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

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.

Flagellin-specific human CAR Tregs for immune regulation in IBD.

Regulatory T cell (Treg) therapy is a promising strategy to treat inflammatory bowel disease (IBD). Data from animal models has shown that Tregs specific for intestinal antigens are more potent than polyclonal Tregs at inhibiting colitis. Flagellins, the major structural proteins of bacterial flagella, are immunogenic antigens frequently targeted in IBD subjects, leading to the hypothesis that flagellin-specific Tregs could be an effective cell therapy for IBD. We developed a novel chimeric antigen receptor (CAR) specific for flagellin derived from Escherichia coli H18 (FliC). We used this CAR to confer FliC-specificity to human Tregs and investigated their therapeutic potential. FliC-CAR Tregs were activated by recombinant FliC protein but not a control flagellin protein, demonstrating CAR specificity and functionality. In a humanized mouse model, expression of the FliC-CAR drove preferential migration to the colon and expression of the activation marker PD1. In the presence of recombinant FliC protein in vitro, FliC-CAR Tregs were significantly more suppressive than control Tregs and promoted the establishment of colon-derived epithelial cell monolayers. These results demonstrate the potential of FliC-CAR Tregs to treat IBD and more broadly show the therapeutic potential of CARs targeting microbial-derived antigens.

Modelling Graft-Versus-Host Disease in Mice Using Human Peripheral Blood Mononuclear Cells.

Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies. Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies. This protocol was validated in: Sci Transl Med (2020), DOI: 10.1126/scitranslmed.aaz3866 Graphical abstract.

Emerging strategies for treating autoimmune disorders with genetically modified Treg cells.

Gene editing of living cells is a cornerstone of present-day medical research that has enabled scientists to address fundamental biologic questions and identify novel strategies to treat diseases. The ability to manipulate adoptive cell therapy products has revolutionized cancer immunotherapy and promises similar results for the treatment of autoimmune diseases, inflammatory disorders, and transplant rejection. Clinical trials have recently deemed polyclonal regulatory T (Treg) cell therapy to be a safe therapeutic option, but questions remain regarding the efficacy of this approach. In this review, we discuss how gene editing technologies are being applied to transform the future of Treg cell therapy, focusing on the preclinical strategies that are currently being investigated to enhance the efficacy, function, and survival of human Treg cells. We explore approaches that may be used to generate immunoregulatory cells ex vivo, detail emerging strategies that are being used to modify these cells (such as using chimeric antigen receptors to confer antigen specificity), and outline concepts that have been explored to repurpose conventional T cells to target and destroy autoreactive and alloreactive lymphocytes. We also describe the key hurdles that currently hinder the clinical adoption of Treg cell therapy and propose potential future avenues of research for this field.

Suppression of Human Dendritic Cells by Regulatory T Cells.

Regulatory T cells (Tregs) suppress immune responses via a variety of mechanisms and can be used as a cellular therapy to induce tolerance. The function of Tregs is commonly assessed in vitro using assays that measure suppression of effector T cell proliferation and/or cytokine production. However, Tregs can also suppress the function of antigen presenting cells, creating a need for methodology to routinely measure this aspect of their function. This protocol describes a method to measure human Treg-mediated suppression of CD80 and CD86 expression on mature, monocyte-derived dendritic cells. Representative data show suppression mediated by polyclonal Tregs as well as antigen-specific Tregs generated using chimeric antigen receptor (CAR) technology. This method can be used in parallel to T cell suppression assays to measure the functional activity of human Tregs.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

A method for expansion and retroviral transduction of mouse regulatory T cells.

Adoptive cell therapy with genetically modified regulatory T cells (Tregs) is under clinical investigation for the treatment of transplant rejection and various autoimmune conditions. A limitation of modelling this approach in mice is the lack of optimized protocols for expanding and transducing mouse Tregs. Here we describe a protocol for purifying, expanding and retrovirally transducing mouse Tregs with a vector encoding a chimeric antigen receptor as a model transgene. We found that isolation of Tregs from C57Bl/6J Foxp3EGFP mice solely based on eGFP expression resulted in sufficiently pure cells; co-sorting of CD25hi cells was not essential. Although expansion with rapamycin reduced Treg expansion, it promoted maximal in vitro suppressive activity. Retroviral transduction of Tregs following 2 days of stimulation with anti-CD3/CD28 beads achieved a transduction efficiency of ~40% and did not impair their suppressive capacity. When injected into a conventional T cell (Tconv)-transfer-induced colitis model, transduced Tregs inhibited colitis progression at ratios as low as 1 Treg to 100 Tconvs, and maintained Foxp3 and transgene expression throughout an 8-week period. This method facilitates the study of transduced Tregs in animal models and will enable the study of genetically engineered Treg therapy for a variety of inflammatory diseases.

