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

Document Type : Original Article

Authors

1 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

2 Immunology, Asthma and Allergy Research Institute (IAARI), Tehran University of Medical Sciences, Tehran, Iran

3 Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran

4 Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

Abstract

Objective: T-cells express two functional forms of the programmed cell death protein 1 (PD-1): membrane (mPD-1) and
soluble (sPD-1). The binding of mPD-1 and its ligand (PD-L1) on tumor cells could lead activated lymphocytes toward
exhaustion. Selective deletion of the transmembrane domain via alternative splicing of exon-3 in PD-1 mRNA could
generate sPD-1. Overexpression of sPD-1 could disrupt the mPD-1/PD-L1 interaction in tumor-specific T cells. We
investigated the effect of secreted sPD-1 from pooled engineered and non-engineered T cell supernatant on survival
and proliferation of lymphocytes in the tumor microenvironment (TME).
Materials and Methods: In this experimental study, we designed two sgRNA sequences upstream and downstream of
exon-3 in the PDCD1 gene. The lentiCRISPRv2 puro vector was used to clone the dual sgRNAs and produce lentiviral
particles to transduce Jurkat T cells. Analysis assays were used to clarify the change in PD-1 expression pattern in the
pooled (engineered and non-engineered) Jurkat cells. Co-culture conditions were established with PD-L1+ cancer cells
and lymphocytes.
Results: CRISPR/Cas9 could delete exon-3 of the PDCD1 gene in the engineered cells based on the tracking of indels
by decomposition (TIDE) and interference of CRISPR edit (ICE) sequencing analysis reports. Our results showed a
12% reduction in mPD-1 positive cell population after CRISPR manipulation and increment in sPD-1 concentration in
the 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% more
than the control.
Conclusion: The CRISPR/Cas9 exon skipping approach could be used in adoptive cell immunotherapies to change
PD-1 expression patterns and overcome exhaustion.

Keywords

Main Subjects

References

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Cell Journal (Yakhteh)
Volume 25, Issue 9 - Serial Number 116
September 2023
Pages 633-644

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