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Given the promising results from phase 1/2 clinical trials of therapy involving regulatory T cells (Tregs), it is critical to develop Treg manufacturing methods that use well-defined reagents.Seeking to maximize expansion of human thymic Tregs activated with anti-CD3/CD28 antibody-coated beads and cultured in serum-free medium, the authors investigated the effect of adjusting process parameters including cell density and cell concentration, and feeding strategy on Treg yield and quality.The authors found that levels of expansion and viability varied with cell density on the day of restimulation. Tregs restimulated at low cell densities (1 × 10These data show the previously undescribed consequences of adjusting cell density on Treg expansion and establish a Good Manufacturing Practice-relevant protocol using non-cell-based activation reagents and serum-free media that supports sustained expansion without loss of viability or phenotype.

For the past century, insulin injections have saved millions of lives, but glycemic instability is still a persistent challenge for people with diabetes, leading to tremendous morbidity and premature mortality. Research in the field of islet transplantation has demonstrated that replacing insulin-producing β cells can restore euglycemia comparable to individuals without diabetes. However, a short supply of cadaveric islet donors, the technically challenging process of isolating islets, and the requirement for chronic immune suppression have impeded widespread clinical adoption. Rather than relying on cadaveric cells, pluripotent stem cells could serve as a virtually unlimited supply of insulin-producing β cells. Protocols have been developed that mimic the normal in vivo development of the human pancreas to generate pancreatic progenitor cells in vitro. Ongoing investigations have yielded progressively more mature β-like cells in vitro that produce insulin but do not yet fully mimic healthy mature β cells. Alongside development of differentiation protocols, other work has provided insight into potential implantation sites for stem cell–derived islet cells including the subcutaneous space, portal vein, and omentum. To optimize implanted cell survival and function, development of immune modulation therapies is ongoing, including selection of immunomodulatory medications and genetic modification of implanted cells to evade immune responses. Further, macroencapsulation or microencapsulation devices could be used to contain and/or immunoprotect implanted cells from the immune response including by using 3-dimensional bioprinting to facilitate the process. Remarkably, ongoing clinical trials have now yielded the first patient relying on differentiated stem cells rather than syringes as their insulin replacement therapy.

A primary goal in transplantation medicine is the induction of a tolerogenic environment for prevention of transplant rejection without the need for long-term pharmacological immunosuppression. Generation of alloantigen-specific regulatory T cells (Tregs) by transduction with chimeric antigen receptors (CARs) is a promising strategy to achieve this goal. This publication reports the preclinical characterization of Tregs (TR101) transduced with a human leukocyte antigen (HLA)-A*02 CAR lentiviral vector (TX200) designated to induce immunosuppression of allograft-specific effector T cells in HLA-A*02-negative recipients of HLA-A*02-positive transplants. In vitro results demonstrated specificity, immunosuppressive function, and safety of TX200-TR101. In NOD scid gamma (NSG) mice, TX200-TR101 prevented graft-versus-host disease (GvHD) in a xenogeneic GvHD model and TX200-TR101 Tregs localized to human HLA-A*02-positive skin transplants in a transplant model. TX200-TR101 persisted over the entire duration of a 3-month study in humanized HLA-A*02 NSG mice and remained stable, without switching to a proinflammatory phenotype. Concomitant tacrolimus did not impair TX200-TR101 Treg survival or their ability to inhibit peripheral blood mononuclear cell (PBMC) engraftment. These data demonstrate that TX200-TR101 is specific, stable, efficacious, and safe in preclinical models, and provide the basis for a first-in-human study.

SummaryWithin the thymus, regulation of the cellular cross-talk directing T cell development is dependent on spatial interactions within specialized niches. To create a holistic, spatially defined map of tissue niches guiding postnatal T cell development we employed the multidimensional imaging platform CO-detection by indEXing (CODEX), as well as CITE-seq and ATAC-seq. We generated age-matched 4–5-month-old postnatal thymus datasets for male and female donors, and identify significant sex differences in both T cell and thymus biology. We demonstrate a crucial role for JAG ligands in directing thymic-like dendritic cell development, reveal important functions of a novel population of ECM-fibroblasts, and characterize the medullary niches surrounding Hassall’s corpuscles. Together, these data represent a unique age-matched spatial multiomic resource to investigate how sex-based differences in thymus regulation and T cell development arise, and provide an essential resource to understand the mechanisms underlying immune function and dysfunction in males and females.