Pharmacological inhibition of RORC2 enhances human Th17-Treg stability and function.

Inflammatory bowel diseases (IBD) are chronic conditions that result from uncontrolled intestinal inflammation. Pathogenic Th17 cells, characterized by production of IL-17A in the absence of IL-10, are thought to contribute to this inflammation, but in humans, antibody-mediated blockade of IL-17A is an ineffective IBD therapy whereas IL-23 blockade is effective. Here, we investigated the effects of pharmacological inhibition of RORC2, the Th17 cell lineage-defining transcription factor, on in vivo-differentiated human Th17 cells and Th17-like Tregs (Th17-Tregs). BMS-336, a small molecule RORC2 inverse agonist, inhibited expression of RORC2-regulated genes in peripheral Th17 cells (CD4+ CD25- CD127+ CXCR3- CCR4+ CCR6+ ) in a dose-dependent manner, with similar inhibitory effects on laminar propria mononuclear cells from IBD and non-IBD subjects. Exposure of peripheral Th17-Tregs (CD4+ CD25hi CD127lo CXCR3- CCR4+ CCR6+ ) to BMS-336 also inhibited IL-17A production and prevented inflammatory cytokine-induced destabilization, as evidenced by preserved FOXP3 expression and epigenetic status of the Treg-specific demethylation region. In parallel, RORC2 inhibition increased the production of IL-10 in Th17-Tregs, resulting in enhanced suppression of inflammatory cytokines from myeloid cells. Thus, via its ability to simultaneously inhibit Th17 cells and enhance the stability and function of Th17-Tregs, pharmacological inhibition of RORC2 is a promising approach to suppress inflammation and promote immune regulation in IBD.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Protease Activated Receptor 4 as a Novel Modulator of Regulatory T Cell Function.

Regulatory T cells (Tregs) are a subpopulation of T cells that maintain immunological tolerance. In inflammatory responses the function of Tregs is tightly controlled by several factors including signaling through innate receptors such as Toll like receptors and anaphylatoxin receptors allowing an effective immune response to be generated. Protease-activated receptors (PARs) are another family of innate receptors expressed on multiple cell types and involved in the pathogenesis of autoimmune disorders. Whether proteases are able to directly modulate Treg function is unknown. Here, we show using two complimentary approaches that signaling through PAR-4 influences the expression of CD25, CD62L, and CD73, the suppressive capacity, and the stability of Tregs, via phosphorylation of FoxO1 and negative regulation of PTEN and FoxP3. Taken together, our results demonstrate an important role of PAR4 in tuning the function of Tregs and open the possibility of targeting PAR4 to modulate immune responses.

Taking regulatory T-cell therapy one step further.

PURPOSE OF REVIEW: Adoptive cell therapy using CD4FOXP3 regulatory T cells (Treg) has emerged as a promising therapeutic strategy to treat autoimmunity and alloimmunity. Preclinical studies suggest that the efficacy of Treg therapy can be improved by modifying the antigen specificity, stability and function of therapeutic Tregs. We review recent innovations that considerably enhance the possibilities of controlling these parameters. RECENT FINDINGS: Antigen-specific Tregs can be generated by genetically modifying polyclonal Tregs to express designated T-cell receptors or single-chain chimeric antigen receptors. The benefits of this approach can be further extended by using novel strategies to fine-tune the antigen-specificity and affinity of Treg in vivo. CRISPR/Cas 9 technology now enables the modification of therapeutic Tregs so they are safer, more stable and long lived. The differentiation and homing properties of Tregs can also be modulated by gene editing or modifying ex-vivo stimulation conditions. SUMMARY: A new wave of innovation has considerably increased the number of strategies that could be used to increase the therapeutic potential of Treg therapy. However, the increased complexity of these approaches may limit their wide accessibility. Third-party therapy with off-the-shelf Treg products could be a solution.

