Inducible and reversible in vivo protein degradation reveals T cell-intrinsic regulatory function of SHP-1
7 affiliations
- 1Department of Pathology & Immunology, Division of Laboratory and Genomic Medicine, Washington University in St. Louis
- 2Department of Microbiology, Immunology, and Cancer Biology, University of Virginia
- 3Beirne B. Carter Center for Immunology Research, University of Virginia
- 4Department of Pathology & Immunology, Division of Immunobiology, Washington University in St. Louis
- 5Bursky Center for Human Immunology and Immunotherapy, Washington University in St. Louis
- 6Unit for Cell Clearance in Health and Disease, VIB Center for Inflammation Research
- 7Department of Biomedical Molecular Biology, Ghent University
Abstract
Scientific problem: Studying the acute, cell-intrinsic functions of signaling proteins in T cells has long been challenging. Existing constitutive or inducible gene deletion cannot overcome developmental adaptation that obscures true phenotypes. Similarly, RNA interference suffers from incomplete knockdown and off-target effects, and pharmacological inhibitors often lack selectivity. Thus, approaches allowing rapid, specific, and reversible protein depletion in defined cell populations in vivo are needed.
Methods: Here, we describe an Inducible and Reversible in vivo Protein Degradation (IRIP) system in T cells by combining auxin-induced degron technology with Cre-driver-mediated, lineage-specific expression of the plant E3 ligase adaptor OsTIR1. We first validated a minimal auxin-inducible degron tag (mAID-tag) inserted into the endogenous Ptpn6 locus encoding the tyrosine phosphatase SHP-1 in murine and human T cell lines. We then generated SHP-1 mAID knock-in mice and crossed them with mice carrying a floxed OsTIR1 construct in the Rosa26 locus, enabling Cre-mediated, T cell-specific, auxin-dependent SHP-1 degradation. We assessed SHP - 1 mAID protein degradation kinetics, specificity, reversibility, tissue restriction, and functional consequences.
Results: The mAID-tagged SHP-1 protein was expressed at wild-type levels and retained full signaling function. Auxin treatment induced complete degradation of SHP-1mAID protein restricted to cells co-expressing OsTIR1, with no loss of untagged SHP-1. Degradation was fully reversible, with SHP-1 protein restored to baseline within 2–3 hours of auxin withdrawal in vitro and 60 hours in vivo . Degradation was confined to the T cell lineage by restricting OsTIR1 expression through a T cell-specific Cre driver. Such targeting could be extended to additional cell populations using a ubiquitous Cre driver. Acute in vivo depletion of SHP-1 caused T cell hyperproliferation upon restimulation, which was reversed upon SHP-1 re-expression.
Conclusion: These findings demonstrate that SHP-1 restrains T cell proliferation in a manner that is direct, cell-intrinsic, acute, and independent of prior developmental conditioning. Our SHP-1 protein targeting approach also resolves a long-standing uncertainty in using the motheaten mouse line or the floxed Ptpn6 locus. Our IRIP system represents a versatile and broadly adaptable platform for dissecting acute physiological roles of proteins whose constitutive loss produces complex and difficult-to-interpret phenotypes.
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BibTeX
@article{khan2026inducible,
title = {{Inducible and reversible in vivo protein degradation reveals T cell-intrinsic regulatory function of SHP-1}},
author = {Tabish H. Khan and Kodi S. Ravichandran and Ulrike M. Lorenz},
journal = {Frontiers in Immunology},
year = {2026},
month = oct,
doi = {10.3389/fimmu.2026.1961691},
url = {https://www.frontiersin.org/articles/10.3389/fimmu.2026.1961691/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Inducible and reversible in vivo protein degradation reveals T cell-intrinsic regulatory function of SHP-1
AU - Tabish H. Khan
AU - Kodi S. Ravichandran
AU - Ulrike M. Lorenz
PY - 2026
DA - 2026/10/21
JO - Frontiers in Immunology
DO - 10.3389/fimmu.2026.1961691
UR - https://www.frontiersin.org/articles/10.3389/fimmu.2026.1961691/full
AB - Scientific problem: Studying the acute, cell-intrinsic functions of signaling proteins in T cells has long been challenging. Existing constitutive or inducible gene deletion cannot overcome developmental adaptation that obscures true phenotypes. Similarly, RNA interference suffers from incomplete knockdown and off-target effects, and pharmacological inhibitors often lack selectivity. Thus, approaches allowing rapid, specific, and reversible protein depletion in defined cell populations in vivo are needed. Methods: Here, we describe an Inducible and Reversible in vivo Protein Degradation (IRIP) system in T cells by combining auxin-induced degron technology with Cre-driver-mediated, lineage-specific expression of the plant E3 ligase adaptor OsTIR1. We first validated a minimal auxin-inducible degron tag (mAID-tag) inserted into the endogenous Ptpn6 locus encoding the tyrosine phosphatase SHP-1 in murine and human T cell lines. We then generated SHP-1 mAID knock-in mice and crossed them with mice carrying a floxed OsTIR1 construct in the Rosa26 locus, enabling Cre-mediated, T cell-specific, auxin-dependent SHP-1 degradation. We assessed SHP - 1 mAID protein degradation kinetics, specificity, reversibility, tissue restriction, and functional consequences. Results: The mAID-tagged SHP-1 protein was expressed at wild-type levels and retained full signaling function. Auxin treatment induced complete degradation of SHP-1mAID protein restricted to cells co-expressing OsTIR1, with no loss of untagged SHP-1. Degradation was fully reversible, with SHP-1 protein restored to baseline within 2–3 hours of auxin withdrawal in vitro and 60 hours in vivo . Degradation was confined to the T cell lineage by restricting OsTIR1 expression through a T cell-specific Cre driver. Such targeting could be extended to additional cell populations using a ubiquitous Cre driver. Acute in vivo depletion of SHP-1 caused T cell hyperproliferation upon restimulation, which was reversed upon SHP-1 re-expression. Conclusion: These findings demonstrate that SHP-1 restrains T cell proliferation in a manner that is direct, cell-intrinsic, acute, and independent of prior developmental conditioning. Our SHP-1 protein targeting approach also resolves a long-standing uncertainty in using the motheaten mouse line or the floxed Ptpn6 locus. Our IRIP system represents a versatile and broadly adaptable platform for dissecting acute physiological roles of proteins whose constitutive loss produces complex and difficult-to-interpret phenotypes.
SN - 1664-3224
ER -