ENH update aim 1 methods section
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tex/thesis.bbl
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tex/thesis.bbl
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\begin{thebibliography}{10}
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\expandafter\ifx\csname url\endcsname\relax
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\def\url#1{\texttt{#1}}\fi
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\expandafter\ifx\csname urlprefix\endcsname\relax\def\urlprefix{URL }\fi
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\providecommand{\bibinfo}[2]{#2}
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\providecommand{\eprint}[2][]{\url{#2}}
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\bibitem{Fesnak2016}
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\bibinfo{author}{Fesnak, A.~D.}, \bibinfo{author}{June, C.~H.} \&
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\bibinfo{author}{Levine, B.~L.}
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\newblock \bibinfo{title}{Engineered t cells: the promise and challenges of
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cancer immunotherapy}.
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\newblock \emph{\bibinfo{journal}{Nature Reviews Cancer}}
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\textbf{\bibinfo{volume}{16}}, \bibinfo{pages}{566--581}
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(\bibinfo{year}{2016}).
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\bibitem{Rosenberg2015}
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\bibinfo{author}{Rosenberg, S.~A.} \& \bibinfo{author}{Restifo, N.~P.}
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\newblock \bibinfo{title}{Adoptive cell transfer as personalized immunotherapy
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for human cancer}.
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\newblock \emph{\bibinfo{journal}{Science}} \textbf{\bibinfo{volume}{348}},
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\bibinfo{pages}{62--68} (\bibinfo{year}{2015}).
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\bibitem{Roddie2019}
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\bibinfo{author}{Roddie, C.}, \bibinfo{author}{O'Reilly, M.},
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\bibinfo{author}{Pinto, J. D.~A.}, \bibinfo{author}{Vispute, K.} \&
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\bibinfo{author}{Lowdell, M.}
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\newblock \bibinfo{title}{Manufacturing chimeric antigen receptor t cells:
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issues and challenges}.
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\newblock \emph{\bibinfo{journal}{Cytotherapy}} (\bibinfo{year}{2019}).
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\bibitem{Dwarshuis2017}
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\bibinfo{author}{Dwarshuis, N.~J.}, \bibinfo{author}{Parratt, K.},
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\bibinfo{author}{Santiago-Miranda, A.} \& \bibinfo{author}{Roy, K.}
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\newblock \bibinfo{title}{Cells as advanced therapeutics: State-of-the-art,
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challenges, and opportunities in large scale biomanufacturing of high-quality
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cells for adoptive immunotherapies}.
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\newblock \emph{\bibinfo{journal}{Advanced Drug Delivery Reviews}}
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\textbf{\bibinfo{volume}{114}}, \bibinfo{pages}{222--239}
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(\bibinfo{year}{2017}).
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\bibitem{Wang2016}
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\bibinfo{author}{Wang, X.} \& \bibinfo{author}{Rivi{\`{e}}re, I.}
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\newblock \bibinfo{title}{Clinical manufacturing of {CAR} t cells: foundation
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of a promising therapy}.
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\newblock \emph{\bibinfo{journal}{Molecular Therapy - Oncolytics}}
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\textbf{\bibinfo{volume}{3}}, \bibinfo{pages}{16015} (\bibinfo{year}{2016}).
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\bibitem{Piscopo2017}
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\bibinfo{author}{Piscopo, N.~J.} \emph{et~al.}
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\newblock \bibinfo{title}{Bioengineering solutions for manufacturing challenges
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in {CAR} t cells}.
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\newblock \emph{\bibinfo{journal}{Biotechnology Journal}}
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\textbf{\bibinfo{volume}{13}}, \bibinfo{pages}{1700095}
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(\bibinfo{year}{2017}).
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\bibitem{Bashour2015}
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\bibinfo{author}{Bashour, K.~T.} \emph{et~al.}
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\newblock \bibinfo{title}{Functional characterization of a t cell stimulation
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reagent for the production of therapeutic chimeric antigen receptor t cells}.
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\newblock \emph{\bibinfo{journal}{Blood}} \textbf{\bibinfo{volume}{126}},
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\bibinfo{pages}{1901--1901} (\bibinfo{year}{2015}).
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\bibitem{Gendron2003}
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\bibinfo{author}{Gendron, S.}, \bibinfo{author}{Couture, J.} \&
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\bibinfo{author}{Aoudjit, F.}
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\newblock \bibinfo{title}{Integrin $\alpha$2$\beta$1inhibits fas-mediated
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apoptosis in t lymphocytes by protein phosphatase 2a-dependent activation of
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the {MAPK}/{ERK} pathway}.
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\newblock \emph{\bibinfo{journal}{Journal of Biological Chemistry}}
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\textbf{\bibinfo{volume}{278}}, \bibinfo{pages}{48633--48643}
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(\bibinfo{year}{2003}).
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\bibitem{Ohtani2008}
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\bibinfo{author}{Ohtani, O.} \& \bibinfo{author}{Ohtani, Y.}
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\newblock \bibinfo{title}{Structure and function of rat lymph nodes}.
