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Chimeric antigen receptor costimulation domains modulate human regulatory T cell function
Angela C. Boroughs, … , Shadmehr Demehri, Marcela V. Maus
Angela C. Boroughs, … , Shadmehr Demehri, Marcela V. Maus
Published March 14, 2019
Citation Information: JCI Insight. 2019;4(8):e126194. https://doi.org/10.1172/jci.insight.126194.
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Research Article Immunology

Chimeric antigen receptor costimulation domains modulate human regulatory T cell function

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Abstract

Tregs are key modulators of inflammation and are important for the maintenance of peripheral tolerance. Adoptive immunotherapy with polyclonal Tregs holds promise in organ transplantation, graft-versus-host disease, and autoimmune diseases but may be enhanced by antigen-specific, long-lived Tregs. We modified primary human Tregs with chimeric antigen receptors (CARs) bearing different costimulatory domains and performed in vitro analyses of their phenotype and function. While neither the presence of a CAR nor the type of costimulation domain influenced Foxp3 expression in Tregs, the costimulation domain of the CARs affected CAR-Treg surface phenotype and functions, such as cytokine production. Furthermore, signaling from the CD28 costimulation domain maintained CAR-Treg suppressor function, whereas 4-1B costimulation did not. In vivo, CAR-Tregs accumulated at sites expressing target antigen and suppressed antigen-specific effector T cell responses; however, only CAR-Tregs with CD28 signaling domains were potent inhibitors of effector T cell–mediated graft rejection in vivo. Our findings support the use of CD28-based CAR-Tregs for tissue-specific immune suppression in the clinic.

Authors

Angela C. Boroughs, Rebecca C. Larson, Bryan D. Choi, Amanda A. Bouffard, Lauren S. Riley, Erik Schiferle, Anupriya S. Kulkarni, Curtis L. Cetrulo, David Ting, Bruce R. Blazar, Shadmehr Demehri, Marcela V. Maus

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Figure 4

4-1BB costimulation decreases CAR-Treg suppressive function.

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4-1BB costimulation decreases CAR-Treg suppressive function.
(A) MLRs of...
(A) MLRs of CFSE-labeled CD19 ζ CAR-Teffs cultured with indicated ratios of violet-labeled CD19 CAR-Tregs. After 3 days, CFSE dilution of the mCherry+ Teff population was measured by flow cytometry to calculate suppression as a percentage of [1 – (number of CFSElo cells in the Treg condition)/(the number of mCherry+ Teffs proliferating with no Tregs present]; (n = 5 human donors)]. Data represent mean ± SEM. *P < 0.05, paired t test performed for 1:1 Treg-to-Teff ratio comparing only BBζ and 28ζ. Supernatants were also collected in the same MLR, except with 28ζ CAR-Teffs as the CFSE-labeled cells. After 24 hours, cytokine levels of (B) TNF-α, (C) GM-CSF, (D) IL-2, and (E) IFN-γ were measured. MLRs comparing Treg suppression of Teff proliferation after activation though (F) the CAR (CFSE-labeled CD19 ζ CAR-Teffs, irradiated Nalm6 targets, 1:2 Teff-to-target cell ratio) or (G) the TCR (CFSE-labeled naive T cells, anti-CD3/anti-CD28 beads, 10:1 cell-to-bead ratio). After 4 days, CFSE dilution was measured). (B–G) Data are representative of 3 human donors, with technical triplicates. Mean and SEM of the triplicates plotted. Tr, Tregs.

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