Tregs integrate native and CAR-mediated costimulatory signals for control of allograft rejection
Isaac Rosado-Sánchez, … , Giorgio Raimondi, Megan K. Levings
Isaac Rosado-Sánchez, … , Giorgio Raimondi, Megan K. Levings
Published September 5, 2023
Citation Information: JCI Insight. 2023;8(19):e167215. https://doi.org/10.1172/jci.insight.167215.
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Research Article Immunology Transplantation

Tregs integrate native and CAR-mediated costimulatory signals for control of allograft rejection

Abstract

Tregs expressing chimeric antigen receptors (CAR-Tregs) are a promising tool to promote transplant tolerance. The relationship between CAR structure and Treg function was studied in xenogeneic, immunodeficient mice, revealing advantages of CD28-encoding CARs. However, these models could underrepresent interactions between CAR-Tregs, antigen-presenting cells (APCs), and donor-specific Abs. We generated Tregs expressing HLA-A2–specific CARs with different costimulatory domains and compared their function in vitro and in vivo using an immunocompetent model of transplantation. In vitro, the CD28-encoding CAR had superior antigen-specific suppression, proliferation, and cytokine production. In contrast, in vivo, Tregs expressing CARs encoding CD28, ICOS, programmed cell death 1, and GITR, but not 4-1BB or OX40, all extended skin allograft survival. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3ζ but no costimulatory domain. These data revealed that exogenous costimulation from APCs can compensate for the lack of a CAR-encoded CD28 domain. Thus, Tregs expressing a CAR with or without CD28 are functionally equivalent in vivo, mediating similar extension of skin allograft survival and controlling the generation of anti–HLA-A2 alloantibodies. This study reveals a dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.

Authors

Isaac Rosado-Sánchez, Manjurul Haque, Kevin Salim, Madeleine Speck, Vivian C.W. Fung, Dominic A. Boardman, Majid Mojibian, Giorgio Raimondi, Megan K. Levings

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Figure 3

In vivo effects of Tregs expressing costimulatory CAR variants on skin rejection.

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In vivo effects of Tregs expressing costimulatory CAR variants on skin r...
BL/6 mice were transplanted with skin grafts from syngeneic or HLA-A2+ BL/6 mice and administered 1 × 106 CAR-Tregs i.v. (A) Skin graft survival curves and (B) levels of anti–HLA-A2 IgG Abs from mice infused with Tregs expressing CARs encoding costimulatory domains from CD28 (left) or TNFR (right) family members. (A) Data from mice receiving no Treg treatment (PBS) or transplanted with syngeneic WT BL/6 grafts are shown in both graphs. (C) Correlation between anti–HLA-A2 IgG Abs in plasma at day 14 and skin graft rejection of mice receiving Tregs bearing CD28 family–based CARs. (D) Correlation between anti–HLA-A2 IgG Abs in plasma at day 14 and skin graft rejection of mice receiving Tregs bearing TNFR family–based CARs. (E) Persistence of CAR-Tregs measured as the percentage of Thy1.1+ CAR-Tregs of total CD45+ T cells in peripheral blood over time. (F and G) Phenotype of Thy1.1+CD4+ CAR-Tregs in peripheral blood over time including expression of (F) CAR (c-Myc+) and (G), FoxP3 alone (left), and FoxP3 with Helios (right). Data are reported as mean ± SEM pooled from 4 individual experiments with n = 3–13 mice per group. Statistical significance was determined using (A) log-rank Mantel-Cox test, (B and E–G) 2-way ANOVA with a Holm-Šidak posttest, and Pearson’s correlation (C and D). *P < 0.05, **P < 0.01, ****P < 0.0001.