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\newblock \emph{\bibinfo{journal}{Archives of Histology and Cytology}}
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\textbf{\bibinfo{volume}{71}}, \bibinfo{pages}{69--76}
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(\bibinfo{year}{2008}).
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\bibitem{Boisvert2007}
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\bibinfo{author}{Boisvert, M.}, \bibinfo{author}{Gendron, S.},
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\bibinfo{author}{Chetoui, N.} \& \bibinfo{author}{Aoudjit, F.}
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\newblock \bibinfo{title}{Alpha2beta1 integrin signaling augments t cell
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receptor-dependent production of interferon-gamma in human t cells}.
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\newblock \emph{\bibinfo{journal}{Molecular Immunology}}
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\textbf{\bibinfo{volume}{44}}, \bibinfo{pages}{3732--3740}
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(\bibinfo{year}{2007}).
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\bibitem{Ben-Horin2004}
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\bibinfo{author}{Ben-Horin, S.} \& \bibinfo{author}{Bank, I.}
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\newblock \bibinfo{title}{The role of very late antigen-1 in immune-mediated
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inflammation}.
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\newblock \emph{\bibinfo{journal}{Clinical Immunology}}
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\textbf{\bibinfo{volume}{113}}, \bibinfo{pages}{119--129}
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(\bibinfo{year}{2004}).
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\bibitem{Forget2014}
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\bibinfo{author}{Forget, M.-A.} \emph{et~al.}
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\newblock \bibinfo{title}{Activation and propagation of tumor-infiltrating
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lymphocytes on clinical-grade designer artificial antigen-presenting cells
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for adoptive immunotherapy of melanoma}.
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\newblock \emph{\bibinfo{journal}{Journal of Immunotherapy}}
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\textbf{\bibinfo{volume}{37}}, \bibinfo{pages}{448--460}
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(\bibinfo{year}{2014}).
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\bibitem{Cheung2018}
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\bibinfo{author}{Cheung, A.~S.}, \bibinfo{author}{Zhang, D. K.~Y.},
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\bibinfo{author}{Koshy, S.~T.} \& \bibinfo{author}{Mooney, D.~J.}
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\newblock \bibinfo{title}{Scaffolds that mimic antigen-presenting cells enable
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ex vivo expansion of primary {T} cells}.
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\newblock \emph{\bibinfo{journal}{Nature Biotechnology}}
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\textbf{\bibinfo{volume}{36}}, \bibinfo{pages}{160--169}
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(\bibinfo{year}{2018}).
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\bibitem{Rio2018}
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\bibinfo{author}{del R{\'{\i}}o, E.~P.}, \bibinfo{author}{Miguel, M.~M.},
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\bibinfo{author}{Veciana, J.}, \bibinfo{author}{Ratera, I.} \&
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\bibinfo{author}{Guasch, J.}
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\newblock \bibinfo{title}{Artificial 3d culture systems for t cell expansion}.
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\newblock \emph{\bibinfo{journal}{{ACS} Omega}} \textbf{\bibinfo{volume}{3}},
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\bibinfo{pages}{5273--5280} (\bibinfo{year}{2018}).
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\bibitem{Delalat2017}
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\bibinfo{author}{Delalat, B.} \emph{et~al.}
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\newblock \bibinfo{title}{{3D printed lattices as an activation and expansion
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platform for T cell therapy}}.
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\newblock \emph{\bibinfo{journal}{Biomaterials}}
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\textbf{\bibinfo{volume}{140}}, \bibinfo{pages}{58--68}
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(\bibinfo{year}{2017}).
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\bibitem{meyer15_immun}
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\bibinfo{author}{Meyer, R.~A.} \emph{et~al.}
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\newblock \bibinfo{title}{Immunoengineering: Biodegradable nanoellipsoidal
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artificial antigen presenting cells for antigen specific t-cell activation
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(small 13/2015)}.
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\newblock \emph{\bibinfo{journal}{Small}} \textbf{\bibinfo{volume}{11}},
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\bibinfo{pages}{1612--1612} (\bibinfo{year}{2015}).
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\bibitem{Lambert2017}
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\bibinfo{author}{Lambert, L.~H.} \emph{et~al.}
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\newblock \bibinfo{title}{{Improving T Cell Expansion with a Soft Touch.}}
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\newblock \emph{\bibinfo{journal}{Nano letters}} \textbf{\bibinfo{volume}{17}},
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\bibinfo{pages}{821--826} (\bibinfo{year}{2017}).
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\bibitem{Xu2014}
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\bibinfo{author}{Xu, Y.} \emph{et~al.}
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\newblock \bibinfo{title}{Closely related t-memory stem cells correlate with in
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vivo expansion of car.cd19-t cells and are preserved by il-7 and il-15.}
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\newblock \emph{\bibinfo{journal}{Blood}} \textbf{\bibinfo{volume}{123}},
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\bibinfo{pages}{3750--3759} (\bibinfo{year}{2014}).
