The Mechanism Of CD19-Specific Chimeric Antigen Receptor Therapy (CAR-T) Induced Down-Regulation Of CD19 Expression In Target Cells

ObjectiveChimeric antigen receptor therapy(CAR-T)is one of the most promising approaches for the treatment of cancer.While studies in recent year have demonstrated remarkable efficacy of CAR-T in the control of hematopoietic tumors,a substantial proportion of patients undergo reoccurrence of diseases.Previous studies from this laboratory have shown that co-culture with CD19-specific CAR-expressing T cells induce the rapid downregulation of CD19 in tumor cells derived from the B cell lineage,leading to the resistance of tumor cells to CAR-T-mediated cytotoxicity.The present study explored the mechanisms behind CAR-T-induced loss of CD19 from the perspective of protein degradation,transcriptional regulation and alternative splicing.Methods1.Peripheral blood mononuclear cells(PBMCs)were isolated from healthy donors through FICOLL density gradient centrifugation.CD19-specific CAR-expressing T cells were prepared through transfection with lentiviral vectors.Co-cultures were subsequently set up by mixing Raji cells with control activated T cells or CD19 CAR-T cells.At various time points,remaining live Raji cells were harvested and analyzed for surface markers by flow cytometry,mRNA expression by RT-PCR,protein expression by Western blot,and transcriptional profile through the third-generation full length sequencing.2.Raji and primary human B cells were stimulated with CD19-specific antibody FMC68,from which CD19 CAR-T is derived.CD19 expression was then examined by flow cytometry,RT-PCR and Western blot.CD19 degradation was monitored following treatment with cycloheximide to block protein synthesis.3.Differentially expressed genes identified by RNA-seq were subjected to KEGG pathway analysis.RT-qPCR was performed to verify the differential expression of spliceosome-related genes,including SRSF8,SNRPA1,NCBP2 and BUD31.4.Differentially expressed genes with alternative splicing were identified using the Metas cape platform.Functional clustering was carried out through Go analysis.The alternative splicing of genes of interest were verified by RT-PCR using primers designed according to the splicing events.5.pCDH-CMV-MCS-EF1-GFP-T2A-Puro,lentiviral constructs were prepared for overexpression of SRSF8,SNRPA1,NCBP2 and BUD31.Knockdown of their expression was achieved using si RNA.Upon introduction of overexpressing constructs or si RNA into Raji cells,RT-PCR and Western blot were performed to detect alternative splicing and protein expression of potential targets and downstream molecules.Results1.Flow cytometry revealed that,in comparison to uninfected control T cells,a large percentage of cells(64.2 – 91.7%)acquired CD19 CAR expression upon infection with the lentiviral construct,indicative of successful establishment of CAR-T cells.While CD19 expression was maintained in Raji cells co-cultured with control activated T cells,the remaining live Raji cells were virtually negative for CD19 expression in co-cultures with CD19 CAR-T cells.Of note,a substantial fraction of them also lost CD22 expression.2.Similar to co-culture with CD19 CAR-T cells,treatment with FMC68 resulted in the loss of surface expression of CD19 in Raji cells as revealed flow cytometry.Loss of CD19 protein in while cells was confirmed by Western blot.In contrast,RT-qPCR demonstrated that CD19 mRNA was only slightly decreased.It is worth mentioning that downregulation of CD19 protein expression was also observed following treatment with HIB19,an antibody recognizing another epitope on CD19,suggesting that this is a target-specific phenomenon.Subsequently,cycloheximide was added to the culture to block protein synthesis.It was found that FMC68 stimulation facilitated the degradation of CD19,leading to a markedly shortened half-life.Moreover,we examined the impact of FMC68 on CD19 expression in primary human B cells.Similar to Raji cells,primary B cells showed reduced CD19 expression upon antibody stimulation,indicating that this is a universal phenomenon for all CD19~+ cells.3.Transcriptional profiling indicated that,in comparison to untreated or activated T cell-treated Raji cells,cells exposed to CD19 CAR-T cells showed a slight but insignificant decrease in the number of CD19 transcripts.KEGG analysis of the differentially expressed genes revealed the upregulation of multiple genes related spliceosome,such as SRSF8,SNRPA1,NCBP2 and BUD31,which was subsequently confirmed by RT-qPCR.Special attention was paid for alternative slicing events related to CD19.Although there was a general reduction of all splicing isoforms,no significant change was observed in the frequency of any specific isoforms.4.To further interrogate the significance of altered expression of splicing factors,GO analysis was performed for the differentially expressed genes with alternative splicing.They were found to be enriched for mRNA metabolism and histone modification.RT-PCR verified that intron retention occurred in CSKMT,SETD5,BTG2,TINF2 and TARDBP mRNA.Given its key role in epigenetic regulation,subsequent studies were focused on SETD5.Co-culture with CD19CAR-T resulted in a significant increase in total and intron 21-retained SETD5 mRNA as well as SETD5 protein.In line with this,there was an increased level of H3K36me3 modification,which is catalyzed by SETD5,in Raji cells treated with CAR-T.5.To identify the slicing factors responsible for SETD5 mRNA alternative splicing,SRSF8,SNRPA1,NCBP2 and BUD31 were overexpressed in Raji cells using lentiviral vectors.Highest levels of alternative splicing of SETD5 mRNA were detected in the presence of SNPRA1,which was accompanied by an increase in SETD5 protein expression and the downstream H3K36me3 modification.On the contrary,knockdown of SNPR1 expression by si RNA led to a reduction in SETD4 protein and H3K36me3 modification.ConclusionsTo summarize,we have demonstrated that stimulation with either CD19-specific CAR or anti-CD19 antibody induces the downregulation or even loss of CD19,which is mainly attributable to the accelerated degradation of CD19 protein.In addition,we have shown that CD19 CAR-T induces elevated splicing activities in Raji cells.In association with this,SETD5 mRNA alternative spicing,protein levels and the downstream H3K36 modifications are all found to be increased.The present study uncovers a new mechanism for the evasion of B lineage tumor cells from CD19 CAR-T-mediated cytotoxicity.The relevance of alternative splicing of SETD5 in therapeutic resistance warrants further investigation.