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.