From 1a03a47131a8d35c4a293718760a5ed03f2a8c13 Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Fri, 23 Jul 2021 11:53:15 -0400 Subject: [PATCH] ENH update aim 1 methods section --- tex/thesis.bbl | 230 ----------------------------------------- tex/thesis.tex | 272 ++++++++++++++++++++++++++++--------------------- 2 files changed, 156 insertions(+), 346 deletions(-) delete mode 100644 tex/thesis.bbl diff --git a/tex/thesis.bbl b/tex/thesis.bbl deleted file mode 100644 index f27d776..0000000 --- a/tex/thesis.bbl +++ /dev/null @@ -1,230 +0,0 @@ -\begin{thebibliography}{10} -\expandafter\ifx\csname url\endcsname\relax - \def\url#1{\texttt{#1}}\fi -\expandafter\ifx\csname urlprefix\endcsname\relax\def\urlprefix{URL }\fi -\providecommand{\bibinfo}[2]{#2} -\providecommand{\eprint}[2][]{\url{#2}} - -\bibitem{Fesnak2016} -\bibinfo{author}{Fesnak, A.~D.}, \bibinfo{author}{June, C.~H.} \& - \bibinfo{author}{Levine, B.~L.} -\newblock \bibinfo{title}{Engineered t cells: the promise and challenges of - cancer immunotherapy}. -\newblock \emph{\bibinfo{journal}{Nature Reviews Cancer}} - \textbf{\bibinfo{volume}{16}}, \bibinfo{pages}{566--581} - (\bibinfo{year}{2016}). - -\bibitem{Rosenberg2015} -\bibinfo{author}{Rosenberg, S.~A.} \& \bibinfo{author}{Restifo, N.~P.} -\newblock \bibinfo{title}{Adoptive cell transfer as personalized immunotherapy - for human cancer}. -\newblock \emph{\bibinfo{journal}{Science}} \textbf{\bibinfo{volume}{348}}, - \bibinfo{pages}{62--68} (\bibinfo{year}{2015}). - -\bibitem{Roddie2019} -\bibinfo{author}{Roddie, C.}, \bibinfo{author}{O'Reilly, M.}, - \bibinfo{author}{Pinto, J. 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K.~Y.}, - \bibinfo{author}{Koshy, S.~T.} \& \bibinfo{author}{Mooney, D.~J.} -\newblock \bibinfo{title}{Scaffolds that mimic antigen-presenting cells enable - ex vivo expansion of primary {T} cells}. -\newblock \emph{\bibinfo{journal}{Nature Biotechnology}} - \textbf{\bibinfo{volume}{36}}, \bibinfo{pages}{160--169} - (\bibinfo{year}{2018}). - -\bibitem{Rio2018} -\bibinfo{author}{del R{\'{\i}}o, E.~P.}, \bibinfo{author}{Miguel, M.~M.}, - \bibinfo{author}{Veciana, J.}, \bibinfo{author}{Ratera, I.} \& - \bibinfo{author}{Guasch, J.} -\newblock \bibinfo{title}{Artificial 3d culture systems for t cell expansion}. -\newblock \emph{\bibinfo{journal}{{ACS} Omega}} \textbf{\bibinfo{volume}{3}}, - \bibinfo{pages}{5273--5280} (\bibinfo{year}{2018}). - -\bibitem{Delalat2017} -\bibinfo{author}{Delalat, B.} \emph{et~al.} -\newblock \bibinfo{title}{{3D printed lattices as an activation and expansion - platform for T cell therapy}}. -\newblock \emph{\bibinfo{journal}{Biomaterials}} - \textbf{\bibinfo{volume}{140}}, \bibinfo{pages}{58--68} - (\bibinfo{year}{2017}). - -\bibitem{meyer15_immun} -\bibinfo{author}{Meyer, R.~A.} \emph{et~al.} -\newblock \bibinfo{title}{Immunoengineering: Biodegradable nanoellipsoidal - artificial antigen presenting cells for antigen specific t-cell activation - (small 13/2015)}. -\newblock \emph{\bibinfo{journal}{Small}} \textbf{\bibinfo{volume}{11}}, - \bibinfo{pages}{1612--1612} (\bibinfo{year}{2015}). - -\bibitem{Lambert2017} -\bibinfo{author}{Lambert, L.~H.} \emph{et~al.} -\newblock \bibinfo{title}{{Improving T Cell Expansion with a Soft Touch.}} -\newblock \emph{\bibinfo{journal}{Nano letters}} \textbf{\bibinfo{volume}{17}}, - \bibinfo{pages}{821--826} (\bibinfo{year}{2017}). - -\bibitem{Xu2014} -\bibinfo{author}{Xu, Y.} \emph{et~al.} -\newblock \bibinfo{title}{Closely related t-memory stem cells correlate with in - vivo expansion of car.cd19-t cells and are preserved by il-7 and il-15.