ADD results for mouse 2 experiment
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x="0 3.6099999 7.7750001"
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<g
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<g
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<g
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<g
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<g
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<g
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<g
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<g
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<g
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@ -2080,5 +1690,397 @@
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<g
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<g
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<g
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<g
|
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<g
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<g
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<g
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<g
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<path
|
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d="m 49.69,20.79 h 11.56 v 82.97 H 49.69 Z"
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<path
|
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d="m 38.13,20.79 h 11.56 v 65.3 H 38.13 Z"
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<path
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<path
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<path
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<path
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</g>
|
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<g
|
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<g
|
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|
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<g
|
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<g
|
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<g
|
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|
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<g
|
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<g
|
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|
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<g
|
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|
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<g
|
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|
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<g
|
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|
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<g
|
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|
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<g
|
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|
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<g
|
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|
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<path
|
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d="m 34.27,16.64 v 91.27"
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|
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|
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<g
|
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|
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|
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|
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|
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<text
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transform="matrix(1,0,0,-1,15.17,47.82)"
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style="font-variant:normal;font-weight:normal;font-size:5px;font-family:Helvetica;-inkscape-font-specification:Helvetica;writing-mode:lr-tb;fill:#4d4d4d;fill-opacity:1;fill-rule:nonzero;stroke:none"
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|
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<text
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transform="matrix(1,0,0,-1,15.17,76.65)"
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style="font-variant:normal;font-weight:normal;font-size:5px;font-family:Helvetica;-inkscape-font-specification:Helvetica;writing-mode:lr-tb;fill:#4d4d4d;fill-opacity:1;fill-rule:nonzero;stroke:none"
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id="text1762"><tspan
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x="0 2.78 5.5599999 6.9499998 9.7299995"
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sodipodi:role="line"
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<text
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transform="matrix(1,0,0,-1,15.17,105.48)"
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style="font-variant:normal;font-weight:normal;font-size:5px;font-family:Helvetica;-inkscape-font-specification:Helvetica;writing-mode:lr-tb;fill:#4d4d4d;fill-opacity:1;fill-rule:nonzero;stroke:none"
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<g
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|
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<g
|
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<path
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d="m 31.53,20.79 h 2.74"
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|
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<path
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d="m 31.53,49.62 h 2.74"
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|
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<path
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d="m 31.53,78.44 h 2.74"
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|
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<path
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d="m 31.53,107.27 h 2.74"
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|
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</g>
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<g
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id="g1780" />
|
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<g
|
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id="g1782" />
|
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<g
|
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|
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<g
|
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id="g1786" />
|
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<g
|
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id="g1788" />
|
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<g
|
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id="g1790" />
|
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<g
|
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<path
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</g>
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<g
|
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|
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<g
|
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<g
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|
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<path
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<path
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<path
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d="m 101.09,13.9 v 2.74"
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</g>
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<g
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<g
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<text
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<text
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x="0 3.6099999 6.2399998 8.7399998 10.13 12.91"
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<text
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transform="matrix(1,0,0,-1,93.24,8.12)"
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style="font-variant:normal;font-weight:normal;font-size:5px;font-family:Helvetica;-inkscape-font-specification:Helvetica;writing-mode:lr-tb;fill:#4d4d4d;fill-opacity:1;fill-rule:nonzero;stroke:none"
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id="text1822"><tspan
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x="0 3.6099999 6.2399998 8.7399998 10.13 12.91"
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<g
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<g
|
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id="g1826" />
|
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<g
|
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id="g1828" />
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<g
|
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id="g1830" />
|
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<g
|
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id="g1832" />
|
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<g
|
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id="g1834" />
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<g
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id="g1836" />
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<g