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.

Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor in vivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. To “benchmark” exhaustion in human Tregs, we used a method known to induce exhaustion in conventional T cells: expression of a tonic-signaling chimeric antigen receptor (TS-CAR). We found that TS-CAR-expressing Tregs rapidly acquired a phenotype that resembled exhaustion and had major changes in their transcriptome, metabolism, and epigenome. Similar to conventional T cells, TS-CAR Tregs upregulated expression of inhibitory receptors and transcription factors such as PD-1, TIM3, TOX and BLIMP1, and displayed a global increase in chromatin accessibility-enriched AP-1 family transcription factor binding sites. However, they also displayed Treg-specific changes such as high expression of 4-1BB, LAP, and GARP. DNA methylation analysis and comparison to a CD8 + T cell-based multipotency index showed that Tregs naturally exist in a relatively differentiated state, with further TS-CAR-induced changes. Functionally, TS-CAR Tregs remained stable and suppressive in vitro but were nonfunctional in vivo, as tested in a model of xenogeneic graft-versus-host disease. These data are the first comprehensive investigation of exhaustion in Tregs and reveal key similarities and differences with exhausted conventional T cells. The finding that human Tregs are susceptible to chronic stimulation–driven dysfunction has important implications for the design of CAR Treg adoptive immunotherapy strategies.

Adoptive immunotherapy with Tregs is a promising approach for prevention or treatment of type 1 diabetes. Islet antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B-chain 10-23 peptide presented in the context of the IAg7MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR re-directed NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Co-transfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In wild type NOD mice, InsB-g7 CAR Tregs stably expressed Foxp3 and prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising new therapeutic approach for the prevention of autoimmune diabetes.Brief SummaryChimeric antigen receptor Tregs specific for an insulin B-chain peptide presented by MHC class II prevent autoimmune diabetes.

Regulatory T cell (Treg) therapy holds promise as a potentially curative approach to establish immune tolerance in transplantation and autoimmune disease. An outstanding question is whether therapeutic Tregs have the potential to transdifferentiate into effector T cells and thus exacerbate rather than suppress immune responses. In mice, the transcription factor Helios is thought to promote Tregs lineage stability in a range of inflammatory contexts. In humans, the role of Helios in Tregs is less clear, in part due to the inability to enrich and study subsets of Helios-positive versus Helios-negative Tregs. Using an in vitro expansion system, we found that loss of high Helios expression and emergence of an intermediate Helios (Heliosmid)-expressing population correlated with Treg destabilization. We then used CRISPR/Cas9 to genetically ablate Helios expression in human naive or memory Tregs and found that Helios-knockout and unedited Tregs were equivalent in their suppressive function and stability in inflammation. Thus, high Helios expression is a marker, but not a driver, of human Treg stability in vitro. These data highlight the importance of monitoring Helios expression in therapeutic Treg manufacturing and provide new insight into the biological function of this transcription factor in human T cells. ### Competing Interest Statement MKL has received research funding from Sangamo Therapeutics, Bristol-Myers Squibb, Pfizer, Takeda, and CRISPR Therapeutics for work unrelated to this study. All other authors declare no competing interests. * aAPC : artificial antigen-presenting cell mTreg : memory regulatory T cell nTreg : naive regulatory T cell Tconv : CD4+ conventional T cell Treg : regulatory T cell TSDR : Treg-specific demethylation region

Regulatory T cells (Tregs) expressing chimeric antigen receptors (CAR) 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 antibodies. We generated mouse Tregs expressing HLA-A2-specific CARs with different costimulatory domains and compared their function in vitro and in vivo. In vitro assays revealed the CD28-encoding CAR had superior antigen-specific suppression, proliferation and cytokine production. In contrast, in vivo protection from skin allograft rejection and alloantibody production was similar between Tregs expressing CARs encoding CD28, ICOS or PD1, but not GITR, 41BB or OX40, co-stimulatory domains. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3ζ but no co-stimulatory domain. These data revealed that exogenous co-stimulation via 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. This study reveals a new dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.

