ADD methods for aim 1
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tex/thesis.tex
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tex/thesis.tex
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@ -29,17 +29,11 @@
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\setlist[description]{font=$\bullet$~\textbf\normalfont}
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\sisetup{per-mode=symbol,list-units=single}
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\DeclareSIUnit\activityunit{U}
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\DeclareSIUnit\carrier{carriers}
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\DeclareSIUnit\cell{cells}
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\DeclareSIUnit\ab{mAbs}
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\DeclareSIUnit\molar{M}
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\DeclareSIUnit\gforce{\times{} g}
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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% acronyms for the lazy
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% add acronyms here
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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{car}{CAR}{chimeric antigen receptor}
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\newacronym[longplural={monoclonal antibodies}]{mab}{mAb}{monoclonal antibody}
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@ -54,13 +48,43 @@
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\newacronym{pdms}{PDMS}{polydimethylsiloxane}
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\newacronym{dc}{DC}{dendritic cell}
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\newacronym{il2}{IL2}{interleukin 2}
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\newacronym{rhil2}{rhIL2}{recombinant human interleukin 2}
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\newacronym{apc}{APC}{antigen presenting cell}
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\newacronym{mhc}{MHC}{major histocompatibility complex}
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\newacronym{elisa}{ELISA}{enzyme-linked immunosorbent assay}
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\newacronym{nmr}{NMR}{nuclear magnetic resonance}
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\newacronym{haba}{HABA}{4-hydroxyazobenene-2-carboxylic-acid}
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\newacronym{pbs}{PBS}{phosphate buffered saline}
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\newacronym{bca}{BCA}{bicinchoninic acid assay}
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\newacronym{bsa}{BSA}{bovine serum albumin}
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\newacronym{stp}{STP}{streptavidin}
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\newacronym{snb}{SNB}{sulfo-nhs-biotin}
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\newacronym{cug}{CuG}{Cultispher G}
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\newacronym{cus}{CuS}{Cultispher S}
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\newacronym{pbmc}{PBMC}{peripheral blood mononuclear cells}
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\newacronym{macs}{MACS}{magnetic activated cell sorting}
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\newacronym{aopi}{AO/PI}{acridine orange/propidium iodide}
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\newacronym{igg}{IgG}{immunoglobulin G}
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\newacronym{crispr}{CRISPR}{clustered regularly interspaced short
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palindromic repeats}
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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% my commands
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% SI units for uber nerds
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% NOTE the \SI macro is depreciated but the arch repo (!!!) hasn't been updated
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% with the latest package yet (texlive-science)
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\sisetup{per-mode=symbol,list-units=single}
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\DeclareSIUnit\IU{IU}
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\DeclareSIUnit\rpm{RPM}
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\DeclareSIUnit\dms{DMS}
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\DeclareSIUnit\cell{cells}
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\DeclareSIUnit\ab{mAb}
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\DeclareSIUnit\molar{M}
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\DeclareSIUnit\gforce{\times{} g}
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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% commands for lazy farts like me
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\newcommand{\mytitle}{
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\Large{
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@ -86,8 +110,14 @@
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\newcommand{\exvivo}{\textit{ex vivo}}
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\newcommand{\cd}[1]{CD{#1}}
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\newcommand{\anti}[1]{anti-{#1}}
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\newcommand{\anticd}[1]{\anti{\cd{#1}}}
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\newcommand{\cdp}[1]{\cd{#1}+}
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\newcommand{\cdn}[1]{\cd{#1}-}
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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% my environments
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% ditto for environments
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\newenvironment{mytitlepage}{
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\begin{singlespace}
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@ -99,7 +129,7 @@
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}
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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% document
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% begin document (proceed with caution)
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\begin{document}
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@ -208,15 +238,15 @@ Thank you to Lex Fridman and Devin Townsend for being awesome and inspirational.