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\bibitem{Fraietta2018}
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\bibinfo{author}{Fraietta, J.~A.} \emph{et~al.}
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\newblock \bibinfo{title}{Determinants of response and resistance to {CD}19
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chimeric antigen receptor ({CAR}) t cell therapy of chronic lymphocytic
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leukemia}.
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\newblock \emph{\bibinfo{journal}{Nature Medicine}}
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\textbf{\bibinfo{volume}{24}}, \bibinfo{pages}{563--571}
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(\bibinfo{year}{2018}).
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\bibitem{Gattinoni2011}
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\bibinfo{author}{Gattinoni, L.} \emph{et~al.}
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\newblock \bibinfo{title}{A human memory t cell subset with stem
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cell{\textendash}like properties}.
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\newblock \emph{\bibinfo{journal}{Nature Medicine}}
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\textbf{\bibinfo{volume}{17}}, \bibinfo{pages}{1290--1297}
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(\bibinfo{year}{2011}).
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\bibitem{Gattinoni2012}
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\bibinfo{author}{Gattinoni, L.}, \bibinfo{author}{Klebanoff, C.~A.} \&
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\bibinfo{author}{Restifo, N.~P.}
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\newblock \bibinfo{title}{{Paths to stemness: building the ultimate antitumour
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T cell.}}
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\newblock \emph{\bibinfo{journal}{Nature reviews. Cancer}}
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\textbf{\bibinfo{volume}{12}}, \bibinfo{pages}{671--84}
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(\bibinfo{year}{2012}).
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\bibitem{Wang2018}
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\bibinfo{author}{Wang, D.} \emph{et~al.}
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\newblock \bibinfo{title}{Glioblastoma-targeted {CD}4+ {CAR} t cells mediate
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superior antitumor activity}.
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\newblock \emph{\bibinfo{journal}{{JCI} Insight}} \textbf{\bibinfo{volume}{3}}
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(\bibinfo{year}{2018}).
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\bibitem{Yang2017}
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\bibinfo{author}{Yang, Y.} \emph{et~al.}
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\newblock \bibinfo{title}{{TCR} engagement negatively affects {CD}8 but not
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{CD}4 {CAR} t cell expansion and leukemic clearance}.
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\newblock \emph{\bibinfo{journal}{Science Translational Medicine}}
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\textbf{\bibinfo{volume}{9}}, \bibinfo{pages}{eaag1209}
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(\bibinfo{year}{2017}).
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\bibitem{Heathman2015}
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\bibinfo{author}{Heathman, T. R.~J.} \emph{et~al.}
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\newblock \bibinfo{title}{Expansion, harvest and cryopreservation of human
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mesenchymal stem cells in a serum-free microcarrier process}.
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\newblock \emph{\bibinfo{journal}{Biotechnology and Bioengineering}}
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\textbf{\bibinfo{volume}{112}}, \bibinfo{pages}{1696--1707}
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(\bibinfo{year}{2015}).
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\bibitem{Sart2011}
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\bibinfo{author}{Sart, S.}, \bibinfo{author}{Errachid, A.},
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\bibinfo{author}{Schneider, Y.-J.} \& \bibinfo{author}{Agathos, S.~N.}
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\newblock \bibinfo{title}{Controlled expansion and differentiation of
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mesenchymal stem cells in a microcarrier based stirred bioreactor}.
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\newblock \emph{\bibinfo{journal}{{BMC} Proceedings}}
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\textbf{\bibinfo{volume}{5}} (\bibinfo{year}{2011}).
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\bibitem{Buck2016}
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\bibinfo{author}{Buck, M.~D.} \emph{et~al.}
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\newblock \bibinfo{title}{{Mitochondrial Dynamics Controls T Cell Fate through
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Metabolic Programming}}.
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\newblock \emph{\bibinfo{journal}{Cell}} \textbf{\bibinfo{volume}{166}},
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\bibinfo{pages}{114} (\bibinfo{year}{2016}).
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\bibitem{van_der_Windt_2012}
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\bibinfo{author}{van~der Windt, G.~J.} \emph{et~al.}
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\newblock \bibinfo{title}{Mitochondrial respiratory capacity is a critical
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regulator of {CD}8+ t cell memory development}.
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\newblock \emph{\bibinfo{journal}{Immunity}} \textbf{\bibinfo{volume}{36}},
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\bibinfo{pages}{68--78} (\bibinfo{year}{2012}).
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\bibitem{Spitzer2016}
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\bibinfo{author}{Spitzer, M.~H.} \& \bibinfo{author}{Nolan, G.~P.}
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\newblock \bibinfo{title}{Mass cytometry: Single cells, many features}.
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\newblock \emph{\bibinfo{journal}{Cell}} \textbf{\bibinfo{volume}{165}},
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\bibinfo{pages}{780--791} (\bibinfo{year}{2016}).