Pleckstrin homology domain leucine-rich repeat protein phosphatases (PHLPP) belong to the protein phosphatase magnesium/manganese-dependent family of Ser/Thr phosphatases. Their general role as tumor suppressors has been documented for over a decade. In recent years, accumulating evidence suggests that PHLPP isozymes have key regulatory roles in both innate and adaptive immunity. In macrophages, PHLPP1 dampens signaling through TLR4 and the IFN-γ receptor by altering cytosolic signaling pathways. Additionally, nuclear-localized PHLPP1 inhibits STAT1-mediated inflammatory gene expression by direct dephosphorylation at Ser 727. PHLPP1 also regulates the migratory and inflammatory capacity of neutrophils in vivo. Furthermore, PHLPP1-mediated dephosphorylation of AKT on Ser 473 is required for both the suppressive function of regulatory T cells and for the pro-apoptotic effects of PHLPP1 in B cell chronic lymphocytic leukemia. In the context of immune homeostasis, PHLPP1 expression is modulated in multiple cell types by inflammatory signals, and the dynamics of its expression have varying effects on the pathogenesis of inflammatory bowel disease and septic shock. In this review, we summarize recent findings on the functions of PHLPP in inflammatory and regulatory signaling in the context of both innate and adaptive immunity.

Type 1 diabetes results from defects in immune self-tolerance that lead to inflammatory infiltrate in pancreatic islets, beta cell dysfunction and T cell-mediated killing of beta cells. Although therapies that broadly inhibit immunity show promise to mitigate autoinflammatory damage caused by effector T cells, these are unlikely to permanently reset tolerance or promote regeneration of the already diminished pool of beta cells. An emerging concept is that certain populations of immune cells may have the capacity to both promote tolerance and support the restoration of beta cells by supporting proliferation, differentiation and/or regeneration. Here we will highlight three immune cell types-macrophages, regulatory T cells and innate lymphoid cells-for which there is evidence of dual roles of immune regulation and tissue regeneration. We explore how findings in this area from other fields might be extrapolated to type 1 diabetes and highlight recent discoveries in the context of type 1 diabetes. We also discuss technological advances that are supporting this area of research and contextualise new therapeutic avenues to consider for type 1 diabetes.

Systemic immunosuppression for the mitigation of immune rejection after organ transplantation causes adverse side effects and constrains the long-term benefits of the transplanted graft. Here we show that protecting the endothelial glycocalyx in vascular allografts via the enzymatic ligation of immunosuppressive glycopolymers under cold-storage conditions attenuates the acute and chronic rejection of the grafts after transplantation in the absence of systemic immunosuppression. In syngeneic and allogeneic mice that received kidney transplants, the steric and immunosuppressive properties of the ligated polymers largely protected the transplanted grafts from ischaemic reperfusion injury, and from immune-cell adhesion and thereby immunocytotoxicity. Polymer-mediated shielding of the endothelial glycocalyx following organ procurement should be compatible with clinical procedures for transplant preservation and perfusion, and may reduce the damage and rejection of transplanted organs after surgery. Protection of the endothelial glycocalyx in vascular allografts via the enzymatic ligation of immunosuppressive glycopolymers prevents allograft rejection after transplantation in the absence of systemic immunosuppression.