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\Gls{act} using \gls{car} T cells have shown promise in treating cancer, but
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manufacturing large numbers of high quality cells remains challenging. Currently
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approved T cell expansion technologies involve anti-CD3 and CD28 \glspl{mab},
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usually mounted on magnetic beads. This method fails to recapitulate many key
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signals found \invivo{} and is also heavily licensed by a few companies,
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limiting its long-term usefulness to manufactures and clinicians. Furthermore,
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we understand that highly potent T cells are generally less-differentiated
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subtypes such as central memory and stem memory T cells. Despite this
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understanding, little has been done to optimize T cell expansion for generating
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these subtypes, including measurement and feedback control strategies that are
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necessary for any modern manufacturing process.
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approved T cell expansion technologies involve \anti-cd{3} and \anti-cd{28}
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\glspl{mab}, usually mounted on magnetic beads. This method fails to
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recapitulate many key signals found \invivo{} and is also heavily licensed by a
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few companies, limiting its long-term usefulness to manufactures and clinicians.
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Furthermore, we understand that highly potent T cells are generally
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less-differentiated subtypes such as central memory and stem memory T cells.
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Despite this understanding, little has been done to optimize T cell expansion
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for generating these subtypes, including measurement and feedback control
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strategies that are necessary for any modern manufacturing process.
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The goal of this thesis was to develop a microcarrier-based \gls{dms} T cell
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expansion system as well as determine biologically-meaningful \glspl{cqa} and
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@ -272,9 +302,9 @@ two genetically-modified \gls{car} T cell therapies against B cell malignancies.
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Despite these successes, \gls{car} T cell therapies are constrained by an
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expensive and difficult-to-scale manufacturing process with little control on
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cell quality and phenotype3,4. State-of-the-art T cell manufacturing techniques
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focus on anti-CD3 and anti-CD28 activation and expansion, typically presented on
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superparamagnetic, iron-based microbeads (Invitrogen Dynabead, Miltenyi MACS
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beads), on nanobeads (Miltenyi TransACT), or in soluble tetramers
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focus on \anticd{3} and \anticd{28} activation and expansion, typically
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presented on superparamagnetic, iron-based microbeads (Invitrogen Dynabead,
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Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or in soluble tetramers
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(Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, Piscopo2017, Bashour2015}.
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These strategies overlook many of the signaling components present in the
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secondary lymphoid organs where T cells expand \invivo{}. Typically, T cells are
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@ -319,8 +349,8 @@ such as bioreactors.
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% TODO probably need to address some of the modeling stuff here as well
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This thesis describes a novel degradable microscaffold-based method derived from
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porous microcarriers functionalized with anti-CD3 and anti-CD28 \glspl{mab} for
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use in T cell expansion cultures. Microcarriers have historically been used
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porous microcarriers functionalized with \anticd{3} and \anticd{28} \glspl{mab}
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for use in T cell expansion cultures. Microcarriers have historically been used
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throughout the bioprocess industry for adherent cultures such as stem cells and
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\gls{cho} cells, but not with suspension cells such as T
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cells\cite{Heathman2015, Sart2011}. The microcarriers chosen to make the DMSs in
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@ -340,10 +370,10 @@ only provide superior expansion, but consistently provide a higher frequency of
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naïve/memory and CD4 T cells (CCR7+CD62L+) across multiple donors. We also
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demonstrate functional cytotoxicity using a CD19 \gls{car} and a superior
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performance, even at a lower \gls{car} T cell dose, of \gls{dms}-expanded
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\gls{car}-T cells \invivo{} in a mouse xenograft model of human B cell \gls{all}.
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Our results indicate that \glspl{dms} provide a robust and scalable platform for
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manufacturing therapeutic T cells with higher naïve/memory phenotype and more
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balanced CD4+ T cell content.
