + + + + + + CD4+ + + + + + + CD62L+CCR7+ + + + + + + CD62L+CCR7+CD45RA+ + + + + + + + + + + + Bead + DMS + + + + + + + + + + Bead + DMS + + + + + + + + + + Bead + DMS + + + + + + + + + + Bead + DMS + + + + + + 0 + 400000 + 800000 + 1200000 + + + + + + + + + Number of cells Injected + + + + + + Dose + + + + + + + + + + + + Low + + + Med + + + High + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + CAR+ + + + + + + CD4+ + + + + + + CD62L+CCR7+ + + + + + + CD62L+CCR7+CD45RA+ + + + + + + + + + + + Bead + DMS + + + + + + + + + + Bead + DMS + + + + + + + + + + Bead + DMS + + + + + + + + + + Bead + DMS + + + + + + 0 + 400000 + 800000 + 1200000 + + + + + + + + + Number of cells Injected + + + + + + Harvest Day + + + + + + + + + + + + 6 + + + 10 + + + 14 + + + + + + + + + + + + + + + diff --git a/tex/references.bib b/tex/references.bib index c18bcbd..51f1b11 100644 --- a/tex/references.bib +++ b/tex/references.bib @@ -2544,6 +2544,45 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec revised = {2008-07-28}, } +@Article{Lozza2008, + author = {Laura Lozza and Laura Rivino and Greta Guarda and David Jarrossay and Andrea Rinaldi and Francesco Bertoni and Federica Sallusto and Antonio Lanzavecchia and Jens Geginat}, + journal = {European Journal of Immunology}, + title = {The strength of T cell stimulation determines {IL}-7 responsiveness, secondary expansion, and lineage commitment of primed human {CD}4+{IL}-7Rhi T cells}, + year = {2008}, + month = {jan}, + number = {1}, + pages = {30--39}, + volume = {38}, + doi = {10.1002/eji.200737852}, + publisher = {Wiley}, +} + +@Article{Lanzavecchia2005, + author = {Antonio Lanzavecchia and Federica Sallusto}, + journal = {Current Opinion in Immunology}, + title = {Understanding the generation and function of memory T cell subsets}, + year = {2005}, + month = {jun}, + number = {3}, + pages = {326--332}, + volume = {17}, + doi = {10.1016/j.coi.2005.04.010}, + publisher = {Elsevier {BV}}, +} + +@Article{Corse2011, + author = {Emily Corse and Rachel A. Gottschalk and James P. Allison}, + journal = {The Journal of Immunology}, + title = {Strength of {TCR}{\textendash}Peptide/{MHC} Interactions and In Vivo T Cell Responses}, + year = {2011}, + month = {apr}, + number = {9}, + pages = {5039--5045}, + volume = {186}, + doi = {10.4049/jimmunol.1003650}, + publisher = {The American Association of Immunologists}, +} + @Comment{jabref-meta: databaseType:bibtex;} @Comment{jabref-meta: grouping: diff --git a/tex/thesis.tex b/tex/thesis.tex index 8233a7c..21d662d 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -2465,6 +2465,9 @@ 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. +% DISCUSSION assuage krish by showing that in the isotype control fig that IL2 +% doesn't activation T cells: https://www.jimmunol.org/content/jimmunol/191/12/5822.full.pdf + 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 @@ -4148,8 +4151,6 @@ other groups in regard to the final tumor burden. \section{discussion} -% FIGURE add CD45RA to this to rule out one of the alternative possibilities -% explaining this data \begin{figure*}[ht!] \begingroup @@ -4163,7 +4164,8 @@ other groups in regard to the final tumor burden. \subcap{fig:mouse_summary_1}{the first mouse experiment} and \subcap{fig:mouse_summary_2}{the second mouse experiment}. The y axis maximum is set to the maximum number of cells injected between both - experiments (\num{1.25e6}). + experiments (\num{1.25e6}). Note that the \gls{car} was quantified using a + separate panel than the rest of the markers. } \label{fig:mouse_summary} \end{figure*} @@ -4180,32 +4182,32 @@ tumor burden was higher than DMS groups across all the total T cell doses tested here. More interestingly, when only CAR-expressing T cell doses between bead and DMS groups were compared, DMS group had significantly higher survival effects over similar or higher CAR expression T cell doses from bead group. All these -results suggest that the higher proportion of memory T cells in DMS groups -(compared to bead group) resulted in highly effective CAR-T cells that can -efficiently kill tumor cells as recently reported in -literature\cite{Fraietta2018, Sommermeyer2015}. +results suggest that the T cells in DMS groups (compared to bead group) resulted +in highly effective CAR-T cells that can efficiently kill tumor cells. -% DISCUSSION cite a bunch of data saying memory and CD4 T cells are better in -% mice When comparing the total number of T cells of different phenotypes, we observed that when comparing low-dose \gls{dms} group to the mid- bead groups (which had -similar numbers of \gls{car} T cells), the number of \ptmem{} T cells injected -was much lower in the \gls{dms} group (\cref{fig:mouse_summary_1}). This could -mean several things. First, the \ptmem{} phenotype may have nothing to do with -the results seen here, at least in this model. Second, the distribution of -\gls{car} T cells across different subtypes of T cells was different between the -\gls{dms} and bead groups (with possibly higher correlation of \gls{car} -expression and the \ptmem{} phenotype). Third, the \ptmem{} phenotype may not be -precise enough, and the functional `memory' phenotype is a subset of \ptmem{} -which may be higher in the \gls{dms} group and explains the discrepancy between -the two methods. +similar numbers of \gls{car} T cells), the number of \ptmem{} (both with and +without CD45RA) T cells injected was much lower in the \gls{dms} group +(\cref{fig:mouse_summary_1}). This could mean several things. First, the +\ptmem{} phenotype may have nothing to do with the results seen here, at least +in this model. While this may have been the case in our hands, this would +contradict previous evidence suggesting that \gls{tn} and \gls{tcm} cells work +better in almost the same model (the only difference being Raji cells in place +of Nalm-6 cells, both of which express CD19)\cite{Sommermeyer2015}. Second, the +distribution of \gls{car} T cells across different subtypes of T cells was +different between the \gls{dms} and bead groups (with possibly higher +correlation of \gls{car} expression and the \ptmem{} phenotype). It is hard to +assess this without strong assumptions as the \gls{car} was quantified using a +separate flow panel relative to the other markers. -% DISCUSSION cite why CD4 or CD8 matters in this model We can also make a similar observation for the number of \pth{} T cells injected (\cref{fig:mouse_summary_1}). In this case, either the \pth{} phenotype doesn't matter in this model (or the \ptk{} population matters much more), or the distribution of \gls{car} is different between CD4 and CD8 T cells in a manner -that favors the \gls{dms} group. +that favors the \gls{dms} group. While in a glioblastoma model and not a B-cell +\gls{all} model, previous groups have shown that \pthp{} T cells are important +for response\cite{Wang2018}. When testing \gls{car} T cells at earlier timepoints relative to day 14 as used in the first \invivo{} experiment, we noted that none of the \gls{car} @@ -4221,10 +4223,11 @@ to perform better at day 6 as it held off the tumor longer, and also slowed the tumor progression relative to the bead group at day 14 (\cref{fig:mouse_timecourse_ivis_plots}). -Taken together, these data suggest that on average, the \gls{dms} platform -produces T cells that have an advantage \invivo{} over beads. While we may not -know the exact mechanism, our data suggests that the responses are -unsurprisingly influenced by the \ptcarp{} of the final product. +Taken together, these data suggest that the \gls{dms} platform produces T cells +that have an advantage \invivo{} over beads. While we may not know the exact +mechanism, our data suggests that the responses are unsurprisingly influenced by +the \ptcarp{} of the final product. Followup experiments would need to be +performed to determine the precise phenotype responsible for these responses. \chapter{conclusions and future work}\label{conclusions} @@ -4287,13 +4290,13 @@ to control and optimize the \gls{dms} system. We determined that altering the \gls{dms} concentration temporally has profound effects on the phenotype and expansion rate. This agrees with other data we obtained in \cref{aim2a} and with what others have generally reported about signal strength and T cell -differentiation\cite{Gattinoni2012}. We did not find any mechanistic -relationship between either integrin signaling or \gls{il15} signaling. In the -case of the former, it may be more likely that the \glspl{dms} surfaces are -saturated to the point of sterically hindering any integrin interactions with -the collagen surface. In the case of \gls{il15} more experiments likely need to -be done in order to plausibly rule out this mechanism and/or determine if it is -involved at all. +differentiation\cite{Gattinoni2012, Lozza2008, Lanzavecchia2005, Corse2011}. We +did not find any mechanistic relationship between either integrin signaling or +\gls{il15} signaling. In the case of the former, it may be more likely that the +\glspl{dms} surfaces are saturated to the point of sterically hindering any +integrin interactions with the collagen surface. In the case of \gls{il15} more +experiments likely need to be done in order to plausibly rule out this mechanism +and/or determine if it is involved at all. % TODO make this tighter and cite paper showing that this makes at least some % sense