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\end{thebibliography}
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tex/thesis.tex
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@ -65,6 +65,10 @@
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\newacronym{macs}{MACS}{magnetic activated cell sorting}
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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{aopi}{AO/PI}{acridine orange/propidium iodide}
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\newacronym{igg}{IgG}{immunoglobulin G}
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\newacronym{igg}{IgG}{immunoglobulin G}
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\newacronym{pe}{PE}{phycoerythrin}
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\newacronym{ptnl}{PTN-L}{Protein L}
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\newacronym{af647}{AF647}{Alexa Fluor 647}
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\newacronym{anova}{ANOVA}{analysis of variance}
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\newacronym{crispr}{CRISPR}{clustered regularly interspaced short
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\newacronym{crispr}{CRISPR}{clustered regularly interspaced short
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palindromic repeats}
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palindromic repeats}
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}
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}
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\newcommand{\invivo}{\textit{in vivo}}
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\newcommand{\invivo}{\textit{in vivo}}
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\newcommand{\invitro}{\textit{in vitro}}
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\newcommand{\invitro}{\textit{in vitro}}
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\newcommand{\exvivo}{\textit{ex vivo}}
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\newcommand{\exvivo}{\textit{ex vivo}}
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\newcommand{\cd}[1]{CD{#1}}
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\newcommand{\cd}[1]{CD{#1}}
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\newcommand{\anti}[1]{anti-{#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{\acd}[1]{\anti{\cd{#1}}}
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\newcommand{\cdp}[1]{\cd{#1}+}
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\newcommand{\cdp}[1]{\cd{#1}+}
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\newcommand{\cdn}[1]{\cd{#1}-}
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\newcommand{\cdn}[1]{\cd{#1}-}
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\newcommand{\catnum}[2]{(#1, #2)}
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\newcommand{\product}[3]{#1 \catnum{#2}{#3}}
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\newcommand{\thermo}{Thermo Fisher}
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\newcommand{\miltenyi}{Miltenyi Biotech}
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\newcommand{\bl}{Biolegend}
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\newcommand{\inlinecode}{\texttt}
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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% ditto for environments
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% ditto for environments
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@ -302,7 +313,7 @@ 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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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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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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cell quality and phenotype3,4. State-of-the-art T cell manufacturing techniques
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focus on \anticd{3} and \anticd{28} activation and expansion, typically
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focus on \acd{3} and \acd{28} activation and expansion, typically
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presented on superparamagnetic, iron-based microbeads (Invitrogen Dynabead,
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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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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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(Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, Piscopo2017, Bashour2015}.
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@ -349,7 +360,7 @@ such as bioreactors.
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% TODO probably need to address some of the modeling stuff here as well
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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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This thesis describes a novel degradable microscaffold-based method derived from
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porous microcarriers functionalized with \anticd{3} and \anticd{28} \glspl{mab}
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porous microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab}
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for use in T cell expansion cultures. Microcarriers have historically been used
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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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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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\gls{cho} cells, but not with suspension cells such as T
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@ -458,7 +469,7 @@ successes, \gls{car} T cell therapies are constrained by an expensive and
|
||||||
difficult-to-scale manufacturing process\cite{Roddie2019, Dwarshuis2017}.
|
difficult-to-scale manufacturing process\cite{Roddie2019, Dwarshuis2017}.
|
||||||
|
|
||||||
Of critical concern, state-of-the-art manufacturing techniques focus only on
|
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
|
typically presented on a microbead (Invitrogen Dynabead, Miltenyi MACS beads) or
|
||||||
nanobead (Miltenyi TransACT), but also in soluble forms in the case of antibody
|
nanobead (Miltenyi TransACT), but also in soluble forms in the case of antibody
|
||||||
tetramers (Expamer)\cite{Wang2016, Piscopo2017, Roddie2019, Bashour2015}. These
|
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
|
Yang2017}, and no method exists to preferentially expand the CD4 population
|
||||||
compared to state-of-the-art systems.
|
compared to state-of-the-art systems.
|
||||||
|
|
||||||
Here we propose a method using microcarriers functionalized with anti-CD3 and
|
Here we propose a method using microcarriers functionalized with \acd{3} and
|
||||||
anti-CD28 \glspl{mab} for use in T cell expansion cultures. Microcarriers have
|
\acd{28} \glspl{mab} for use in T cell expansion cultures. Microcarriers have
|
||||||
historically been used throughout the bioprocess industry for adherent cultures
|
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
|
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
|
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
|
aspects of the \invivo{} lymph node microenvironment. We compared compare this
|
||||||
system to state-of-the-art T cell activation technologies for both expansion
|
system to state-of-the-art T cell activation technologies for both expansion
|
||||||
potential and memory cell formation. The governing hypothesis was that
|
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
|
provide superior expansion and memory phenotype compared to state-of-the-art
|
||||||
bead-based T cell expansion technology.
|
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
|
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,
|
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
|
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
|
volume ratio. \product{\Gls{snb}}{\thermo}{21217} was dissolved at
|
||||||
\SI{10}{\micro\molar} in sterile ultrapure water, and the true concentration was
|
approximately \SI{10}{\micro\molar} in sterile ultrapure water, and the true
|
||||||
then determined using the \gls{haba} assay (see below).
|
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
|
\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
|
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
|
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
|
\SI{1000}{\gforce} for \SI{1}{\minute}, and removing all liquid back down to the
|
||||||
reaction volume.