} -\newblock \emph{\bibinfo{journal}{Blood}} \textbf{\bibinfo{volume}{123}}, - \bibinfo{pages}{3750--3759} (\bibinfo{year}{2014}). - -\bibitem{Fraietta2018} -\bibinfo{author}{Fraietta, J.~A.} \emph{et~al.} -\newblock \bibinfo{title}{Determinants of response and resistance to {CD}19 - chimeric antigen receptor ({CAR}) t cell therapy of chronic lymphocytic - leukemia}. -\newblock \emph{\bibinfo{journal}{Nature Medicine}} - \textbf{\bibinfo{volume}{24}}, \bibinfo{pages}{563--571} - (\bibinfo{year}{2018}). - -\bibitem{Gattinoni2011} -\bibinfo{author}{Gattinoni, L.} \emph{et~al.} -\newblock \bibinfo{title}{A human memory t cell subset with stem - cell{\textendash}like properties}. -\newblock \emph{\bibinfo{journal}{Nature Medicine}} - \textbf{\bibinfo{volume}{17}}, \bibinfo{pages}{1290--1297} - (\bibinfo{year}{2011}). - -\bibitem{Gattinoni2012} -\bibinfo{author}{Gattinoni, L.}, \bibinfo{author}{Klebanoff, C.~A.} \& - \bibinfo{author}{Restifo, N.~P.} -\newblock \bibinfo{title}{{Paths to stemness: building the ultimate antitumour - T cell.}} -\newblock \emph{\bibinfo{journal}{Nature reviews. Cancer}} - \textbf{\bibinfo{volume}{12}}, \bibinfo{pages}{671--84} - (\bibinfo{year}{2012}). - -\bibitem{Wang2018} -\bibinfo{author}{Wang, D.} \emph{et~al.} -\newblock \bibinfo{title}{Glioblastoma-targeted {CD}4+ {CAR} t cells mediate - superior antitumor activity}. -\newblock \emph{\bibinfo{journal}{{JCI} Insight}} \textbf{\bibinfo{volume}{3}} - (\bibinfo{year}{2018}). - -\bibitem{Yang2017} -\bibinfo{author}{Yang, Y.} \emph{et~al.} -\newblock \bibinfo{title}{{TCR} engagement negatively affects {CD}8 but not - {CD}4 {CAR} t cell expansion and leukemic clearance}. -\newblock \emph{\bibinfo{journal}{Science Translational Medicine}} - \textbf{\bibinfo{volume}{9}}, \bibinfo{pages}{eaag1209} - (\bibinfo{year}{2017}). - -\bibitem{Heathman2015} -\bibinfo{author}{Heathman, T. R.~J.} \emph{et~al.} -\newblock \bibinfo{title}{Expansion, harvest and cryopreservation of human - mesenchymal stem cells in a serum-free microcarrier process}. -\newblock \emph{\bibinfo{journal}{Biotechnology and Bioengineering}} - \textbf{\bibinfo{volume}{112}}, \bibinfo{pages}{1696--1707} - (\bibinfo{year}{2015}). - -\bibitem{Sart2011} -\bibinfo{author}{Sart, S.}, \bibinfo{author}{Errachid, A.}, - \bibinfo{author}{Schneider, Y.-J.} \& \bibinfo{author}{Agathos, S.~N.} -\newblock \bibinfo{title}{Controlled expansion and differentiation of - mesenchymal stem cells in a microcarrier based stirred bioreactor}. -\newblock \emph{\bibinfo{journal}{{BMC} Proceedings}} - \textbf{\bibinfo{volume}{5}} (\bibinfo{year}{2011}). - -\bibitem{Buck2016} -\bibinfo{author}{Buck, M.~D.} \emph{et~al.} -\newblock \bibinfo{title}{{Mitochondrial Dynamics Controls T Cell Fate through - Metabolic Programming}}. -\newblock \emph{\bibinfo{journal}{Cell}} \textbf{\bibinfo{volume}{166}}, - \bibinfo{pages}{114} (\bibinfo{year}{2016}). - -\bibitem{van_der_Windt_2012} -\bibinfo{author}{van~der Windt, G.~J.} \emph{et~al.} -\newblock \bibinfo{title}{Mitochondrial respiratory capacity is a critical - regulator of {CD}8+ t cell memory development}. -\newblock \emph{\bibinfo{journal}{Immunity}} \textbf{\bibinfo{volume}{36}}, - \bibinfo{pages}{68--78} (\bibinfo{year}{2012}). - -\bibitem{Spitzer2016} -\bibinfo{author}{Spitzer, M.~H.} \& \bibinfo{author}{Nolan, G.~P.