TRAIL splice variant TRAILshort disrupts T cell receptor signaling and promotes immune tolerance in vivo
Shahrzad Jalali, Sekar Natesampillai, Zilin Nie, Ying Zhang, Ismail Can, Aswath P. Chandrasekar, Brianna M. Hameister, Cristina Correia, Tuantuan V. Zhao, Dong-Gi Mun, Enrique Garcia-Rivera, Robert Matson, Ashton Krogman, Mark A. Maynes, Robin Batchelor, Dileep D. Monie, Hu Li, Atta Behfar, Saad S. Kenderian, Akhilesh Pandey, Stephen M. Ansell, Timucin Taner, Cornelia Weyand, Daniel D. Billadeau, Andrew D. Badley
Shahrzad Jalali, Sekar Natesampillai, Zilin Nie, Ying Zhang, Ismail Can, Aswath P. Chandrasekar, Brianna M. Hameister, Cristina Correia, Tuantuan V. Zhao, Dong-Gi Mun, Enrique Garcia-Rivera, Robert Matson, Ashton Krogman, Mark A. Maynes, Robin Batchelor, Dileep D. Monie, Hu Li, Atta Behfar, Saad S. Kenderian, Akhilesh Pandey, Stephen M. Ansell, Timucin Taner, Cornelia Weyand, Daniel D. Billadeau, Andrew D. Badley
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Research Article AIDS/HIV Immunology Oncology

TRAIL splice variant TRAILshort disrupts T cell receptor signaling and promotes immune tolerance in vivo

Abstract

TRAIL is a TNF family ligand that trimerizes TRAIL-R1 (DR4) or TRAIL-R2 (DR5) to induce apoptosis, necroptosis, and/or NF-κB activation in receptor-bearing cells. We previously identified TRAILshort as a splice variant of TRAIL that lacks cysteine 230, cannot trimerize, and acts as a dominant-negative ligand that blocks TRAIL-mediated apoptosis. TRAILshort is expressed on cell surfaces and within extracellular vesicles, enabling it to confer TRAIL resistance to both producing and bystander cells. In this study, we showed that elevated TRAILshort levels were associated with chronic viral infections, cancer, and autoimmune diseases, suggesting a link to impaired immune regulation. Using unbiased phosphoproteomics and mechanistic studies, we demonstrated that TRAILshort binding to DR5 recruited and activated the phosphatase Src homology region 2 domain–containing phosphatase 1 (SHP-1), leading to zeta-chain-associated protein kinase 70 (ZAP-70) dephosphorylation, disruption of ZAP-70–CD3ζ interactions, and impaired T cell receptor signaling, thereby reducing T cell activation, proliferation, and cytokine production in response to antigen or CD3/CD28 ligation. Genetic or pharmacologic SHP-1 inhibition reverses these effects. In humanized mouse models, TRAILshort promoted the persistence of transformed mouse embryonic fibroblasts (MEFs) and L428 and antagonized CD19-directed CAR T cell activity, revealing TRAILshort as an immunomodulator of T cell function with therapeutic implications, including blocking TRAILshort to restore T cell immunity or delivering TRAILshort to enforce tolerance.

Authors

Shahrzad Jalali, Sekar Natesampillai, Zilin Nie, Ying Zhang, Ismail Can, Aswath P. Chandrasekar, Brianna M. Hameister, Cristina Correia, Tuantuan V. Zhao, Dong-Gi Mun, Enrique Garcia-Rivera, Robert Matson, Ashton Krogman, Mark A. Maynes, Robin Batchelor, Dileep D. Monie, Hu Li, Atta Behfar, Saad S. Kenderian, Akhilesh Pandey, Stephen M. Ansell, Timucin Taner, Cornelia Weyand, Daniel D. Billadeau, Andrew D. Badley

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Figure 5

TRAILshort treatment induces the recruitment and activation of SHP-1 at the DR5 receptor.

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TRAILshort treatment induces the recruitment and activation of SHP-1 at ...
(A) CD3+ T cell lysates were immunoprecipitated with FAS-Fc, DR5-Fc, or TRAILshort-Fc, resolved by SDS-PAGE, and analyzed by in-gel digestion and proteomics. (B) Proteomics analysis identified DR5, ZAP-70, and SHP-1 as proteins pulled down by TRAILshort-Fc. (C) STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis revealed interactions among DR5, SHP-1, and TCR signaling molecules associated with TRAILshort-Fc. (D) Western blot showing full-length TRAIL and TRAILshort in EVs isolated from 293T cells transfected with TRAIL or TRAILshort constructs. (E) Western blot analysis of T cells from 2 healthy donors treated with purified EVs (D) at a 1:1,000 cell-to-EV ratio and probed for total SHP-1 and p–SHP-1 (Y536). (F) Western blot band quantification demonstrated increased p–SHP-1 expression in cells treated with TRAILshort EV (*P < 0.05, by paired, 2-tailed t test with Holm adjustment). Data are presented as the mean ± SEM. (G) Jurkat cells were treated with no stimulus, BSA, or TRAILshort, followed by immunoprecipitation with DR5 or isotype control antibodies. Immunoblotting for p–SHP-1 and total SHP-1 showed increased DR5-associated p–SHP-1 after TRAILshort treatment. (H) DR5-KO 293T cells were transfected with DR5 alone, DR5 plus Flag-tagged WT SHP-1, or Flag-tagged mutant SHP-1 (D419A). Cells were treated with TRAILshort peptide or BSA, followed by anti-Flag immunoprecipitation. Immunoblotting shows mutant SHP-1 (D419A) associated with DR5 under both conditions, whereas WT SHP-1 interacted with DR5 only after TRAILshort treatment, consistent with TRAILshort-induced SHP-1 recruitment and activation through DR5.