|
reaction volume.
|
||||||
|
|
||||||
To coat with \gls{stp}, \SI{40}{\ug\per\mL} \gls{stp} (Jackson Immunoresearch
|
To coat with \gls{stp}, \SI{40}{\ug\per\mL} \product{\gls{stp}}{Jackson
|
||||||
016-000-114) was added and allowed to react for \SI{60}{\minute} at
|
Immunoresearch}{ 016-000-114} was added and allowed to react for
|
||||||
\SI{700}{RPM} of agitation. After the reaction, supernatant was taken for the
|
\SI{60}{\minute} at \SI{700}{RPM} of agitation. After the reaction, supernatant
|
||||||
\gls{bca} assay, and the carriers were washed analogously to the previous wash
|
was taken for the \gls{bca} assay, and the carriers were washed analogously to
|
||||||
step to remove the biotin, except two washes were done and the agitation time
|
the previous wash step to remove the biotin, except two washes were done and the
|
||||||
was \SI{30}{\minute}. Biotinylated \glspl{mab} against human CD3 (Biolegend
|
agitation time was \SI{30}{\minute}. Biotinylated \glspl{mab} against human CD3
|
||||||
317320) and CD28 (Biolegend 302904) were combined in a 1:1 mass ratio and added
|
\catnum{\bl}{317320} and CD28 \catnum{\bl}{302904} were combined in a 1:1 mass
|
||||||
to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along with the
|
ratio and added to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along
|
||||||
\glspl{mab}, sterile \gls{bsa} (Sigma A9576) was added to a final concentration
|
with the \glspl{mab}, sterile \product{\gls{bsa}}{Sigma}{A9576} was added to a
|
||||||
of \SI{2}{\percent} in order to prevent non-specific binding of the antibodies
|
final concentration of \SI{2}{\percent} in order to prevent non-specific binding
|
||||||
to the reaction tubes. \glspl{mab} were allowed to bind to the carriers for
|
of the antibodies to the reaction tubes. \glspl{mab} were allowed to bind to the
|
||||||
\SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, supernatants were
|
carriers for \SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding,
|
||||||
sampled to quantify remaining antibody concentration using an \anti{\gls{igg}}
|
supernatants were sampled to quantify remaining \gls{mab} concentration using an
|
||||||
\gls{elisa} kit (Abcam 157719). Fully functionalized \glspl{dms} were washed in
|
\product{\anti{\gls{igg}} \gls{elisa} kit}{Abcam}{157719}. Fully functionalized
|
||||||
sterile \gls{pbs} analogous to the previous washing step to remove excess
|
\glspl{dms} were washed in sterile \gls{pbs} analogous to the previous washing
|
||||||
\gls{stp}. They were washed once again in the cell culture media to be used for
|
step to remove excess \gls{stp}. They were washed once again in the cell culture
|
||||||
the T cell expansion.
|
media to be used for the T cell expansion.
|
||||||
|
|
||||||
The concentration of the final \gls{dms} suspension was found by taking a
|
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
|
\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}
|
\subsection{dms quality control assays}
|
||||||
|
|
||||||
Biotin was quantified using the \gls{haba} assay (\gls{haba}/avidin premix from
|
Biotin was quantified using the \product{\gls{haba} assay}{Sigma}{H2153-1VL}. In
|
||||||
Sigma as product H2153-1VL). In the case of quantifying sulfo-NHS-biotin prior
|
the case of quantifying \gls{snb} prior to adding it to the microcarriers, the
|
||||||
to adding it to the microcarriers, the sample volume was quenched in a 1:1
|
sample volume was quenched in a 1:1 volumetric ratio with \SI{1}{\molar} NaOH
|
||||||
volumetric ratio with \SI{1}{\molar} NaOH and allowed to react for
|
and allowed to react for \SI{1}{\minute} in order to prevent the reactive ester
|
||||||
\SI{1}{\minute} in order to prevent the reactive ester linkages from binding to
|
linkages from binding to the avidin proteins in the \gls{haba}/avidin premix.
|
||||||
the avidin proteins in the \gls{haba}/avidin premix. All quantifications of
|
All quantifications of \gls{haba} were performed on an Eppendorf D30
|
||||||
\gls{haba} were performed on an Eppendorf D30 Spectrophotometer using \SI{70}{\ul}
|
Spectrophotometer using \product{\SI{70}{\ul} cuvettes}{BrandTech}{759200}. The
|
||||||
uCuvettes (BrandTech 759200). The extinction coefficient at \SI{500}{\nm} for
|
extinction coefficient at \SI{500}{\nm} for \gls{haba}/avidin was assumed to be
|
||||||
\gls{haba}/avidin was assumed to be \SI{34000}{\per\cm\per\molar}.
|
\SI{34000}{\per\cm\per\molar}.