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\gls{car}-T cells \invivo{} in a mouse xenograft model of human B cell
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\gls{all}. Our results indicate that \glspl{dms} provide a robust and scalable
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platform for manufacturing therapeutic T cells with higher naïve/memory
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phenotype and more balanced CD4+ T cell content.
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\section*{hypothesis}
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@ -437,11 +467,11 @@ lymphoid organs where T cells normally expand. Typically, T cells are activated
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under close cell-cell contact via \glspl{apc} such as \glspl{dc}, which present
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peptide-\glspl{mhc} to T cells as well as a variety of other costimulatory
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signals. These close quarters allow for efficient autocrine/paracrine signaling
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among the expanding T cells, which secrete IL2 and other cytokines to assist
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their own growth. Additionally, the lymphoid tissues are comprised of \gls{ecm}
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components such as collagen, which provide signals to upregulate proliferation,
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cytokine production, and pro-survival pathways\cite{Gendron2003, Ohtani2008,
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Boisvert2007, Ben-Horin2004}.
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among the expanding T cells, which secrete gls{il2} and other cytokines to
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assist their own growth. Additionally, the lymphoid tissues are comprised of
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\gls{ecm} components such as collagen, which provide signals to upregulate
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proliferation, cytokine production, and pro-survival pathways\cite{Gendron2003,
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Ohtani2008, Boisvert2007, Ben-Horin2004}.
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A variety of solutions have been proposed to make the T cell expansion process
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more physiological. One strategy is to use modified feeder cell cultures to
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@ -590,18 +620,216 @@ technology for T cell manufacturing:
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\chapter{aim 1}\label{aim1}
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\section{introduction}
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The first aim was to develop a microcarrier system that mimics several key
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aspects of the \invivo{} lymph node microenvironment. We compared compare this
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system to state-of-the-art T cell activation technologies for both expansion
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potential and memory cell formation. The governing hypothesis was that
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microcarriers functionalized with anti-CD3 and anti-CD28 \glspl{mab} will
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provide superior expansion and memory phenotype compared to state-of-the-art
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bead-based T cell expansion technology.
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% TODO this doesn't flow that well and is repetitive with what comes above
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Microcarriers have been used throughout the bioprocess industry for adherent
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cell cultures such as \gls{cho} cells and stem cells, as they are able to
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achieve much greater surface area per unit volume than traditional 2D
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cultures\cite{Heathman2015, Sart2011}. Adding adhesive \glspl{mab} to the
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microcarriers will adapt them for suspension cell cultures such as T cells.
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Consequently, the large macroporous structure will allow T cells to cluster more
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closely, which in turn will enable better autocrine and paracrine signaling.
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Specifically, two cytokines that are secreted by T cells, IL-2 and IL-15, are
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known to drive expansion and memory phenotype respectively\cite{Buck2016}.
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Therefore, the proposed microcarrier system should enable greater expansion and
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better retention of memory phenotype compared to current bead-based methods.
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\section{methods}
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\subsection{dms functionalization}
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Gelatin microcarriers (\gls{cus} or \gls{cug}, GE Healthcare, DG-2001-OO and
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DG-0001-OO) were suspended at \SI{20}{\mg\per\ml} in 1X \gls{pbs} and
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autoclaved. All subsequent steps were done aseptically, and all reactions were
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carried out at \SI{20}{\mg\per\ml} carriers at room temperature and agitated
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using an orbital shaker with a \SI{3}{\mm} orbit diameter. After autoclaving,
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the microcarriers were washed using sterile \gls{pbs} three times in a 10:1
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volume ratio. \gls{snb} (Thermo Fisher 21217) was dissolved at approximately
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\SI{10}{\micro\molar} in sterile ultrapure water, and the true concentration was
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then determined using the \gls{haba} assay (see below).
