ITPK1 sensitizes tumor cells to IgA-dependent neutrophil killing in vivo [CRISPR genome-wide]. ITPK1 sensitizes tumor cells to IgA-dependent neutrophil killing in vivo [CRISPR genome-wide]

Neutrophils can efficiently trigger cytotoxicity towards tumor cells and other target cells upon engagement of the IgA receptor CD89. However, the cell-intrinsic factors that influence the induction of cell death upon exposure to neutrophil effector mechanisms in vivo remain largely unknown. To uncover genetic regulators that influence target cell sensitivity to IgA-induced neutrophil-mediated killing, we used a human CD89 (hCD89) transgenic mouse model in which IgA-mediated killing of Her2-positive CD47-deficient murine target cells is mediated by neutrophils. Using a genome-wide in vivo screening approach, we demonstrate that deletion of the gene encoding inositol-tetrakisphosphate 1 kinase, ITPK1, increases survival of target cells in anti-Her2 IgA-treated mice. Moreover, we show that this effect depends on neutrophil activity and on the ITPK1 kinase domain. Notably, ITPK1 deficiency did not measurably impact survival of IgA-opsonized target cells in in vitro systems, underscoring the importance of in vivo screening systems to uncover physiologically relevant regulators of neutrophil killing. Overall design: For the genome-wide screen, sensBa/F3 cells were lentivirally transduced with the pooled Brie CRISPR KO library (Addgene #73632/3) in the lenti-Guide-Puro (Addgene #52963) vector at a transduction rate of approximately 50% and a coverage of >1800 fold. Following transduction, the cells were cultured for >7 days in the presence of 5ug/ml puromycin. Before injection into mice, a library reference sample was taken. Next, hCD89-transgenic mice and littermate controls were injected intraperitoneally (i.p.) with 2x10^7 cells in 200uL PBS. Directly after target cell injection, mice received PBS (control treatment) or 100ug anti-hHer2 IgA1 antibody in PBS by intraperitoneal injection. 16h after injection, mice were euthanized and the peritoneal cavity was washed with PBS containing 5 mM EDTA to obtain target cells and murine immune cell infiltrates. Next, cells were stained with APC-conjugated anti-hHer2 and anti-hCD19 antibodies and target cells were positively selected for by MACS using anti-APC magnetic beads (MiltenyiBiotec #130-090-855), according to the manufacturer’s protocol. Thereafter, DNA was isolated from enriched target cells using the DNeasy Blood & Tissue Kit (QIAGEN #69504). In the genome-wide screen 3-4 samples within each group were pooled based on recovered DNA amounts, yielding 3 anti-hHer2-treated samples and 3 PBS-treated genomic DNA samples. SgRNA sequences in the pooled or unpooled sample groups were amplified using NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs #M0541S), following the manufacturer’s instructions and using primers containing sample barcodes. After PCR, prepared libraries were pooled at equimolar amounts and sgRNA distributions were analyzed by deep sequencing using the Illumina HiSeq2500 platform. In brief, sequence reads were aligned to the Brie mouse library, reads containing mismatches were removed, sgRNA distributions were quantified, and genes were scored using MAGeCK RRA (version 0.5.6)

嗜中性粒细胞（Neutrophils）在结合IgA受体CD89（IgA receptor CD89）后，可有效触发对肿瘤细胞及其他靶细胞的细胞毒性作用。然而，在体内暴露于嗜中性粒细胞效应机制时，影响细胞死亡诱导的细胞内在因素仍尚未明确。为揭示影响靶细胞对IgA诱导的嗜中性粒细胞介导杀伤作用敏感性的遗传调控因子，我们采用了人源CD89（hCD89）转基因小鼠模型，该模型中IgA介导的Her2阳性CD47缺陷型小鼠靶细胞杀伤作用由嗜中性粒细胞介导。
采用全基因组体内筛选方法，我们证实编码肌醇四磷酸1激酶（inositol-tetrakisphosphate 1 kinase, ITPK1）的基因缺失，可提高抗Her2 IgA处理小鼠体内靶细胞的存活率。此外，我们证实该效应依赖于嗜中性粒细胞活性及ITPK1的激酶结构域。值得注意的是，ITPK1缺失在体外系统中并未对IgA调理的靶细胞存活率产生可检测的影响，这凸显了体内筛选系统在发现嗜中性粒细胞杀伤相关生理调控因子方面的重要性。
实验整体设计：针对全基因组筛选，我们将sensBa/F3细胞以约50%的转导效率、>1800倍的覆盖度，在lenti-Guide-Puro（Addgene #52963）载体中与混合Brie CRISPR敲除文库（Addgene #73632/3）进行慢病毒转导。转导完成后，将细胞在含有5μg/ml嘌呤霉素的培养基中培养>7天。在注射至小鼠体内前，采集文库参照样本。随后，将hCD89转基因小鼠及同窝对照小鼠经腹腔（intraperitoneal, i.p.）注射2×10^7个细胞，注射体积为200μL磷酸盐缓冲液（PBS）。靶细胞注射后即刻，小鼠经腹腔注射PBS（对照处理）或100μg抗hHer2 IgA1抗体的PBS溶液。注射后16小时，处死小鼠，用含有5mM EDTA的PBS冲洗腹腔，以收集靶细胞及小鼠免疫细胞浸润物。随后，使用APC标记的抗hHer2及抗hCD19抗体对细胞进行染色，并按照制造商的方案，使用抗APC磁珠（MiltenyiBiotec #130-090-855）通过磁珠激活细胞分选（Magnetic Activated Cell Sorting, MACS）技术阳性分选靶细胞。之后，使用DNeasy血液与组织试剂盒（QIAGEN #69504）从富集的靶细胞中提取DNA。在全基因组筛选中，根据回收的DNA量将每组内的3-4个样本合并，最终获得3个抗hHer2处理样本及3个PBS处理的基因组DNA（deoxyribonucleic acid, DNA）样本。使用NEBNext高保真2X聚合酶链式反应（Polymerase Chain Reaction, PCR）预混液（New England Biolabs #M0541S），按照制造商的说明书，使用带有样本条码的引物对合并或未合并的样本组中的单引导RNA（Single Guide RNA, sgRNA）序列进行扩增。PCR完成后，将制备好的文库以等摩尔浓度混合，并使用Illumina HiSeq2500测序平台进行深度测序，以分析sgRNA的分布情况。简言之，将测序读段比对至Brie小鼠文库，移除带有错配的读段，定量sgRNA的分布，并使用MAGeCK RRA（版本0.5.6）对基因进行评分。