} -\newblock \bibinfo{title}{Mass cytometry: Single cells, many features}. -\newblock \emph{\bibinfo{journal}{Cell}} \textbf{\bibinfo{volume}{165}}, - \bibinfo{pages}{780--791} (\bibinfo{year}{2016}). - -\end{thebibliography} diff --git a/tex/thesis.tex b/tex/thesis.tex index 4d1b140..5d8b076 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -65,6 +65,10 @@ \newacronym{macs}{MACS}{magnetic activated cell sorting} \newacronym{aopi}{AO/PI}{acridine orange/propidium iodide} \newacronym{igg}{IgG}{immunoglobulin G} +\newacronym{pe}{PE}{phycoerythrin} +\newacronym{ptnl}{PTN-L}{Protein L} +\newacronym{af647}{AF647}{Alexa Fluor 647} +\newacronym{anova}{ANOVA}{analysis of variance} \newacronym{crispr}{CRISPR}{clustered regularly interspaced short palindromic repeats} @@ -105,17 +109,24 @@ } \newcommand{\invivo}{\textit{in vivo}} - \newcommand{\invitro}{\textit{in vitro}} - \newcommand{\exvivo}{\textit{ex vivo}} \newcommand{\cd}[1]{CD{#1}} \newcommand{\anti}[1]{anti-{#1}} -\newcommand{\anticd}[1]{\anti{\cd{#1}}} +\newcommand{\acd}[1]{\anti{\cd{#1}}} \newcommand{\cdp}[1]{\cd{#1}+} \newcommand{\cdn}[1]{\cd{#1}-} +\newcommand{\catnum}[2]{(#1, #2)} +\newcommand{\product}[3]{#1 \catnum{#2}{#3}} + +\newcommand{\thermo}{Thermo Fisher} +\newcommand{\miltenyi}{Miltenyi Biotech} +\newcommand{\bl}{Biolegend} + +\newcommand{\inlinecode}{\texttt} + %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % ditto for environments @@ -302,7 +313,7 @@ two genetically-modified \gls{car} T cell therapies against B cell malignancies. Despite these successes, \gls{car} T cell therapies are constrained by an expensive and difficult-to-scale manufacturing process with little control on cell quality and phenotype3,4. State-of-the-art T cell manufacturing techniques -focus on \anticd{3} and \anticd{28} activation and expansion, typically +focus on \acd{3} and \acd{28} activation and expansion, typically presented on superparamagnetic, iron-based microbeads (Invitrogen Dynabead, Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or in soluble tetramers (Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, Piscopo2017, Bashour2015}. @@ -349,7 +360,7 @@ such as bioreactors. % TODO probably need to address some of the modeling stuff here as well This thesis describes a novel degradable microscaffold-based method derived from -porous microcarriers functionalized with \anticd{3} and \anticd{28} \glspl{mab} +porous microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab} for use in T cell expansion cultures. Microcarriers have historically been used throughout the bioprocess industry for adherent cultures such as stem cells and \gls{cho} cells, but not with suspension cells such as T @@ -458,7 +469,7 @@ successes, \gls{car} T cell therapies are constrained by an expensive and difficult-to-scale manufacturing process\cite{Roddie2019, Dwarshuis2017}. Of critical concern, state-of-the-art manufacturing techniques focus only on -Signal 1 and Signal 2-based activation via anti-CD3 and anti-CD28 \glspl{mab}, +Signal 1 and Signal 2-based activation via \acd{3} and \acd{28} \glspl{mab}, typically presented on a microbead (Invitrogen Dynabead, Miltenyi MACS beads) or nanobead (Miltenyi TransACT), but also in soluble forms in the case of antibody tetramers (Expamer)\cite{Wang2016, Piscopo2017, Roddie2019, Bashour2015}. These @@ -497,8 +508,8 @@ cytokine release properties and ability to resist exhaustion\cite{Wang2018, Yang2017}, and no method exists to preferentially expand the CD4 population compared to state-of-the-art systems. -Here we propose a method using microcarriers functionalized with anti-CD3 and -anti-CD28 \glspl{mab} for use in T cell expansion