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Reversing T Cell Exhaustion by Converting Membrane PD-1 to Its Soluble form in Jurkat Cells; Applying The CRISPR/Cas9
Zeinab Yousefi-Najafabadi1,2, Zohreh Mehmandoostli2, Yazdan Asgari1
1Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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.
Insights
Engineered T-cells can reduce membrane PD-1 (mPD-1) and increase soluble PD-1 (sPD-1) via CRISPR/Cas9 exon skipping. This enhances lymphocyte survival and proliferation in the tumor microenvironment, offering a new immunotherapy approach.
Area of Science:
- Immunology and Cancer Research
- Molecular Biology and Gene Editing
Background:
- T-cells express membrane PD-1 (mPD-1) and soluble PD-1 (sPD-1), crucial for immune regulation.
- mPD-1/PD-L1 interaction can lead to T-cell exhaustion within the tumor microenvironment (TME).
- sPD-1 can potentially disrupt mPD-1/PD-L1 binding, mitigating T-cell exhaustion.
Purpose of the Study:
- To investigate the effect of secreted sPD-1 from engineered T-cells on lymphocyte survival and proliferation in the TME.
- To assess the efficacy of CRISPR/Cas9-mediated exon skipping to alter PD-1 expression patterns.
Main Methods:
- Designed sgRNAs targeting exon-3 of the PDCD1 gene for CRISPR/Cas9 editing.
- Generated lentiviral particles to transduce Jurkat T cells with dual sgRNAs.
- Co-cultured engineered T-cells with PD-L1+ cancer cells and analyzed lymphocyte responses.
Main Results:
- CRISPR/Cas9 successfully deleted exon-3, reducing mPD-1 positive cells by 12% and increasing sPD-1 concentration.
- Increased sPD-1 levels positively impacted lymphocyte proliferation when co-cultured with PD-L1+ cancer cells.
- Lymphocyte survival increased by 12.5% when cultured with supernatant from engineered T-cells compared to controls.
Conclusions:
- CRISPR/Cas9 exon skipping is a viable strategy to modify PD-1 expression.
- This approach can enhance sPD-1 production, potentially overcoming T-cell exhaustion in adoptive cell immunotherapies.
- The study highlights a novel method to improve anti-tumor immune responses.