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id="g1838">
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<text
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transform="matrix(0,1,1,0,9.79,30.71)"
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style="font-variant:normal;font-weight:normal;font-size:6px;font-family:Helvetica;-inkscape-font-specification:Helvetica;writing-mode:lr-tb;fill:#000000;fill-opacity:1;fill-rule:nonzero;stroke:none"
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x="0 4.3319998 8.6639996 12 13.668 17.334 19.271999 22.608 25.608 27.275999 28.608 31.944 35.279999 36.948002 38.945999 42.282001 43.950001 45.618 49.950001 54.282001 57.618 61.122002"
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y="0"
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sodipodi:role="line"
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<g
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|
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<g
|
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id="g1846" />
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<g
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id="g1848" />
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<g
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id="g1850" />
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<g
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id="g1852" />
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<g
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id="g1854" />
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<g
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id="g1856" />
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<g
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id="g1858">
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<path
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d="m 122.17,46.55 h 28.25 V 78 h -28.25 z"
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style="fill:#ffffff;fill-opacity:1;fill-rule:nonzero;stroke:none"
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id="path1860" />
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</g>
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<g
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id="g1862" />
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<g
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id="g1864" />
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<g
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id="g1866" />
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<g
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id="g1868">
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<text
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transform="matrix(1,0,0,-1,125.01,69.53)"
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style="font-variant:normal;font-weight:normal;font-size:6px;font-family:Helvetica;-inkscape-font-specification:Helvetica;writing-mode:lr-tb;fill:#000000;fill-opacity:1;fill-rule:nonzero;stroke:none"
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id="text1872"><tspan
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x="0 4.9980001 8.3339996 10.002 13.338 16.674"
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y="0"
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</g>
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<g
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<g
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id="g1876" />
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<g
|
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id="g1878" />
|
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<g
|
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id="g1880" />
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<g
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id="g1882">
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<path
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d="m 125.72,58.01 h 6.5 v 6.5 h -6.5 z"
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style="fill:#f8766d;fill-opacity:1;fill-rule:nonzero;stroke:none"
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id="path1884" />
|
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</g>
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<g
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id="g1886" />
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<g
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id="g1888" />
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<g
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<g
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id="g1892">
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<path
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d="m 125.72,50.09 h 6.5 v 6.5 h -6.5 z"
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</g>
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<g
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id="g1896" />
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<g
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id="g1898" />
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<g
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id="g1900" />
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<g
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id="g1902">
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<text
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transform="matrix(1,0,0,-1,135.92,59.47)"
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style="font-variant:normal;font-weight:normal;font-size:5px;font-family:Helvetica;-inkscape-font-specification:Helvetica;writing-mode:lr-tb;fill:#000000;fill-opacity:1;fill-rule:nonzero;stroke:none"
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id="text1906"><tspan
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x="0 3.335 6.1149998 8.8950005"
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y="0"
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</g>
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<g
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<g
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<text
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transform="matrix(1,0,0,-1,135.92,51.55)"
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id="text1920"><tspan
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x="0 3.6099999 7.7750001"
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y="0"
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sodipodi:role="line"
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id="tspan1918">DMS</tspan></text>
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</g>
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id="g1922" />
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id="g1942" />
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id="g1944" />
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</svg>
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|
Before Width: | Height: | Size: 79 KiB After Width: | Height: | Size: 78 KiB |
|
|
@ -102,6 +102,9 @@
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|||
\newacronym{tnfa}{TNF$\upalpha$}{tumor necrosis factor-$\upalpha$}
|
||||
\newacronym{sql}{SQL}{structured query language}
|
||||
\newacronym{fcs}{FCS}{flow cytometry standard}
|
||||
\newacronym{ivis}{ivis}{in vivo imaging system}
|
||||
\newacronym{iacuc}{IACUC}{institutional animal care and use committee}
|
||||
|
||||
|
||||
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
|
||||
% SI units for uber nerds
|
||||
|
|
@ -2232,18 +2235,18 @@ day 0 (-7 day relative to T cell injection), 1e6 firefly luciferase-expressing
|
|||
via tail vein into each mouse. At day 7, saline or CAR T cells at the indicated
|
||||
doses from either bead or DMS-expanded T cell cultures (for 14 days) were
|
||||
injected into each mouse via tail vein. Tumor burden was quantified
|
||||
longitudinally via IVIS Spectrum In Vivo Imaging System (Perkin Elmer). Briefly,
|
||||
200ug/mice luciferin at 15 mg/ml in PBS was injected intraperitoneally under
|
||||
isoflurane anesthesia into each mouse and waited for at least 10 minutes before
|
||||
imaging. Mice were anesthetized again and imaged using the IVIS. Mice from each
|
||||
longitudinally via an \gls{ivis} Spectrum (Perkin Elmer). Briefly, 200ug/mice
|
||||
luciferin at 15 mg/ml in PBS was injected intraperitoneally under isoflurane
|
||||
anesthesia into each mouse and waited for at least 10 minutes before imaging.
|
||||
Mice were anesthetized again and imaged using the \gls{ivis}. Mice from each
|
||||
treatment group/dose were anesthetized, injected, and imaged together, and
|
||||
exposure time of the IVIS was limited to avoid saturation based on the signal
|
||||
from the saline group. IVIS images were processed by normalizing them to common
|
||||
minimum and maximum photon counts and total flux was estimated in terms of
|
||||
photons/second. Endpoint for each mouse was determined by IACUC euthanasia
|
||||
criteria (hunched back, paralysis, blindness, lethargy, and weight loss).
