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Regulatory T cells (Tregs) play an important role in mediating immune tolerance after stem cell transplantation, and a reduction in Tregs has been associated with the development of graft-versus-host disease (GvHD). Treg reconstitution is being actively investigated for the prevention of GvHD, as recently presented on the GvHD Hub, and although most clinical trials have used polyclonal Tregs, antigen-specific Tregs engineered using chimeric antigen receptors (CARs) may be a more potent approach.1
CAR T cell therapy, which is showing great promise as a cancer immunotherapeutic, was originally developed using conventional T cells. Tregs have different signaling requirements to conventional T cells. Although multiple studies have used CAR Tregs with the CD28 or 4-1BB domains, the most optimal co-receptor signaling domain for potent CAR Treg function is yet unclear.2 In this study, published in Science Translational Medicine by Nicholas Dawson and colleagues, the functional effects of different co-receptor signaling domains on antigen-specific CAR Tregs were investigated both in vitro and in an in vivo GvHD model.2
CARs were designed according to the schematic shown in Figure 1.
Figure 1. CAR construct design2
CAR constructs were cloned into a lentiviral vector that encoded a truncated nerve growth factor receptor (NGFR) as a transduction marker. Flow cytometry-isolated CD4+ CD25+ CD127− or CD4+ CD25+ CD127− CD45RA+ human Tregs were transduced. Seven days later, NGFR+ cells were isolated by magnetic sorting (CAR Tregs).
Function of the various constructs was assessed in vivo using a well-established HLA-A2 mismatched xenogeneic GvHD mouse model. Immunodeficient NOD/SCID gamma (NSG) mice were injected with 107 HLA-A2+ peripheral blood mononuclear cells (PBMCs) and either 2.5 × 106 CAR Tregs (low ratio) or 5 × 106 CAR Tregs (high ratio). Control groups were injected with either saline or PBMCs without CAR Tregs. The ability of the CAR Treg variants to provide protection from GvHD was assessed by survival and GvHD score (a scale from 0–3 based on weight loss, skin integrity, fur maintenance, activity, and deteriorating posture).
In vitro activity of CAR constructs was evaluated in several ways:
In all, nine different CAR Treg variants were tested against an existing CAR construct comprising an intracellular CD28 domain and extracellular single-chain variable fragment (scFv) specific for the human leukocyte antigen (HLA)-A2 (CD28wt CAR Treg). The nine examined CARs consisted of alternative co-stimulatory domains selected from CD28 or tumor necrosis factor receptor (TNFR) family proteins based on their functional relevance and success in conventional T cells (Table 1).
Table 1. Signaling domains for CAR Treg variants2
CAR T, chimeric antigen receptor T cells; CTLA-4, cytotoxic T lymphocyte-associated protein 4; GITR, glucocorticoid-induced TNFR-related protein; ICOS, inducible T cell costimulatory; IL-10, interleukin-10; PD-1, programmed cell death protein 1; PIK3, phosphoinositide 3-kinase; TNFR, tumor necrosis factor receptor. |
|||
Construct |
Transmembrane domain |
Intracellular co-receptor signaling domain |
Rationale for selection |
CD28wt |
CD28 |
CD28 |
Acute anti-tumor activity |
CD28mut |
CD28 |
CD28 (Y173F) |
Mutant with reduced PI3K pathway activity, likely beneficial for Treg function |
ICOS |
ICOS |
ICOS (CD278) |
Important for Treg survival, may be involved in IL-10 production |
CTLA-4wt |
CD28 |
CTLA-4 (CD152) |
Essential for Treg function |
CTLA-4mut |
CD28 |
CTLA-4 (Y165G) |
Mutant with increased cell surface expression |
PD-1 |
CD28 |
PD1 (CD279) |
Required for peripheral Treg production and maintenance |
OX40 |
OX40 |
OX40 (CD134) |
Promote Tregs in some contexts |
GITR |
GITR |
GITR (CD357) |
Promote Tregs in some contexts |
4-1BB |
4-1BB |
4-1BB (CD137) |
Important for Treg and CAR T cell survival |
TNFR2 |
TNFR2 |
TNFR2 (CD120b) |
Stimulates Treg proliferation and expression |
CAR Tregs were stimulated with HLA-A2-expressing K562 cells to assess their ability to activate Tregs.
High levels of the transcription factor forkhead box protein 3 (FOXP3) and demethylation of the Treg-specific demethylation region (TDSR) are distinct characteristics of Tregs, while Helios expression is considered important for Treg stability. The long-term effect of Treg activation was assessed by monitoring cell number, expression of FOXP3 and Helios, and TDSR methylation over 12 days.
Evidence suggests that antigen-presenting cells play a key role in T cell suppression, so in addition to measuring suppression of CD4+ and CD8+ cells, the authors evaluated expression of co-stimulatory molecules on DCs. CAR Treg stimulation was performed via a combination of anti-CD3/-CD28 bead exposure and HLA-A2 CAR Treg in vitro pre-stimulation. These in vitro results were compared with data from the in vivo GvHD model.
Although CAR Tregs showed activity in the in vivo GvHD mouse model, it is unknown how well these results translate to human trials. The authors acknowledge that the xenogeneic system does not represent a normal immune response and recommend that the performance of CAR Tregs should be further tested in syngeneic models.
This comprehensive study supports the use of the CD28wt co-stimulatory domain for potent CAR Treg function. There was a clear survival advantage for CD28wt CAR Tregs in vivo, which could not be entirely predicted by the in vitro results. Survival benefit and the ability of CD28wt CAR Tregs to proliferate in response to HLA-A2 was supported by transcriptome analysis showing enrichment of genes involved in DNA replication and cell proliferation pathways. The findings of this study might be valuable for CAR design optimization for future Treg therapies.
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