cultures. Microcarriers have +Here we propose a method using microcarriers functionalized with \acd{3} and +\acd{28} \glspl{mab} for use in T cell expansion cultures. Microcarriers have historically been used throughout the bioprocess industry for adherent cultures such as stem cells and \gls{cho} cells, but not with suspension cells such as T cells\cite{Heathman2015, Sart2011}. The carriers have a macroporous structure @@ -625,7 +636,7 @@ The first aim was to develop a microcarrier system that mimics several key aspects of the \invivo{} lymph node microenvironment. We compared compare this system to state-of-the-art T cell activation technologies for both expansion potential and memory cell formation. The governing hypothesis was that -microcarriers functionalized with anti-CD3 and anti-CD28 \glspl{mab} will +microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab} will provide superior expansion and memory phenotype compared to state-of-the-art bead-based T cell expansion technology. @@ -652,9 +663,9 @@ autoclaved. All subsequent steps were done aseptically, and all reactions were carried out at \SI{20}{\mg\per\ml} carriers at room temperature and agitated using an orbital shaker with a \SI{3}{\mm} orbit diameter. After autoclaving, the microcarriers were washed using sterile \gls{pbs} three times in a 10:1 -volume ratio. \gls{snb} (Thermo Fisher 21217) was dissolved at approximately -\SI{10}{\micro\molar} in sterile ultrapure water, and the true concentration was -then determined using the \gls{haba} assay (see below). +volume ratio. \product{\Gls{snb}}{\thermo}{21217} was dissolved at +approximately \SI{10}{\micro\molar} in sterile ultrapure water, and the true +concentration was then determined using the \gls{haba} assay (see below). \SI{5}{\ul\of{\ab}\per\mL} \gls{pbs} was added to carrier suspension and allowed to react for \SI{60}{\minute} at \SI{700}{\rpm} of agitation. After the reaction, the amount of biotin remaining in solution was quantified using the @@ -665,23 +676,23 @@ entailed adding sterile \gls{pbs} in a 10:1 volumetric ratio, agitating at \SI{1000}{\gforce} for \SI{1}{\minute}, and removing all liquid back down to the reaction volume. -To coat with \gls{stp}, \SI{40}{\ug\per\mL} \gls{stp} (Jackson Immunoresearch -016-000-114) was added and allowed to react for \SI{60}{\minute} at -\SI{700}{RPM} of agitation. After the reaction, supernatant was taken for the -\gls{bca} assay, and the carriers were washed analogously to the previous wash -step to remove the biotin, except two washes were done and the agitation time -was \SI{30}{\minute}. Biotinylated \glspl{mab} against human CD3 (Biolegend -317320) and CD28 (Biolegend 302904) were combined in a 1:1 mass ratio and added -to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along with the -\glspl{mab}, sterile \gls{bsa} (Sigma A9576) was added to a final concentration -of \SI{2}{\percent} in order to prevent non-specific binding of the antibodies -to the reaction tubes. \glspl{mab} were allowed to bind to the carriers for -\SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, supernatants were -sampled to quantify remaining antibody concentration using an \anti{\gls{igg}} -\gls{elisa} kit (Abcam 157719). Fully functionalized \glspl{dms} were washed in -sterile \gls{pbs} analogous to the previous washing step to remove excess -\gls{stp}. They were washed once again in the cell culture media to be used for -the T cell expansion. +To coat with \gls{stp}, \SI{40}{\ug\per\mL} \product{\gls{stp}}{Jackson + Immunoresearch}{ 