CD19和CD22编码外显子的异常剪接损害了对B细胞恶性肿瘤的免疫治疗反应。在这里，我们展示了编码人CD20的MS4A1基因也产生了几个具有不同5'非翻译区（5'-UTR）的mRNA亚型。通过RNA-seq，在正常B细胞分化和B淋巴恶性肿瘤的不同阶段，检测到了四个变体（V1-4），其中V1和V3远远是数量最多的。在B细胞激活和EB病毒感染过程中，剪接重定向从V1到V3，与CD20阳性的增加相一致。同样，在弥漫性大B细胞淋巴瘤中只有V3而不是V1与CD20蛋白水平相关，暗示V1可能缺乏翻译能力。事实上，较长的V1亚型被发现包含上游开放阅读框（uORFs）和一个螺状结构，二者协同抑制了多聚体援用。通过调控具有偏好剪接的morpholino寡核苷酸改变CD20亚型，我们增强了一系列B细胞系中的CD20表达和抗CD20抗体利妥昔单抗引起的细胞毒性。此外，用V3 mRNA重构CD20敲除细胞导致CD20阳性的恢复，而用V1重构细胞中CD20蛋白的水平无法检测。令人惊讶的是，体外CD20导向CAR T细胞能够杀死V3和V1表达的细胞，但双特异性T细胞结合剂莫舒奈单抗只对V3表达的细胞有效。为了确定CD20剪接是否涉及免疫治疗抵抗，我们对四个莫舒奈单抗后复发的滤泡性淋巴瘤进行了RNA-seq分析，并发现其中两个中CD20的下调伴随着V3向V1的转变。因此，剪接介导的表位丧失机制扩展到了CD20导向的免疫治疗。《2023年美国血液学会版权所有。

Aberrant skipping of coding exons in CD19 and CD22 compromises responses to immunotherapy for B-cell malignancies. Here, we show that the MS4A1 gene encoding human CD20 also produces several mRNA isoforms with distinct 5' untranslated regions (5'-UTR). Four variants (V1-4) were detectable by RNA-seq in distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant by far. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform was found to contain upstream open reading frames (uORFs) and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching Morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, while V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed CAR T cells were able to kill both V3- and V1-expressing cells, but the bispecific T cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on four post-mosunetuzumab follicular lymphoma relapses and discovered that in two of them downregulation of CD20 was accompanied by the V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.Copyright © 2023 American Society of Hematology.