|
||||
Mice were euthanized according to these endpoint criteria using carbon dioxide
|
||||
asphyxiation.
|
||||
exposure time of the \gls{ivis} was limited to avoid saturation based on the
|
||||
signal from the saline group. \gls{ivis} images were processed by normalizing
|
||||
them to common minimum and maximum photon counts and total flux was estimated in
|
||||
terms of photons/second. Endpoint for each mouse was determined by \gls{iacuc}
|
||||
euthanasia criteria (hunched back, paralysis, blindness, lethargy, and weight
|
||||
loss). Mice were euthanized according to these endpoint criteria using carbon
|
||||
dioxide asphyxiation.
|
||||
|
||||
\subsection{statistics}
|
||||
|
||||
|
|
@ -2384,6 +2387,15 @@ case of beads (\cref{fig:mouse_dosing_qc_mem}).
|
|||
|
||||
\subsection{Beads and DMSs perform similarly at earlier timepoints}
|
||||
|
||||
We then asked how T cells harvested using either beads or \gls{dms} performed
|
||||
when harvested at earlier timepoints {\#}{levine paper with early timepoints}.
|
||||
We performed the same experiments as described in
|
||||
\cref{fig:mouse_dosing_overview} with the modification that T cells were only
|
||||
grown and harvested after \SI{6}{\day}, \SI{10}{\day}, or \SI{14}{\day} of
|
||||
expansion (\cref{fig:mouse_timecourse_overview}). T cells were frozen after
|
||||
harvest, and all timepoints were thawed at the same time prior to injection. All
|
||||
other characteristics of the experiment were the same.
|
||||
|
||||
\begin{figure*}[ht!]
|
||||
\begingroup
|
||||
|
||||
|
|
@ -2397,6 +2409,22 @@ case of beads (\cref{fig:mouse_dosing_qc_mem}).
|
|||
\label{fig:mouse_timecourse_overview}
|
||||
\end{figure*}
|
||||
|
||||
% TODO find literature saying that CAR T cells grow slower
|
||||
As was the case with the first \invivo{} experiment, T cells activated with
|
||||
\glspl{dms} expanded much more efficiently compared to those expanded with beads
|
||||
(\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} of T
|
||||
cells harvested at each timepoint, we noted that the bead group had much higher
|
||||
\ptcar{} expression at earlier timpoints compared to \gls{dms}, while they
|
||||
equalized at later timepoints (\cref{fig:mouse_timecourse_qc_car}). In addition,
|
||||
overall \ptcar{} expression decreased at later timepoints, indicating that
|
||||
\gls{car} transduced T cells either grow slower or die faster compared to
|
||||
untransduced cells. The \pthp{} of the harvested T cells was higher overall in
|
||||
\gls{dms} expanded T cells but decreased with increasing timepoints
|
||||
(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6
|
||||
between bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{}
|
||||
at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere
|
||||
(\cref{fig:mouse_timecourse_qc_mem}) {\#}{levine paper mem over time}.
|
||||
|
||||
\begin{figure*}[ht!]
|
||||
\begingroup
|
||||
|
||||
|
|
@ -2420,6 +2448,21 @@ case of beads (\cref{fig:mouse_dosing_qc_mem}).
|
|||
\label{fig:mouse_timecourse_qc}
|
||||
\end{figure*}
|
||||
|
||||
We analyzed the tumor burden using \gls{ivis} which showed that mice that
|
||||
received T cells from any group performed better than those that received only
|
||||
saline (\cref{fig:mouse_timecourse_ivis}). Note that unlike the previous
|
||||
experiment, many of the mice survived until day 40 at which point \gls{gvhd}
|
||||
began to take effect (after euthanizing the mice at day 42, most had small or no
|
||||
spleen). When comparing bead and \gls{dms} groups, the \gls{dms} T cells still
|
||||
seemed superior to the bead group, at least initially (note that in this case
|
||||
they had similar numbers of \ptcar{} cells). At day 6, both \gls{dms} and bead
|
||||
groups seemed to eradicate the tumor initially, after which it came back after
|
||||
day 21 for the bead and day 28 for the \gls{dms} group. The day 10 groups
|
||||
performed somewhere in between, where they increased linearly unlike the day 6
|
||||
groups but not as quickly as the day 14 groups. In the case of the \gls{dms} day
|
||||
10 group, it also appeared like a few mice actually performed better than all
|
||||
other groups in regard to the final tumor burden.
|
||||
|
||||
\begin{figure*}[ht!]
|
||||
\begingroup
|
||||
|
||||
|
|
|
|||
Loading…
Reference in New Issue