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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 5

Foxp3+ CAR-Tregs degranulate and mediate target cell cytolysis.

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Foxp3+ CAR-Tregs degranulate and mediate target cell cytolysis.
(A) Luci...
(A) Luciferase-based killing assay of CD19+ target cells (Nalm6 CBG-GFP) at indicated T cell–to-target ratios for 16 hours. ****P < 0.0001, by paired t test of Tconvs compared with Tregs (10:1 ratio, n = 3 human donors). (B) Whiskers plot depicting the percentage of CD107a+ of total mCherry+ (CAR) or mCherry– (UT) cells over a 6-hour coculture with live Nalm6 cells measured on nonfixed cells by flow cytometry (n = 3 human donors). (C) After the 6-hour coculture with Nalm6 in the presence of CD107a antibodies, T cells were fixed, permeabilized, and stained for intracellular Foxp3. Representative donor flow plots of CD107a versus Foxp3. Data are representative of n = 3 human donors. (D) GZMB expression of sorted CAR-Tregs after 24-hour Nalm6 stimulation, measured by digital droplet PCR and expressed as a relative ratio to the internal control gene TBP (n = 3 human donors). (E) Luciferase-based killing assays of Nalm6 CBG-GFP with granzyme/perforin inhibitors CMA or Z-AAD-CMK added to the media (1:3 T cell-to-target ratio, 16-hour incubation time). Data are representative of 3 donors with technical triplicates (repeated with n = 3 human donors). Data represent mean ± SEM for A, D, and E. *P < 0.05, by paired t test comparing Tr Δζ and Tr 28ζ (D), and **adj-P < 0.01, by unpaired t test with Holm-Bonferroni method adjustment for 2 tests (E). Tc, Tconvs; Tr, Treg.

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