Mis-splicing derived neoantigens and cognate T cell receptors in splicing factor mutant leukemias

Mutations in RNA splicing factors are prevalent across cancers and generate recurrently mis-spliced mRNA isoforms. Here we identified a series of bona fide neoantigens translated from highly stereotyped splicing alterations promoted by neomorphic, leukemia-associated somatic mutations in the splicing machinery. We utilized feature-barcoded peptide-MHC dextramers to isolate neoantigen-specific T cell receptors (TCR) from both healthy donors and patients with leukemia. While circulating neoantigen-specific CD8+ T cells were identified in patients with active disease, they were dysfunctional with reduced inflammatory response gene signatures. In contrast, donor CD8+ T cells with tumor-reactive TCRs were present following curative allogeneic hematopoietic cell transplant. T cells engineered with TCRs recognizing an SRSF2 mutant-induced neoantigen in CLK3 resulted in specific recognition and cytotoxicity of SRSF2 mutant leukemia. These data identify RNA mis-splicing derived neoantigens and neoantigen-specific TCRs across patients and provide proof-of-concept to genetically redirect T cells to public mis-splicing derived neoantigens in myeloid leukemias. We isolated bulk T cells from patient PBMCs via negative selection and stained the T cells with a dextramer pool. We sorted live, CD3+, CD8+ dextramer+ cells and performed RNA-, TCR-, and dextramer feature barcode sequencing (Fig. S5A). For certain samples where dextramer+ cell numbers were limiting, live CD3+ CD8+ dextramer- cells were also sorted, stained with cell hashing antibodies, and doped into the dextramer+ populations. Using this approach, we single-cell profiled CD8+ T-cells from nine samples across five distinct HLA-A*02:01+ SRSF2 mutant myeloid leukemia patients (Fig. 5A, Fig.S5B, and Table S3) yielding 75,343 high-quality T cells.

RNA剪接因子（RNA splicing factors）突变在各类癌症中广泛存在，并可产生反复出现的错误剪接mRNA异构体。本研究鉴定了一系列由剪接机器中白血病相关的新形态体细胞突变驱动的高度定型剪接改变所翻译产生的真实新抗原（neoantigen）。我们利用特征条形码标记的肽-MHC多聚体（dextramer），从健康供者与白血病患者体内分离识别新抗原的T细胞受体（TCR）。尽管在活动性疾病患者体内可检测到循环中的新抗原特异性CD8+ T细胞，但这类细胞存在功能失调，且炎症应答相关基因特征表达下调。相比之下，接受根治性异基因造血细胞移植后的患者体内，可检测到携带肿瘤反应性TCR的供者来源CD8+ T细胞。经工程化改造、表达识别CLK3基因中SRSF2突变诱导的新抗原的TCR的T细胞，可特异性识别并杀伤携带SRSF2突变的白血病细胞。本研究证实了RNA错误剪接来源的新抗原及新抗原特异性TCR在患者群体中的存在，并为通过基因重定向T细胞靶向髓系白血病中公共的错误剪接来源新抗原提供了概念验证依据。我们通过阴性分选从患者外周血单个核细胞（PBMC）中分离总T细胞，并用多聚体混合试剂对T细胞进行染色。随后分选活的CD3+、CD8+且多聚体阳性的细胞，进行转录组测序、TCR测序及多聚体特征条形码测序（补充图S5A）。对于部分多聚体阳性细胞数量有限的样本，我们同时分选活的CD3+、CD8+且多聚体阴性的细胞，用细胞哈希抗体进行染色，并将其掺入多聚体阳性细胞群中。通过该策略，我们对来自5名不同的HLA-A*02:01阳性且携带SRSF2突变的髓系白血病患者的9份样本中的CD8+ T细胞开展了单细胞测序分析（图5A、补充图S5B及表S3），最终获得75343个高质量T细胞。