|
||||||
|
|
||||||
\gls{stp} binding to the carriers was quantified indirectly using a \gls{bca}
|
\gls{stp} binding to the carriers was quantified indirectly using a
|
||||||
kit (Thermo Fisher 23227) according to the manufacturer’s instructions, with the
|
\product{\gls{bca} kit }{\thermo}{23227} according to the manufacturer’s
|
||||||
exception that the standard curve was made with known concentrations of purified
|
instructions, with the exception that the standard curve was made with known
|
||||||
\gls{stp} instead of \gls{bsa}. Absorbance at \SI{592}{\nm} was
|
concentrations of purified \gls{stp} instead of \gls{bsa}. Absorbance at
|
||||||
quantified using a Biotek plate reader.
|
\SI{592}{\nm} was quantified using a Biotek plate reader.
|
||||||
|
|
||||||
\Gls{mab} binding to the microcarriers was quantified indirectly using an
|
\Gls{mab} binding to the microcarriers was quantified indirectly using an
|
||||||
\gls{elisa} assay per the manufacturer’s instructions, with the exception that
|
\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.
|
\gls{elisa} standard curve.
|
||||||
|
|
||||||
Open biotin binding sites on the \glspl{dms} after \gls{stp} coating was
|
Open biotin binding sites on the \glspl{dms} after \gls{stp} coating was
|
||||||
quantified indirectly using FITC-biotin (Thermo Fisher B10570). Briefly,
|
quantified indirectly using \product{FITC-biotin}{\thermo}{B10570}.
|
||||||
\SI{400}{\pmol\per\ml} FITC-biotin were added to \gls{stp}-coated carriers and
|
Briefly, \SI{400}{\pmol\per\ml} FITC-biotin were added to \gls{stp}-coated
|
||||||
allowed to react for 20 min at room temperature under constant agitation. The
|
carriers and allowed to react for \SI{20}{\minute} at room temperature under
|
||||||
supernatant was quantified against a standard curve of FITC-biotin using a
|
constant agitation. The supernatant was quantified against a standard curve of
|
||||||
Biotek plate reader.
|
FITC-biotin using a Biotek plate reader.
|
||||||
|
|
||||||
\Gls{stp} binding was verified after the \gls{stp}-binding step visually by
|
\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\%
|
adding biotin-FITC to the \gls{stp}-coated \glspl{dms}, resuspending in
|
||||||
agarose gel, and imaging on a lightsheet microscope (Zeiss Z.1). \Gls{mab}
|
\SI{1}{\percent} agarose gel, and imaging on a \product{lightsheet
|
||||||
binding was verified visually by first staining with \anti{gls{igg}}-FITC
|
microscope}{Zeiss}{Z.1}. \Gls{mab} binding was verified visually by first
|
||||||
(Biolegend 406001), incubating for \SI{30}{\minute}, washing with \gls{pbs}, and
|
staining with \product{\anti{gls{igg}}-FITC}{\bl}{406001}, incubating for
|
||||||
imaging on a confocal microscope.
|
\SI{30}{\minute}, washing with \gls{pbs}, and imaging on a confocal microscope.
|
||||||
|
|
||||||
\subsection{t cell culture}
|
\subsection{t cell culture}
|
||||||
|
|
||||||
Cryopreserved primary human T cells were either obtained as sorted CD3
|
% TODO verify countess product number
|
||||||
subpopulations (Astarte Biotech) or isolated from \glspl{pbmc} (Zenbio) using a
|
Cryopreserved primary human T cells were either obtained as sorted
|
||||||
negative selection \gls{macs} kit for the CD3 subset (Miltenyi Biotech
|
\product{\cdp{3} T cells}{Astarte Biotech}{1017} or isolated from
|
||||||
130-096-535). T cells were activated using \glspl{dms} or \SI{3.5}{\um} CD3/CD28
|
\product{\glspl{pbmc}}{Zenbio}{SER-PBMC} using a negative selection
|
||||||
magnetic beads (Miltenyi Biotech 130-091-441). In the case of beads, T cells
|
\product{\cdp{3} \gls{macs} kit}{\miltenyi}{130-096-535}. T cells were activated
|
||||||
were activated at the manufacturer recommended cell:bead ratio of 2:1. In the
|
using \glspl{dms} or \product{\SI{3.5}{\um} CD3/CD28 magnetic
|
||||||
case of \glspl{dms}, cells were activated using \SI{2500}{\dms\per\cm\squared}
|
beads}{\miltenyi}{130-091-441}. In the case of beads, T cells were activated
|
||||||
unless otherwise noted. Initial cell density was
|
at the manufacturer recommended cell:bead ratio of 2:1. In the case of
|
||||||
\SIrange{2e6}{2.5e6}{\cell\per\ml} to in a 96 well plate with \SI{300}{\ul}
|
\glspl{dms}, cells were activated using \SI{2500}{\dms\per\cm\squared} unless
|
||||||
volume. All media was serum-free Cell Therapy Systems OpTmizer (Thermo Fisher)
|
otherwise noted. Initial cell density was \SIrange{2e6}{2.5e6}{\cell\per\ml} to
|
||||||
or TexMACS (Miltentyi Biotech 170-076-307) supplemented with
|
in a 96 well plate with \SI{300}{\ul} volume. Serum-free media was either
|
||||||
\SIrange{100}{400}{\IU\per\ml} \gls{rhil2} (Peprotech 200-02). Cell cultures
|
\product{OpTmizer}{\thermo}{A1048501} or
|
||||||
were expanded for \SI{14}{\day} as counted from the time of initial seeding and
|
\product{TexMACS}{\miltenyi}{170-076-307} supplemented with
|
||||||
activation. Cell counts and viability were assessed using trypan blue or
|
\SIrange{100}{400}{\IU\per\ml} \product{\gls{rhil2}}{Peprotech}{200-02}. Cell
|
||||||
\gls{aopi} and a Countess Automated Cell Counter (Thermo Fisher). Media was
|
cultures were expanded for \SI{14}{\day} as counted from the time of initial
|
||||||
added to cultures every \SIrange{2}{3}{\day} depending on media color or a
|
seeding and activation. Cell counts and viability were assessed using
|
||||||
\SI{300}{\mg\per\deci\liter} minimum glucose threshold. Media glucose was
|
\product{trypan blue}{\thermo}{T10282} or \product{\gls{aopi}}{Nexcelom
|
||||||
measured using a ChemGlass glucometer.