016-000-114} was added and allowed to react for +\SI{60}{\minute} at \SI{700}{RPM} of agitation. After the reaction, supernatant +was taken for the \gls{bca} assay, and the carriers were washed analogously to +the previous wash step to remove the biotin, except two washes were done and the +agitation time was \SI{30}{\minute}. Biotinylated \glspl{mab} against human CD3 +\catnum{\bl}{317320} and CD28 \catnum{\bl}{302904} were combined in a 1:1 mass +ratio and added to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along +with the \glspl{mab}, sterile \product{\gls{bsa}}{Sigma}{A9576} was added to a +final concentration of \SI{2}{\percent} in order to prevent non-specific binding +of the antibodies to the reaction tubes. \glspl{mab} were allowed to bind to the +carriers for \SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, +supernatants were sampled to quantify remaining \gls{mab} concentration using an +\product{\anti{\gls{igg}} \gls{elisa} kit}{Abcam}{157719}. Fully functionalized +\glspl{dms} were washed in sterile \gls{pbs} analogous to the previous washing +step to remove excess \gls{stp}. They were washed once again in the cell culture +media to be used for the T cell expansion. The concentration of the final \gls{dms} suspension was found by taking a \SI{50}{\uL} sample, plating in a well, and imaging the entire well. The image @@ -699,21 +710,21 @@ was then manually counted to obtain a concentration. Surface area for \subsection{dms quality control assays} -Biotin was quantified using the \gls{haba} assay (\gls{haba}/avidin premix from -Sigma as product H2153-1VL). In the case of quantifying sulfo-NHS-biotin prior -to adding it to the microcarriers, the sample volume was quenched in a 1:1 -volumetric ratio with \SI{1}{\molar} NaOH and allowed to react for -\SI{1}{\minute} in order to prevent the reactive ester linkages from binding to -the avidin proteins in the \gls{haba}/avidin premix. All quantifications of -\gls{haba} were performed on an Eppendorf D30 Spectrophotometer using \SI{70}{\ul} -uCuvettes (BrandTech 759200). The extinction coefficient at \SI{500}{\nm} for -\gls{haba}/avidin was assumed to be \SI{34000}{\per\cm\per\molar}. +Biotin was quantified using the \product{\gls{haba} assay}{Sigma}{H2153-1VL}. In +the case of quantifying \gls{snb} prior to adding it to the microcarriers, the +sample volume was quenched in a 1:1 volumetric ratio with \SI{1}{\molar} NaOH +and allowed to react for \SI{1}{\minute} in order to prevent the reactive ester +linkages from binding to the avidin proteins in the \gls{haba}/avidin premix. +All quantifications of \gls{haba} were performed on an Eppendorf D30 +Spectrophotometer using \product{\SI{70}{\ul} cuvettes}{BrandTech}{759200}. The +extinction coefficient at \SI{500}{\nm} for \gls{haba}/avidin was assumed to be +\SI{34000}{\per\cm\per\molar}. -\gls{stp} binding to the carriers was quantified indirectly using a \gls{bca} -kit (Thermo Fisher 23227) according to the manufacturer’s instructions, with the -exception that the standard curve was made with known concentrations of purified -\gls{stp} instead of \gls{bsa}. Absorbance at \SI{592}{\nm} was -quantified using a Biotek plate reader. +\gls{stp} binding to the carriers was quantified indirectly using a +\product{\gls{bca} kit }{\thermo}{23227} according to the manufacturer’s +instructions, with the exception that the standard curve was made with known +concentrations of purified \gls{stp} instead of \gls{bsa}. Absorbance at +\SI{592}{\nm} was quantified