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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 3

CAR-Tregs can be activated through their CAR or their TCR.

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CAR-Tregs can be activated through their CAR or their TCR.
T cells were ...
T cells were left unstimulated or stimulated with irradiated CD19-K562 (CAR stim) or anti-CD3-K562 (TCR stim) at a 1:1 ratio for 20 hours on day 14 followed by surface staining of (A) CD69 and (B) latent-associated peptide (LAP), measured as a percentage, after gating live mCherry+CD4+ T cells, except in the case of the UT groups, which were only gated on live CD4+ T cells (n = 3 human donors). Supernatants were saved 20 hours after T cell/K562 coculture to measure (C) IL-2, (D) TNF-α, and (E) IFN-γ in parallel. Cytokine levels were normalized to Tconv UT stimulated with K562-OKT3 (baseline quantification, IL-2, 7.03 ng/ml; TNF-α, 1.69 ng/ml; IFN-γ, 2.58 ng/ml; n = 3 human donors). (F) IL-10 detected in the supernatants of T cell and CD19-K562 cocultures (n = 5 human donors, baseline quantification of Tc UT stimulated with K562-OKT3 was 380 pg/ml). No cytokines were detected in the wells with K562 cells alone or with T cells alone. Live T cell number counted over time, expressed as the log2 fold change of the starting cell number before K562 stimulation (day 14) after (G) CD19 or (H) anti-CD3 stimulation (n = 3 human donors). *adj-P < 0.05, **adj-P < 0.01, ***adj-P < 0.001, by paired t test with Holm-Bonferroni method adjustment for 3 tests (D and E). Significance was determined between functional CAR T cells in F by 1-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, by paired t test for G and H, with significance calculated at day 8 between Tr Δζ and Tr 28ζ. Data represents box-and-whisker plots (A–E) or mean ± SEM (F and G). Tc, Tconvs; Tr, Treg. Dots within bars represent individual data points (F).

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