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\SI{5}{\ul\of{\ab}\per\mL} \gls{pbs} was added to carrier suspension and allowed
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to react for \SI{60}{\minute} at \SI{700}{\rpm} of agitation. After the
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reaction, the amount of biotin remaining in solution was quantified using the
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\gls{haba} assay (see below). The carriers were then washed three times, which
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entailed adding sterile \gls{pbs} in a 10:1 volumetric ratio, agitating at
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\SI{900}{\rpm} for \SI{10}{\minute}, adding up to a 15:1 volumetric ratio
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(relative to reaction volume) of sterile \gls{pbs}, centrifuging at
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\SI{1000}{\gforce} for \SI{1}{\minute}, and removing all liquid back down to the
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reaction volume.
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To coat with \gls{stp}, \SI{40}{\ug\per\mL} \gls{stp} (Jackson Immunoresearch
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016-000-114) was added and allowed to react for \SI{60}{\minute} at
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\SI{700}{RPM} of agitation. After the reaction, supernatant was taken for the
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\gls{bca} assay, and the carriers were washed analogously to the previous wash
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step to remove the biotin, except two washes were done and the agitation time
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was \SI{30}{\minute}. Biotinylated \glspl{mab} against human CD3 (Biolegend
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317320) and CD28 (Biolegend 302904) were combined in a 1:1 mass ratio and added
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to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along with the
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\glspl{mab}, sterile \gls{bsa} (Sigma A9576) was added to a final concentration
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of \SI{2}{\percent} in order to prevent non-specific binding of the antibodies
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to the reaction tubes. \glspl{mab} were allowed to bind to the carriers for
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\SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, supernatants were
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sampled to quantify remaining antibody concentration using an \anti{\gls{igg}}
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\gls{elisa} kit (Abcam 157719). Fully functionalized \glspl{dms} were washed in
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sterile \gls{pbs} analogous to the previous washing step to remove excess
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\gls{stp}. They were washed once again in the cell culture media to be used for
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the T cell expansion.
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The concentration of the final \gls{dms} suspension was found by taking a
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\SI{50}{\uL} sample, plating in a well, and imaging the entire well. The image
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was then manually counted to obtain a concentration. Surface area for
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\si{\ab\per\um\squared} was calculated using the properties for \gls{cus} and
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\gls{cug} as given by the manufacturer {Table X}.
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%TODO this bit belongs in the next aim
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% In the case of the \gls{doe} experiment where
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% variable mAb surface density was utilized, the anti-CD3/anti-CD28 mAb mixture
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% was further combined with a biotinylated isotype control to reduce the overall
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% fraction of targeted mAbs (for example the 60\% mAb surface density corresponded
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% to 3 mass parts anti-CD3, 3 mass parts anti-CD8, and 4 mass parts isotype
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% control).
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\subsection{dms quality control assays}
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Biotin was quantified using the \gls{haba} assay (\gls{haba}/avidin premix from
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Sigma as product H2153-1VL). In the case of quantifying sulfo-NHS-biotin prior
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to adding it to the microcarriers, the sample volume was quenched in a 1:1
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volumetric ratio with \SI{1}{\molar} NaOH and allowed to react for
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\SI{1}{\minute} in order to prevent the reactive ester linkages from binding to
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the avidin proteins in the \gls{haba}/avidin premix. All quantifications of
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\gls{haba} were performed on an Eppendorf D30 Spectrophotometer using \SI{70}{\ul}
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uCuvettes (BrandTech 759200). The extinction coefficient at \SI{500}{\nm} for
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\gls{haba}/avidin was assumed to be \SI{34000}{\per\cm\per\molar}.
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\gls{stp} binding to the carriers was quantified indirectly using a \gls{bca}
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kit (Thermo Fisher 23227) according to the manufacturer’s instructions, with the
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exception that the standard curve was made with known concentrations of purified
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\gls{stp} instead of \gls{bsa}. Absorbance at \SI{592}{\nm} was
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quantified using a Biotek plate reader.