using a Biotek plate reader. \Gls{mab} binding to the microcarriers was quantified indirectly using an \gls{elisa} assay per the manufacturer’s instructions, with the exception that @@ -721,103 +732,132 @@ the same antibodies used to coat the carriers were used as the standard for the \gls{elisa} standard curve. Open biotin binding sites on the \glspl{dms} after \gls{stp} coating was -quantified indirectly using FITC-biotin (Thermo Fisher B10570). Briefly, -\SI{400}{\pmol\per\ml} FITC-biotin were added to \gls{stp}-coated carriers and -allowed to react for 20 min at room temperature under constant agitation. The -supernatant was quantified against a standard curve of FITC-biotin using a -Biotek plate reader. +quantified indirectly using \product{FITC-biotin}{\thermo}{B10570}. +Briefly, \SI{400}{\pmol\per\ml} FITC-biotin were added to \gls{stp}-coated +carriers and allowed to react for \SI{20}{\minute} at room temperature under +constant agitation. The supernatant was quantified against a standard curve of +FITC-biotin using a Biotek plate reader. \Gls{stp} binding was verified after the \gls{stp}-binding step visually by -adding biotin-FITC to the \gls{stp}-coated \glspl{dms}, resuspending in 1\% -agarose gel, and imaging on a lightsheet microscope (Zeiss Z.1). \Gls{mab} -binding was verified visually by first staining with \anti{gls{igg}}-FITC -(Biolegend 406001), incubating for \SI{30}{\minute}, washing with \gls{pbs}, and -imaging on a confocal microscope. +adding biotin-FITC to the \gls{stp}-coated \glspl{dms}, resuspending in +\SI{1}{\percent} agarose gel, and imaging on a \product{lightsheet + microscope}{Zeiss}{Z.1}. \Gls{mab} binding was verified visually by first +staining with \product{\anti{gls{igg}}-FITC}{\bl}{406001}, incubating for +\SI{30}{\minute}, washing with \gls{pbs}, and imaging on a confocal microscope. \subsection{t cell culture} -Cryopreserved primary human T cells were either obtained as sorted CD3 -subpopulations (Astarte Biotech) or isolated from \glspl{pbmc} (Zenbio) using a -negative selection \gls{macs} kit for the CD3 subset (Miltenyi Biotech -130-096-535). T cells were activated using \glspl{dms} or \SI{3.5}{\um} CD3/CD28 -magnetic beads (Miltenyi Biotech 130-091-441). In the case of beads, T cells -were activated at the manufacturer recommended cell:bead ratio of 2:1. In the -case of \glspl{dms}, cells were activated using \SI{2500}{\dms\per\cm\squared} -unless otherwise noted. Initial cell density was -\SIrange{2e6}{2.5e6}{\cell\per\ml} to in a 96 well plate with \SI{300}{\ul} -volume. All media was serum-free Cell Therapy Systems OpTmizer (Thermo Fisher) -or TexMACS (Miltentyi Biotech 170-076-307) supplemented with -\SIrange{100}{400}{\IU\per\ml} \gls{rhil2} (Peprotech 200-02). Cell cultures -were expanded for \SI{14}{\day} as counted from the time of initial seeding and -activation. Cell counts and viability were assessed using trypan blue or -\gls{aopi} and a Countess Automated Cell Counter (Thermo Fisher). Media was -added to cultures every \SIrange{2}{3}{\day} depending on media color or a -\SI{300}{\mg\per\deci\liter} minimum glucose threshold. Media glucose was -measured using a ChemGlass glucometer. +% TODO verify countess product number +Cryopreserved primary human T cells were either obtained as sorted +\product{\cdp{3} T cells}{Astarte Biotech}{1017} or isolated from +\product{\glspl{pbmc}}{Zenbio}{SER-PBMC} using a negative selection +\product{\cdp{3} \gls{macs} kit}{\miltenyi}{130-096-535}. T cells were activated +using \glspl{dms} or \product{\SI{3.5}{\um} CD3/CD28 magnetic + beads}{\miltenyi}{130-091-441}. In the case of beads, T cells were activated +at the manufacturer recommended cell:bead ratio of 2:1. In the case of +\glspl{dms}, cells were activated using \SI{2500}{\dms\per\cm\squared} unless +otherwise noted. Initial cell density was \SIrange{2e6}{2.5e6}{\cell\per\ml} to +in a 96 well plate with \SI{300}{\ul} volume. Serum-free media was either +\product{OpTmizer}{\thermo}{A1048501} or +\product{TexMACS}{\miltenyi}{170-076-307} supplemented with +\SIrange{100}{400}{\IU\per\ml} \product{\gls{rhil2}}{Peprotech}{200-02}. Cell +cultures were expanded for \SI{14}{\day} as counted from the time of initial +seeding and activation. Cell counts and viability were assessed using +\product{trypan blue}{\thermo}{T10282} or \product{\gls{aopi}}{Nexcelom + Bioscience}{CS2-0106-5} and a \product{Countess Automated Cell Counter}{Thermo + Fisher}{Countess 3 FL}. Media was added to cultures every \SIrange{2}{3}{\day} +depending on media color or a \SI{300}{\mg\per\deci\liter} minimum glucose +threshold. Media glucose was measured using a \product{GlucCell glucose + meter}{Chemglass}{CLS-1322-02}. -% this belongs in aim 2 +% TODO this belongs in aim 2 % In order to remove \glspl{dms} from % culture, collagenase D (Sigma Aldrich) was sterile filtered in culture media and % added to a final concentration of \SI{50}{\ug\per\ml} during media addition. -Cells on the \glspl{dms} were visualized by adding \SI{0.5}{\ul} \gls{stp}-PE -(Biolegend 405204) and \SI{2}{ul} anti-CD45-AF647 (Biolegend 368538), incubating -for an hour, and imaging on a spinning disk confocal microscope. +Cells on the \glspl{dms} were visualized by adding \SI{0.5}{\ul} +\product{\gls{stp}-\gls{pe}}{\bl}{405204} and \SI{2}{ul} +\product{\acd{45}-\gls{af647}}{\bl}{368538}, incubating for \SI{1}{\hour}, and +imaging on a spinning disk confocal microscope. \subsection{chemotaxis assay} +% TODO not sure about the transwell product number Migratory function was assayed using a transwell chemotaxis assay as previously -described62. Briefly, \SI{3e5}{\cell} were loaded into a transwell plate -(\SI{5}{\um} pore size, Corning) with the basolateral chamber loaded with -\SI{600}{\ul} media and 0, 250, or \SI{1000}{\ng\per\mL} CCL21 (Peprotech -250-13). The plate was incubated for \SI{4}{\hour} after loading, and the -basolateral chamber of each transwell was quantified for total cells using -countbright beads (Thermo Fisher C36950). The final readout was normalized using -the \SI{0}{\ng\per\mL} concentration as background. +described\cite{Hromas1997}. Briefly, \SI{3e5}{\cell} were loaded into a +\product{transwell plate with \SI{5}{\um} pore size}{Corning}{3421} with the +basolateral chamber loaded with \SI{600}{\ul} media and 0, 250, or +\SI{1000}{\ng\per\mL} \product{CCL21}{Peprotech}{250-13}. The plate was +incubated for \SI{4}{\hour} after loading, and the basolateral chamber of each +transwell was quantified for total cells using \product{countbright + beads}{\thermo}{C36950}. The final readout was normalized using the +\SI{0}{\ng\per\mL} concentration as background. \subsection{degranulation assay} -Cytotoxicity of expanded CAR T cells was assessed using a degranulation assay as -previously described63. Briefly, \num{3e5} T cells were incubated with -\num{1.5e5} target cells consisting of either K562 wild type cells (ATCC) or -CD19- expressing K562 cells transformed with \gls{crispr} (kindly provided by Dr.