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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 2

Foxp3 expression is stable after transduction, bead expansion, and restimulation.

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Foxp3 expression is stable after transduction, bead expansion, and resti...
(A) Intracellular staining of Foxp3 and CD25 as a percentage of total CD3+CD4+mCherry+ after sorting (day 0), bead expansion, and rest (day 14) and on day 23, 9 days after the addition of irradiated anti-CD3 K562 (TCR stim) or CD19-K562 (CAR stim) (n = 6 human donors). Methylation status using direct bisulfite modification and pyrosequencing of (B) the TSDR (n = 2 human female donors) and (C) the CTLA4 promotor (n = 3 human donors) on day 0 after sort, day 14, and day 23 with CAR stimulation. Surface expression of (D) CTLA4, (E) LAP, and (F) CD39, 9 days after TCR or CAR stimulation with irradiated K562 cells (n ≥ 3 human donors). All data are represented as box-and-whisker plots. *Adj-P < 0.05 by paired t test for CAR stimulation versus TCR stimulation (D) and between Tr 28ζ and Tr BBζ (E) with Holm-Bonferroni method adjustment for 3 and 2 tests, respectively. Blue bars for D0 represent UT Tregs and UT Tconvs immediately after sort. Tc, Tconvs; Tr, Treg.

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