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\Gls{mab} binding to the microcarriers was quantified indirectly using an
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\gls{elisa} assay per the manufacturer’s instructions, with the exception that
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the same antibodies used to coat the carriers were used as the standard for the
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\gls{elisa} standard curve.
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Open biotin binding sites on the \glspl{dms} after \gls{stp} coating was
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quantified indirectly using FITC-biotin (Thermo Fisher B10570). Briefly,
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\SI{400}{\pmol\per\ml} FITC-biotin were added to \gls{stp}-coated carriers and
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allowed to react for 20 min at room temperature under constant agitation. The
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supernatant was quantified against a standard curve of FITC-biotin using a
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Biotek plate reader.
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\Gls{stp} binding was verified after the \gls{stp}-binding step visually by
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adding biotin-FITC to the \gls{stp}-coated \glspl{dms}, resuspending in 1\%
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agarose gel, and imaging on a lightsheet microscope (Zeiss Z.1). \Gls{mab}
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binding was verified visually by first staining with \anti{gls{igg}}-FITC
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(Biolegend 406001), incubating for \SI{30}{\minute}, washing with \gls{pbs}, and
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imaging on a confocal microscope.
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\subsection{t cell culture}
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Cryopreserved primary human T cells were either obtained as sorted CD3
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subpopulations (Astarte Biotech) or isolated from \glspl{pbmc} (Zenbio) using a
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negative selection \gls{macs} kit for the CD3 subset (Miltenyi Biotech
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130-096-535). T cells were activated using \glspl{dms} or \SI{3.5}{\um} CD3/CD28
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magnetic beads (Miltenyi Biotech 130-091-441). In the case of beads, T cells
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were activated at the manufacturer recommended cell:bead ratio of 2:1. In the
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case of \glspl{dms}, cells were activated using \SI{2500}{\dms\per\cm\squared}
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unless otherwise noted. Initial cell density was
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\SIrange{2e6}{2.5e6}{\cell\per\ml} to in a 96 well plate with \SI{300}{\ul}
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volume. All media was serum-free Cell Therapy Systems OpTmizer (Thermo Fisher)
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or TexMACS (Miltentyi Biotech 170-076-307) supplemented with
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\SIrange{100}{400}{\IU\per\ml} \gls{rhil2} (Peprotech 200-02). Cell cultures
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were expanded for \SI{14}{\day} as counted from the time of initial seeding and
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activation. Cell counts and viability were assessed using trypan blue or
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\gls{aopi} and a Countess Automated Cell Counter (Thermo Fisher). Media was
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added to cultures every \SIrange{2}{3}{\day} depending on media color or a
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\SI{300}{\mg\per\deci\liter} minimum glucose threshold. Media glucose was
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measured using a ChemGlass glucometer.
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% this belongs in aim 2
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% In order to remove \glspl{dms} from
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% culture, collagenase D (Sigma Aldrich) was sterile filtered in culture media and
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% added to a final concentration of \SI{50}{\ug\per\ml} during media addition.
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Cells on the \glspl{dms} were visualized by adding \SI{0.5}{\ul} \gls{stp}-PE
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(Biolegend 405204) and \SI{2}{ul} anti-CD45-AF647 (Biolegend 368538), incubating
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for an hour, and imaging on a spinning disk confocal microscope.
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\subsection{chemotaxis assay}
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Migratory function was assayed using a transwell chemotaxis assay as previously
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described62. Briefly, \SI{3e5}{\cell} were loaded into a transwell plate
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(\SI{5}{\um} pore size, Corning) with the basolateral chamber loaded with
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\SI{600}{\ul} media and 0, 250, or \SI{1000}{\ng\per\mL} CCL21 (Peprotech
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250-13). The plate was incubated for \SI{4}{\hour} after loading, and the
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basolateral chamber of each transwell was quantified for total cells using
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countbright beads (Thermo Fisher C36950). The final readout was normalized using
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the \SI{0}{\ng\per\mL} concentration as background.
