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Abstract
Regulatory T cells (Tregs) play essential roles in maintaining immunological self-tolerance and preventing autoimmunity. The adoptive transfer of antigen-specific Tregs has been expected to be a potent therapeutic method for autoimmune diseases, severe allergy, and rejection in organ transplantation. However, effective Treg therapy has not yet been established because of the difficulty in preparing a limited number of antigen-specific Tregs. Chimeric antigen receptor (CAR) T cells have been shown to be a powerful therapeutic method for treating B cell lymphomas, but application of CAR to Treg-mediated therapy has not yet been established. Here, we generated CD19-targeted CAR (CD19-CAR) Tregs from human PBMCs (hPBMCs) and optimized the fraction of the Treg source as CD4+CD25+CD127loCD45RA+CD45RO-. CD19-CAR Tregs could be expanded in vitro while maintaining Treg properties, including high expression of the latent form of TGF-β. CD19-CAR Tregs suppressed IgG antibody production and differentiation of B cells via a TGF-β-dependent mechanism. Unlike conventional CD19-CAR CD8+ T cells, CD19-CAR Tregs suppressed antibody production in immunodeficient mice that were reconstituted with hPBMCs, reducing the risk of graft-versus-host disease. Therefore, the adoptive transfer of CD19-CAR Tregs may provide a novel therapeutic method for treating autoantibody-mediated autoimmune diseases.
Highlights
Tregs suppress excess immunity against a variety of antigens, including self-antigens, commensal bacteria–derived antigens, and environmental allergens
We mainly used the CD19-targeted second generation Chimeric antigen receptor (CAR) (CD19-CAR), which consists of the FMC63 extracellular single-chain variable fragment domain fused to the CD28 hinge, transmembrane, and cytoplasmic domains and the CD3ζ cytoplasmic domain (1928z); the Venus gene is expressed via internal ribosome entry site–2 (IRES2) to detect gene transduction (Figure 1A) [29]
The naive/resting Treg fraction was first enriched from healthy donor PBMCs by magnetic negative selection and purified by FACS based on marker expression, with CD4+CD25+CD127loCD45RA+CD45RO– cells, which were considered to be naive/resting Tregs (CD45RA+ Tregs) (Figure 1B)
Summary
Tregs suppress excess immunity against a variety of antigens, including self-antigens, commensal bacteria–derived antigens, and environmental allergens. Some clinical trials of adoptive transfer of ex vivo expanded polyclonal Tregs have been performed to suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation [2,3,4,5] and type 1 diabetes (T1D) [6,7,8]. There are several strategies to generate antigen-specific Tregs, which overexpress a transgenic T cell receptor (TCR) or expand when stimulated with antigen These antigen-specific Tregs indicate superior suppression of nonspecific or polyclonal Tregs for several diseases, such as GvHD [11], hemophilia A [12], and experimental autoimmune encephalomyelitis (EAE) [13]. In vitro generation of induced antigen-specific Tregs (iTregs) in the presence of vitamin C, which stabilizes Foxp expression, has been used to suppress GvHD [14], but methods for generating stabilized human iTregs have not yet been established [15]
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