Our understanding of programmed cell death 1 (PD‐1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD‐1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD‐1 ligation on short‐term T cell signaling, long‐term T cell function, and the structural consequences of PD‐1 ligation with PD‐1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short‐term assay to examine the signaling cascade triggered by PD‐1. We describe a phospho flow cytometry method to determine how PD‐1 ligation alters the level of CD3ζ phosphorylation on Tyr142, which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate‐bound assay that is useful to examine the long‐term consequences of PD‐1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen‐based assay to evaluate T cell responses to therapeutic agents targeting the PD‐1/PD‐L axis, as well as immune synapse formation in the presence of PD‐1 engagement. Finally, in Basic Protocol 4 we outline a tetramer‐based method useful to interrogate the quality of PD‐1/PD‐L interactions. These protocols can be easily adapted for mouse studies and other inhibitory receptors. They provide a valuable resource to investigate PD‐1 signaling in T cells and the functional consequences of various PD‐1‐based therapeutics on T cell responses. © 2020 Wiley Periodicals LLC.
Regulatory T cells (Tregs) play an important role in controlling autoimmunity and limiting tissue damage and inflammation. IL2‐inducible T cell kinase (Itk) is part of the Tec family of tyrosine kinases and is a critical component of T cell receptor mediated signaling. Here, we showed that either genetic ablation of Itk signaling or inhibition of Itk signaling pathways resulted in increased frequency of “noncanonical” CD4+CD25−FOXP3+Tregs (ncTregs), as well as of “canonical” CD4+CD25+FOXP3+Tregs (canTregs). Using in vivo models, we showed that ncTregs can avert the formation of acute graft‐versus‐host disease (GVHD), in part by reducing conventional T cell proliferation, proinflammatory cytokine production, and tissue damage. This reduction in GVHD occurred without disruption of graft‐versus‐leukaemia (GVL) effects. RNA sequencing revealed that a number of effector, cell adhesion, and migration molecules were upregulated in
- NSF-PAR ID:
- 10361135
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Clinical and Translational Medicine
- Volume:
- 11
- Issue:
- 12
- ISSN:
- 2001-1326
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Basic Protocol 1 : PD‐1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cellsBasic Protocol 2 : Plate‐based ligand binding assay to study PD‐1 function in human T cellsSupport Protocol 1 : T cell proliferation assay in the presence of PD‐1 ligationBasic Protocol 3 : In vitro APC/T cell co‐culture system to evaluate therapeutic interventions targeting the PD‐1/PD‐L1 axisSupport Protocol 2 : Microscopy‐based approach to evaluate the consequences of PD‐1 ligation on immune synapse formationBasic Protocol 4 : Tetramer‐based approach to study PD‐1/PD‐L1 interactions -
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Abstract Herein, this work reports the first synthetic vaccine adjuvants that attenuate potency in response to small, 1–2 °C changes in temperature about their lower critical solution temperature (LCST). Adjuvant additives significantly increase vaccine efficacy. However, adjuvants also cause inflammatory side effects, such as pyrexia, which currently limits their use. To address this, a thermophobic vaccine adjuvant engineered to attenuate potency at temperatures correlating to pyrexia is created. Thermophobic adjuvants are synthesized by combining a rationally designed trehalose glycolipid vaccine adjuvant with thermoresponsive poly‐
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Abstract Background The
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