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\subsection{degranulation assay}
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Cytotoxicity of expanded CAR T cells was assessed using a degranulation assay as
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previously described63. Briefly, \num{3e5} T cells were incubated with
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\num{1.5e5} target cells consisting of either K562 wild type cells (ATCC) or
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CD19- expressing K562 cells transformed with \gls{crispr} (kindly provided by Dr.\
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Yvonne Chen, UCLA)64. Cells were seeded in a flat bottom 96 well plate with
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\SI{1}{\ug\per\ml} anti-CD49d (eBioscience 16-0499-81), \SI{2}{\micro\molar}
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monensin (eBioscience 00-4505-51), and \SI{1}{\ug\per\ml} anti-CD28 (eBioscience
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302914) (all \glspl{mab} functional grade) with \SI{250}{\ul} total volume.
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After \SI{4}{\hour} incubation at \SI{37}{\degreeCelsius}, cells were stained
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for CD3, CD4, and CD107a and analyzed on a BD LSR Fortessa. Readout was
|
||||
calculated as the percent \cdp{107a} cells of the total CD8 fraction.
|
||||
|
||||
\subsection{car expression}
|
||||
|
||||
% TODO add acronym for PE
|
||||
\gls{car} expression was quantified as previously described65. Briefly, cells
|
||||
were washed once and stained with biotinylated Protein L (Thermo Fisher 29997).
|
||||
After a subsequent wash, cells were stained with PE-\gls{stp} (Biolegend
|
||||
405204), washed again, and analyzed on a BD Accuri. Readout was percent PE+
|
||||
cells as compared to secondary controls (PE-\gls{stp} with no Protein L).
|
||||
|
||||
\subsection{car plasmid and lentiviral transduction}
|
||||
|
||||
The anti-CD19-CD8-CD137-CD3z \gls{car} with the EF1$\upalpha$ promotor29 was
|
||||
synthesized (Aldevron) and subcloned into a FUGW lentiviral transfer plasmid
|
||||
(Emory Viral Vector Core). Lentiviral vectors were synthesized by the Emory
|
||||
Viral Vector Core or the Cincinnati Children's Hospital Medical Center Viral
|
||||
Vector Core. To transduce primary human T cells, retronectin (Takara T100A) was
|
||||
coated onto non-TC treated 96 well plates and used to immobilize lentiviral
|
||||
vector particles according to the manufacturer's instructions. Briefly,
|
||||
retronectin solution was adsorbed overnight at \SI{4}{\degreeCelsius} and
|
||||
blocked the next day using \gls{bsa}. Prior to transduction, lentiviral
|
||||
supernatant was spinoculated at \SI{2000}{\gforce} for \SI{2}{\hour} at
|
||||
\SI{4}{\degreeCelsius}. T cells were activated in 96 well plates using beads or
|
||||
DMSs for \SI{24}{\hour}, and then cells and beads/\glspl{dms} were transferred
|
||||
onto lentiviral vector coated plates and incubated for another \SI{24}{\hour}.
|
||||
Cells and beads/\glspl{dms} were removed from the retronectin plates using
|
||||
vigorous pipetting and transferred to another 96 well plate wherein expansion
|
||||
continued.
|
||||
|
||||
% TODO add statistics section (anova, regression, and causal inference)
|
||||
|
||||
\section{results}
|
||||
\section{discussion}
|
||||
|
||||
\chapter{Aim 2}\label{aim2}
|
||||
\chapter{aim 2}\label{aim2}
|
||||
|
||||
\section{introduction}
|
||||
\section{methods}
|
||||
\section{results}
|
||||
\section{discussion}
|
||||
|
||||
\chapter{Aim 3}\label{aim3}
|
||||
\chapter{aim 3}\label{aim3}
|
||||
|
||||
\section{introduction}
|
||||
\section{methods}
|
||||
|
|
Loading…
Reference in New Issue