|
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
|
% In order to remove \glspl{dms} from
|
||||||
% culture, collagenase D (Sigma Aldrich) was sterile filtered in culture media and
|
% 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.
|
% 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
|
Cells on the \glspl{dms} were visualized by adding \SI{0.5}{\ul}
|
||||||
(Biolegend 405204) and \SI{2}{ul} anti-CD45-AF647 (Biolegend 368538), incubating
|
\product{\gls{stp}-\gls{pe}}{\bl}{405204} and \SI{2}{ul}
|
||||||
for an hour, and imaging on a spinning disk confocal microscope.
|
\product{\acd{45}-\gls{af647}}{\bl}{368538}, incubating for \SI{1}{\hour}, and
|
||||||
|
imaging on a spinning disk confocal microscope.
|
||||||
|
|
||||||
\subsection{chemotaxis assay}
|
\subsection{chemotaxis assay}
|
||||||
|
|
||||||
|
% TODO not sure about the transwell product number
|
||||||
Migratory function was assayed using a transwell chemotaxis assay as previously
|
Migratory function was assayed using a transwell chemotaxis assay as previously
|
||||||
described62. Briefly, \SI{3e5}{\cell} were loaded into a transwell plate
|
described\cite{Hromas1997}. Briefly, \SI{3e5}{\cell} were loaded into a
|
||||||
(\SI{5}{\um} pore size, Corning) with the basolateral chamber loaded with
|
\product{transwell plate with \SI{5}{\um} pore size}{Corning}{3421} with the
|
||||||
\SI{600}{\ul} media and 0, 250, or \SI{1000}{\ng\per\mL} CCL21 (Peprotech
|
basolateral chamber loaded with \SI{600}{\ul} media and 0, 250, or
|
||||||
250-13). The plate was incubated for \SI{4}{\hour} after loading, and the
|
\SI{1000}{\ng\per\mL} \product{CCL21}{Peprotech}{250-13}. The plate was
|
||||||
basolateral chamber of each transwell was quantified for total cells using
|
incubated for \SI{4}{\hour} after loading, and the basolateral chamber of each
|
||||||
countbright beads (Thermo Fisher C36950). The final readout was normalized using
|
transwell was quantified for total cells using \product{countbright
|
||||||
the \SI{0}{\ng\per\mL} concentration as background.
|
beads}{\thermo}{C36950}. The final readout was normalized using the
|
||||||
|
\SI{0}{\ng\per\mL} concentration as background.
|
||||||
|
|
||||||
\subsection{degranulation assay}
|
\subsection{degranulation assay}
|
||||||
|
|
||||||
Cytotoxicity of expanded CAR T cells was assessed using a degranulation assay as
|
Cytotoxicity of expanded \gls{car} T cells was assessed using a degranulation
|
||||||
previously described63. Briefly, \num{3e5} T cells were incubated with
|
assay as previously described\cite{Schmoldt1975}. Briefly, \num{3e5} T cells
|
||||||
\num{1.5e5} target cells consisting of either K562 wild type cells (ATCC) or
|
were incubated with \num{1.5e5} target cells consisting of either \product{K562
|
||||||
CD19- expressing K562 cells transformed with \gls{crispr} (kindly provided by Dr.\
|
wild type cells}{ATCC}{CCL-243} or CD19- expressing K562 cells transformed
|
||||||
Yvonne Chen, UCLA)64. Cells were seeded in a flat bottom 96 well plate with
|
with \gls{crispr} (kindly provided by Dr.\ Yvonne Chen, UCLA)\cite{Zah2016}.
|
||||||
\SI{1}{\ug\per\ml} anti-CD49d (eBioscience 16-0499-81), \SI{2}{\micro\molar}
|
Cells were seeded in a flat bottom 96 well plate with \SI{1}{\ug\per\ml}
|
||||||
monensin (eBioscience 00-4505-51), and \SI{1}{\ug\per\ml} anti-CD28 (eBioscience
|
\product{\acd{49d}}{eBioscience}{16-0499-81}, \SI{2}{\micro\molar} \product{monensin}{eBioscience}{
|
||||||
302914) (all \glspl{mab} functional grade) with \SI{250}{\ul} total volume.
