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Updated: Jul 11, 2025
Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies
Published on: February 15, 2021
RIPK1 deficiency prevents thymic NK1.1 expression and subsequent iNKT cell development
Thomas Hägglöf1,2, Raksha Parthasarathy1, Nathaniel Liendo1,3
1Department of Microbiology, Immunology & Molecular Genetics, University of Texas Health at San Antonio, San Antonio, TX, United States.
Abstract:
Receptor Interacting Protein Kinase 1 (RIPK1) and caspase-8 (Casp8) jointly orchestrate apoptosis, a key mechanism for eliminating developing T cells which have autoreactive or improperly arranged T cell receptors. Mutations in the scaffolding domain of Ripk1 gene have been identified in humans with autoinflammatory diseases like Cleavage Resistant RIPK1 Induced Autoinflammatory (CRIA) and Inflammatory Bowel Disease. RIPK1 protein also contributes to conventional T cell differentiation and peripheral T cell homeostasis through its scaffolding domain in a cell death independent context. Ripk1 deficient mice do not survive beyond birth, so we have studied the function of this kinase in vivo against a backdrop Ripk3 and Casp8 deficiency which allows the mice to survive to adulthood. These studies reveal a key role for RIPK1 in mediating NK1.1 expression, including on thymic iNKT cells, which is a key requirement for thymic stage 2 to stage 3 transition as well as iNKT cell precursor development. These results are consistent with RIPK1 mediating responses to TcR engagement, which influence NK1.1 expression and iNKT cell thymic development. We also used in vivo and in vitro stimulation assays to confirm a role for both Casp8 and RIPK1 in mediating iNKT cytokine effector responses. Finally, we also noted expanded and hyperactivated iNKT follicular helper (iNKTFH) cells in both DKO (Casp8-, Ripk3- deficient) and TKO mice (Ripk1-, Casp8-, Ripk3- deficient). Thus, while RIPK1 and Casp8 jointly facilitate iNKT effector function, RIPK1 uniquely influenced thymic iNKT cell development most likely by regulating molecular responses to T cell receptor engagement. iNKT developmental and functional aberrances were not evident in mice expressing a kinase-dead version of RIPK1 (RIPK1kd), indicating that the scaffolding function of this protein exerts the critical regulation of iNKT cells. Our findings suggest that small molecule inhibitors of RIPK1 could be used to regulate iNKT cell development and effector function to alleviate autoinflammatory conditions in humans.
Insights
Receptor Interacting Protein Kinase 1 (RIPK1) and caspase-8 (Casp8) are crucial for T cell development and function. RIPK1
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Receptor Interacting Protein Kinase 1 (RIPK1) and caspase-8 (Casp8) are key regulators of apoptosis and T cell development.
- Mutations in RIPK1 are linked to human autoinflammatory diseases, including Cleavage Resistant RIPK1 Induced Autoinflammatory (CRIA) and Inflammatory Bowel Disease.
- RIPK1 also plays a cell-death-independent role in T cell differentiation and homeostasis.
Purpose of the Study:
- To investigate the in vivo function of RIPK1 in T cell development and homeostasis, particularly in the context of RIPK3 and Caspase-8 deficiency.
- To elucidate the role of RIPK1 in iNKT cell development, NK1.1 expression, and cytokine effector responses.
- To determine whether RIPK1's kinase or scaffolding function is critical for iNKT cell regulation.
Main Methods:
- Utilized Ripk3 and Casp8 deficient mice to enable survival of Ripk1 deficient mice to adulthood for in vivo studies.
- Conducted in vivo and in vitro stimulation assays to assess iNKT cell development, NK1.1 expression, and cytokine production.
- Compared iNKT cell phenotypes in wild-type, DKO (Casp8-, Ripk3- deficient), and TKO (Ripk1-, Casp8-, Ripk3- deficient) mice, as well as mice expressing kinase-dead RIPK1 (RIPK1kd).
Main Results:
- RIPK1 is essential for NK1.1 expression on thymic iNKT cells, a critical step for their development and transition from stage 2 to stage 3.
- Both RIPK1 and Casp8 mediate iNKT cell cytokine effector responses.
- Expanded and hyperactivated iNKT follicular helper (iNKTFH) cells were observed in DKO and TKO mice, indicating a role for RIPK1 and Casp8 in regulating iNKTFH cell homeostasis.
- Kinase-dead RIPK1 (RIPK1kd) did not show developmental or functional iNKT cell aberrations, highlighting the importance of RIPK1's scaffolding function.
Conclusions:
- RIPK1 uniquely regulates thymic iNKT cell development, likely through its scaffolding function in response to T cell receptor engagement.
- RIPK1 and Casp8 jointly regulate iNKT cell effector functions.
- Targeting RIPK1 with small molecule inhibitors may offer a therapeutic strategy for autoinflammatory conditions by modulating iNKT cell development and function.
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