Reversing T Cell Exhaustion by Converting Membrane PD-1 to Its Soluble Form in Jurkat Cells; Applying the CRISPR/Cas9 Exon Skipping Strategy

Objective: T-cells express two functional forms of the programmed cell death protein 1 (PD-1): membrane (mPD-1) andsoluble (sPD-1). The binding of mPD-1 and its ligand (PD-L1) on tumor cells could lead activated lymphocytes towardexhaustion. Selective deletion of the transmembrane domain via alternative splicing of exon-3 in PD-1 mRNA couldgenerate sPD-1. Overexpression of sPD-1 could disrupt the mPD-1/PD-L1 interaction in tumor-specific T cells. Weinvestigated the effect of secreted sPD-1 from pooled engineered and non-engineered T cell supernatant on survivaland proliferation of lymphocytes in the tumor microenvironment (TME).Materials and Methods: In this experimental study, we designed two sgRNA sequences upstream and downstream ofexon-3 in the PDCD1 gene. The lentiCRISPRv2 puro vector was used to clone the dual sgRNAs and produce lentiviralparticles to transduce Jurkat T cells. Analysis assays were used to clarify the change in PD-1 expression pattern in thepooled (engineered and non-engineered) Jurkat cells. Co-culture conditions were established with PD-L1+ cancer cellsand lymphocytes.Results: CRISPR/Cas9 could delete exon-3 of the PDCD1 gene in the engineered cells based on the tracking of indelsby decomposition (TIDE) and interference of CRISPR edit (ICE) sequencing analysis reports. Our results showed a12% reduction in mPD-1 positive cell population after CRISPR manipulation and increment in sPD-1 concentration inthe supernatant. The increased sPD-1 confirmed its positive effect on proliferation of lymphocytes co-cultured with PDL1+cancer cells. The survival percent of lymphocytes co-cultured with the pooled cells supernatant was 12.5% morethan the control.Conclusion: The CRISPR/Cas9 exon skipping approach could be used in adoptive cell immunotherapies to changePD-1 expression patterns and overcome exhaustion.