|
00-4505-51}, and \SI{1}{\ug\per\ml} \product{\acd{28}}{eBioscience}{302914} (all
|
||||||
After \SI{4}{\hour} incubation at \SI{37}{\degreeCelsius}, cells were stained
|
functional grade \glspl{mab}) with \SI{250}{\ul} total volume. After
|
||||||
for CD3, CD4, and CD107a and analyzed on a BD LSR Fortessa. Readout was
|
\SI{4}{\hour} incubation at \SI{37}{\degreeCelsius}, cells were stained for CD3,
|
||||||
calculated as the percent \cdp{107a} cells of the total CD8 fraction.
|
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}
|
\subsection{car expression}
|
||||||
|
|
||||||
% TODO add acronym for PE
|
\gls{car} expression was quantified as previously described\cite{Zheng2012}.
|
||||||
\gls{car} expression was quantified as previously described65. Briefly, cells
|
Briefly, cells were washed once and stained with \product{biotinylated
|
||||||
were washed once and stained with biotinylated Protein L (Thermo Fisher 29997).
|
\gls{ptnl}}{\thermo}{29997}. After a subsequent wash, cells were stained with
|
||||||
After a subsequent wash, cells were stained with PE-\gls{stp} (Biolegend
|
\product{\gls{pe}-\gls{stp}}{\bl}{405204}, washed again, and analyzed on a
|
||||||
405204), washed again, and analyzed on a BD Accuri. Readout was percent PE+
|
BD Accuri. Readout was percent \gls{pe}+ cells as compared to secondary controls
|
||||||
cells as compared to secondary controls (PE-\gls{stp} with no Protein L).
|
(\gls{pe}-\gls{stp} with no \gls{ptnl}).
|
||||||
|
|
||||||
\subsection{car plasmid and lentiviral transduction}
|
\subsection{car plasmid and lentiviral transduction}
|
||||||
|
|
||||||
The anti-CD19-CD8-CD137-CD3z \gls{car} with the EF1$\upalpha$ promotor29 was
|
The anti-CD19-CD8-CD137-CD3z \gls{car} with the EF1$\upalpha$
|
||||||
synthesized (Aldevron) and subcloned into a FUGW lentiviral transfer plasmid
|
promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a
|
||||||
(Emory Viral Vector Core). Lentiviral vectors were synthesized by the Emory
|
\product{FUGW}{Addgene}{14883} kindly provided by the Emory Viral Vector Core.
|
||||||
Viral Vector Core or the Cincinnati Children's Hospital Medical Center Viral
|
Lentiviral vectors were synthesized by the Emory Viral Vector Core or the
|
||||||
Vector Core. To transduce primary human T cells, retronectin (Takara T100A) was
|
Cincinnati Children's Hospital Medical Center Viral Vector Core. To transduce
|
||||||
coated onto non-TC treated 96 well plates and used to immobilize lentiviral
|
primary human T cells, \product{retronectin}{Takara}{T100A} was coated onto
|
||||||
vector particles according to the manufacturer's instructions. Briefly,
|
non-TC treated 96 well plates and used to immobilize lentiviral vector particles
|
||||||
retronectin solution was adsorbed overnight at \SI{4}{\degreeCelsius} and
|
according to the manufacturer's instructions. Briefly, retronectin solution was
|
||||||
blocked the next day using \gls{bsa}. Prior to transduction, lentiviral
|
adsorbed overnight at \SI{4}{\degreeCelsius} and blocked the next day using
|
||||||
supernatant was spinoculated at \SI{2000}{\gforce} for \SI{2}{\hour} at
|
\gls{bsa}. Prior to transduction, lentiviral supernatant was spinoculated at
|
||||||
\SI{4}{\degreeCelsius}. T cells were activated in 96 well plates using beads or
|
\SI{2000}{\gforce} for \SI{2}{\hour} at \SI{4}{\degreeCelsius}. T cells were
|
||||||
DMSs for \SI{24}{\hour}, and then cells and beads/\glspl{dms} were transferred
|
activated in 96 well plates using beads or \glspl{dms} for \SI{24}{\hour}, and
|
||||||
onto lentiviral vector coated plates and incubated for another \SI{24}{\hour}.
|
then cells and beads/\glspl{dms} were transferred onto lentiviral vector coated
|
||||||
Cells and beads/\glspl{dms} were removed from the retronectin plates using
|
plates and incubated for another \SI{24}{\hour}. Cells and beads/\glspl{dms}
|
||||||
vigorous pipetting and transferred to another 96 well plate wherein expansion
|
were removed from the retronectin plates using vigorous pipetting and
|
||||||
continued.
|
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{results}
|
||||||
\section{discussion}
|
\section{discussion}
|
||||||
|
|
Loading…
Reference in New Issue