Regulatory T cell-derived extracellular vesicles modify dendritic cell function.

Regulatory T cells (Treg) are a subpopulation of T cells that maintain tolerance to self and limit other immune responses. They achieve this through different mechanisms including the release of extracellular vesicles (EVs) such as exosomes as shown by us, and others. One of the ways that Treg derived EVs inhibit target cells such as effector T cells is via the transfer of miRNA. Another key target for the immunoregulatory function of Tregs is the dendritic cells (DCs). In this study we demonstrate directly, and for the first time, that miRNAs are transferred from Tregs to DCs via Treg derived EVs. In particular two miRNAs, namely miR-150-5p and miR-142-3p, were increased in DCs following their interaction with Tregs and Treg derived exosomes. One of the consequences for DCs following the acquisition of miRNAs contained in Treg derived EVs was the induction of a tolerogenic phenotype in these cells, with increased IL-10 and decreased IL-6 production being observed following LPS stimulation. Altogether our findings provide data to support the idea that intercellular transfer of miRNAs via EVs may be a novel mechanism by which Tregs regulate DC function and could represent a mechanism to inhibit immune reactions in tissues.

Reduced TCR Signaling Contributes to Impaired Th17 Responses in Tolerant Kidney Transplant Recipients.

BACKGROUND: The development of spontaneous kidney transplant tolerance has been associated with numerous B cell-related immune alterations. We have previously shown that tolerant recipients exhibit reduced B-cell receptor signalling and higher IL-10 production than healthy volunteers. However, it is unclear whether cluster of differentiation (CD)4 T cells from tolerant recipients also display an anti-inflammatory profile that could contribute to graft maintenance. METHODS: CD4 T cells were isolated from kidney transplant recipients who were identified as being tolerant recipients, patients with chronic rejection or healthy volunteers. CD4 T cells from the 3 groups were compared in terms of their gene expression profile, phenotype, and functionally upon activation. RESULTS: Gene expression analysis of transcription factors and signalling proteins, in addition to surface proteins expression and cytokine production, revealed that tolerant recipients possessed fewer Th17 cells and exhibited reduced Th17 responses, relative to patients with chronic rejection or healthy volunteers. Furthermore, impaired T-cell receptor signalling and altered cytokine cooperation by monocytes contributed to the development of Th17 cells in tolerant recipients. CONCLUSIONS: These data suggest that defective proinflammatory Th17 responses may contribute to the prolonged graft survival and stable graft function, which is observed in tolerant recipients in the absence of immunosuppressive agents.

What Is Direct Allorecognition?

Direct allorecognition is the process by which donor-derived major histocompatibility complex (MHC)-peptide complexes, typically presented by donor-derived 'passenger' dendritic cells, are recognised directly by recipient T cells. In this review, we discuss the two principle theories which have been proposed to explain why individuals possess a high-precursor frequency of T cells with direct allospecificity and how self-restricted T cells recognise allogeneic MHC-peptide complexes. These theories, both of which are supported by functional and structural data, suggest that T cells recognising allogeneic MHC-peptide complexes focus either on the allopeptides bound to the allo-MHC molecules or the allo-MHC molecules themselves. We discuss how direct alloimmune responses may be sustained long term, the consequences of this for graft outcome and highlight novel strategies which are currently being investigated as a potential means of reducing rejection mediated through this pathway.

MicroRNAs affect dendritic cell function and phenotype.

MicroRNA (miRNA) are small, non-coding RNA molecules that have been linked with immunity through regulating/modulating gene expression. A role for these molecules in T-cell and B-cell development and function has been well established. An increasing body of literature now highlights the importance of specific miRNA in dendritic cell (DC) development as well as their maturation process, antigen presentation capacity and cytokine release. Given the unique role of DC within the immune system, linking the innate and adaptive immune responses, understanding how specific miRNA affect DC function is of importance for understanding disease. In this review we summarize recent developments in miRNA and DC research, highlighting the requirement of miRNA in DC lineage commitment from bone marrow progenitors and for the development of subsets such as plasmacytoid DC and conventional DC. In addition, we discuss how infections and tumours modulate miRNA expression and consequently DC function.