Supplementary Figure 5: AAV-CRISPR CD8+ T cell screens of membrane bound proteome knockouts in GBM.
(a) Schematics of hybrid AAV-SB-CRIPSR CD8+ T cell screen in a syngeneic mouse model of GBM, showing steps of naïve CD8+ T cell isolation, AAV library transduction, GBM cell transplantation, adoptive T cell transfer, brain and tumor isolation, and sgRNA readout by deep sequencing. (b) Representative in vivo imaging illustrating the growth of GL261-FLuc tumors in the brains of C57BL/6 mice. (c) Representative photos of mice after GBM transplantation and T cell treatment. Red circles indicated macrocephaly suggestive of growing brain tumors. (d) Survival plot of mice after GBM engraftment and adoptive transfer. C57BL/6J mice were transplanted with 1.2 x 106 GL261-FLuc into the lateral ventricle (LV). 4 x 106 OT-I;Cas9β CD8+ T cells were injected after 10 days of tumor engraftment. Survival significance was assessed by a log-rank Mantel-Cox test. The p-values and number of mice used in each group was indicated in the plots. The group of mice receiving adoptive transfer of T cells has significantly enhanced survival, where the effect of AAV-Surf is slightly stronger than Vector. DPI, days post tumor implantation. (e) Representative H&E stained brain sections from PBS, AAV-Vector and AAV-Surf groups. Areas within red dashed lines indicate brain tumors. Scale bar, 2 mm for whole brain sections, and 100 μm for zoom-in sections. These are representative images at the endpoint of survival thus not quantitative for comparison in terms of tumor burden. One experiment, n = 3 individual mice for PBS, n = 5 mice for vector, n = 11 mice for AAV-Surf. (f) Scatterplot of brain vs. cell sgRNA library representation of AAV-Surf shorter term screen experiment (max survival 20 days post injection). The most enriched sgRNAs in the brain are highlighted. Purple dash line, y = x curve; blue dash line, linear regression of the distribution of the 1,000 NTCs between the brain and cell samples. (g) Scatterplot of brain vs. cell sgRNA library representation of AAV-Surf longer term screen experiment (max survival 92 days post injection). The most enriched sgRNAs in the brain are highlighted. Purple dash line, y = x curve; blue dash line, linear regression of the distribution of the 1,000 NTCs between the brain and cell samples. The p-values and number of mice used in each group are indicated in the plots and/or in a supplemental excel table.