\ -Yvonne Chen, UCLA)64. Cells were seeded in a flat bottom 96 well plate with -\SI{1}{\ug\per\ml} anti-CD49d (eBioscience 16-0499-81), \SI{2}{\micro\molar} -monensin (eBioscience 00-4505-51), and \SI{1}{\ug\per\ml} anti-CD28 (eBioscience -302914) (all \glspl{mab} functional grade) with \SI{250}{\ul} total volume. -After \SI{4}{\hour} incubation at \SI{37}{\degreeCelsius}, cells were stained -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. +Cytotoxicity of expanded \gls{car} T cells was assessed using a degranulation +assay as previously described\cite{Schmoldt1975}. Briefly, \num{3e5} T cells +were incubated with \num{1.5e5} target cells consisting of either \product{K562 +wild type cells}{ATCC}{CCL-243} or CD19- expressing K562 cells transformed +with \gls{crispr} (kindly provided by Dr.\ Yvonne Chen, UCLA)\cite{Zah2016}. +Cells were seeded in a flat bottom 96 well plate with \SI{1}{\ug\per\ml} +\product{\acd{49d}}{eBioscience}{16-0499-81}, \SI{2}{\micro\molar} \product{monensin}{eBioscience}{ +00-4505-51}, and \SI{1}{\ug\per\ml} \product{\acd{28}}{eBioscience}{302914} (all +functional grade \glspl{mab}) with \SI{250}{\ul} total volume. After +\SI{4}{\hour} incubation at \SI{37}{\degreeCelsius}, cells were stained for CD3, +CD4, and CD107a and analyzed on a BD LSR Fortessa. Readout was calculated as the +percent \cdp{107a} cells of the total \cdp{8} 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). +\gls{car} expression was quantified as previously described\cite{Zheng2012}. +Briefly, cells were washed once and stained with \product{biotinylated + \gls{ptnl}}{\thermo}{29997}. After a subsequent wash, cells were stained with +\product{\gls{pe}-\gls{stp}}{\bl}{405204}, washed again, and analyzed on a +BD Accuri. Readout was percent \gls{pe}+ cells as compared to secondary controls +(\gls{pe}-\gls{stp} with no \gls{ptnl}). \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. +The anti-CD19-CD8-CD137-CD3z \gls{car} with the EF1$\upalpha$ +promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a +\product{FUGW}{Addgene}{14883} kindly provided by the 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, \product{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 \glspl{dms} 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) +\subsection{statistical analysis} + +For 1-way \gls{anova} analysis with Tukey multiple comparisons test, +significance was assessed using the \inlinecode{stat\_compare\_means} function +with the \inlinecode{t.test} method from the \inlinecode{ggpubr} library in R. +For 2-way \gls{anova} analysis, the significance of main and interaction effects +was determined using the car library in R. + +% TODO not all of this stuff applied to my regressions +For least-squares linear regression, statistical significance was evaluated the +\inlinecode{lm} function in R. Stepwise regression models were obtained using +the \inlinecode{stepAIC} function from the \inlinecode{MASS} package with +forward and reverse stepping. All results with categorical variables are +reported relative to baseline reference. Each linear regression was assessed for +validity using residual plots (to assess constant variance and independence +assumptions), QQplots and Shapiro-Wilk normality test (to assess normality +assumptions), Box-Cox plots (to assess need for power transformations), and +lack-of-fit tests where replicates were present (to assess model fit in the +context of pure error). Statistical significance was evaluated at $\upalpha$ = +0.05. + +% TODO add meta-analysis section \section{results} \section{discussion}