FOCUSImmunologyThe authors of this study began by designinga mouse genome-scale dgRNA librarytargeting more than 22,000 genes, which willbe delivered to CD8+ T cells isolated fromCas9-transgenic mice to conduct CRISPRa.Using an immunogenic mouse tumor model,researchers co-cultured dgRNA-transducedtumor-targeting CD8+ T cells with their targettumor cells. After four hours, the authorsmeasured the levels of CD107a, a moleculeexpressed after degranulation. Then, the researchersused fluorescence-activated cellsorting to isolate CD8+ T cells with CD107a.Genetic sequencing revealed which dgR-NAs were most significantly enriched in theCD107a+ population. “If the gene is highlyenriched, the signal will be really strong. Wepicked the targets with the strongest signals todo our initial validation,” Ye said. One of thescreen’s top hits, the PRODH2 gene, led to increaseddegranulation and more rapid proliferationin CD8+ T when overexpressed comparedto control cells. Could PRODH2 serve asa functional booster for human CAR-T cells?Metabolic Reprogramming SuperchargesCAR-T CellsIndeed, Chen’s team confirmed thatPRODH2 overexpression in human CAR-Tcells, either by CRISPR knock-in or traditionallentiviral delivery, enhanced tumorkilling and proliferation. These findingsIMAGE COURTESY OF FLICKRLeukemia is a cancer of the body’s blood-forming tissues, usually involving white blood cells. CAR-T therapyagainst B-cell acute lymphocytic leukemia (ALL) is convenient because CAR-T cells can be designed toindiscriminately target and kill all B cells, which are considered to be effectively non-essential.were validated in three in vitro cellular models:leukemia, multiple myeloma, and breastcancer. These effects were replicated in vivo,using human tumor xenograft models forthe same three cancers in mice. PRODH2overexpression led to reduced tumor growthand greater survival in CAR-T cell therapy.But why? The authors performed variousprofiling techniques to gain insights into themechanism underlying how PRODH2 overexpressionenhances CAR-T cell antitumorefficacy. mRNA sequence analyses showedthat PRODH2 knock-in significantly alteredgene expression of the cell cycle, activation/effector function, and metabolism-relatedprograms in CAR-T cells.ABOUT THE AUTHORPRODH2’s effects on CAR-T cell antitumorefficacy seemed to be driven by metabolicreprogramming related to proline, anamino acid building block. “If we overexpressPRODH2, then proline metabolismwill be reprogrammed,” Ye said. Metabolomicsdata of PRODH2-overexpressing CAR-Tcells revealed increased biochemical activityof the pathway and alterations in other intersectingmetabolic pathways, such as the metabolismof arginine, another amino acid. Infact, the cancer-killing ability was improvedwhen direct substrates of PRODH2 weresupplied to PRODH2-knockin CAR-T cells,but not in control CAR-T that normally lackthe enzyme. This confirmed that the metabolicactivity of PRODH2 was responsiblefor enhanced cytotoxic activity.Hope for the FutureChen’s team established a novel, genome-wideGOF screening technique inprimary CD8+ T cells that can identify desperately-neededfunctional boosters in a robustand unbiased manner. The beauty of thescreen is its versatility. “This doesn’t have tobe T-cell or cancer-specific—ours is a flexibleand broad platform that can be utilizedto perform screens on virtually any othertype of immune cells,” Chen said. “This platformcan be a broadly enabling technologyfor us and everyone else in the world to utilizeGOF screens in various systems, includingstem cells, NK cells, macrophages, andeven other cells relevant to other diseases.”In the future, the authors wish to validate theother targets identified in their screen. Theyhope to ultimately translate their work intoclinical practice by improving the anti-cancerefficacy of CAR-T therapies. ■SHUDIPTO WAHEDSHUDIPTO WAHED is a sophomore in Benjamin Franklin from Pittsburgh, Pennsylvania, interested instudying Molecular Biophysics & Biochemistry. Shudipto conducts research on protein engineering inthe Ring Lab at Yale’s School of Medicine. Outside of YSM, Shudipto is a senator for the Yale CollegeCouncil and an analyst in the Yale Student Investment Group.THE AUTHOR WOULD LIKE TO THANK Associate Professor Sidi Chen and Associate Research ScientistLupeng Ye for their time and enthusiasm about their research.FURTHER READINGYe, L., Park, J. J., Peng, L., Yang, Q., Chow, R. D., Dong, M. B., … & Chen, S. (2022). A genome-scale gainof-functionCRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-Ttherapy. Cell Metabolism, 34(4): 595-614, https://doi.org/10.1016/j.cmet.2022.02.009“Car T Cells: Timeline of Progress.” Memorial Sloan Kettering Cancer Center, 2022, https://www.mskcc.org/timeline/car-t-timeline-progress.18 Yale Scientific Magazine May 2022 www.yalescientific.org
Wave PhysicsFOCUSPUTTINGORDER INDISORDERFocusing deliveryof energy intodiffusive systemsfor applications inneuronal controland tissue imagingBYEUNSOOHYUNwww.yalescientific.orgART BY NOORA SAID“Why is the sky blue?” is perhaps oneof the first, most vexing questions akid can ask their parents. After all,how do you explain to a three-yearoldthat the answer lies in the scattering of light inopaque diffusive systems?In our day-to-day lives, scattering occurswhen particles pass through a medium—suchas air or water, for example—and collide withother particles, resulting in a change in theirtrajectory. “Scattering of light is very common,”said Hui Cao, a Professor of Applied Physics atYale University, focusing on mesoscopic physics,nanophotonics, and biophotonics.May 2022 Yale Scientific Magazine 19
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