Transforming growth factor-beta (TGF-β) family proteins mediate many vital biological functions in growth, development and regulation of the immune system. TGF-β itself controls immune homeostasis and inflammation, including conversion of naive CD4+ T cells into Foxp3+ regulatory T cells (Tregs) in the presence of IL-2 and T cell receptor ligands. The helminth parasite Heligmosomoides polygyrus exploits this pathway through a structurally novel TGF-β mimic (Hp-TGM), which binds to mammalian TGF-β receptors and induces Tregs. Here, we performed detailed comparisons of Hp-TGM with mammalian TGF-β. Compared to TGF-β, Hp-TGM induced greater numbers of Foxp3+ Tregs (iTregs), with more intense Foxp3 expression. Both ligands upregulated Treg functional markers CD73, CD103 and PD-L1, but Hp-TGM induced significantly higher CD39 expression than did TGF-β. Interestingly, in contrast to canonical TGF-β signalling through Smad2/3, Hp-TGM stimulation was slower and more sustained. Gene expression profiles induced by TGF-β and Hp-TGM were remarkably similar, and both types of iTregs suppressed T cell responses in vitro and EAE-driven inflammation in vivo. In vitro, both types of iTregs were equally stable under inflammatory conditions, but Hp-TGM-induced iTregs were more stable in vivo during DSS-induced colitis, with greater retention of Foxp3 expression and lower conversion to a ROR-γt+ phenotype. Altogether, results from this study suggest that the parasite cytokine mimic, Hp-TGM, may deliver a qualitatively different signal to CD4+ T cells with downstream consequences for the long-term stability of iTregs. These data highlight the potential of Hp-TGM as a new modulator of T cell responses in vitro and in vivo.

Regulatory T-cell (Treg) therapy has shown promise in treating autoimmune diseases, transplant rejection, or graft-versus-host disease in early clinical trials. These trials have demonstrated that cell therapy using polyclonal Tregs is feasible and safe, however, the field has been limited by the lack of polyclonal cell specificity and consequent large cell numbers required, and the difficulty in generating autologous products for some patients. Thus, the field is moving toward 'next generation' Treg cell therapies that include genetic modification strategies to engineer specificity and/or modify function, as well as methods to generate Tregs in vitro. In this review, we describe how genetic modification of Tregs using viral transduction or gene editing may be incorporated into Treg manufacturing protocols. We also describe how Tregs may be generated via FOXP3 gene editing or overexpression, or by differentiation from pluripotent stem cells. The application of these various types of engineered Tregs is discussed.

Immune homeostasis in the intestine is tightly controlled by FOXP3+ regulatory T cells (Tregs), defects of which are linked to the development of chronic conditions, such as inflammatory bowel disease (IBD). As a mechanism of immune evasion, several species of intestinal parasites boost Treg activity. The parasite Heligmosomoides polygyrus is known to secrete a molecule (Hp‐TGM) that mimics the ability of TGF‐β to induce FOXP3 expression in CD4+ T cells. The study aimed to investigate whether Hp‐TGM could induce human FOXP3+ Tregs as a potential therapeutic approach for inflammatory diseases. CD4+ T cells from healthy volunteers were expanded in the presence of Hp‐TGM or TGF‐β. Treg induction was measured by flow cytometric detection of FOXP3 and other Treg markers, such as CD25 and CTLA‐4. Epigenetic changes were detected using ChIP‐Seq and pyrosequencing of FOXP3. Treg phenotype stability was assessed following inflammatory cytokine challenge and Treg function was evaluated by cellular co‐culture suppression assays and cytometric bead arrays for secreted cytokines. Hp‐TGM efficiently induced FOXP3 expression (> 60%), in addition to CD25 and CTLA‐4, and caused epigenetic modification of the FOXP3 locus to a greater extent than TGF‐β. Hp‐TGM‐induced Tregs had superior suppressive function compared with TGF‐β‐induced Tregs, and retained their phenotype following exposure to inflammatory cytokines. Furthermore, Hp‐TGM induced a Treg‐like phenotype in in vivo differentiated Th1 and Th17 cells, indicating its potential to re‐program memory cells to enhance immune tolerance. These data indicate Hp‐TGM has potential to be used to generate stable human FOXP3+ Tregs to treat IBD and other inflammatory diseases., In this study, we showed a parasite‐derived TGF‐β mimic protein can induce stable FOXP3+ Tregs from both naive and pre‐committed human memory CD4+ T cells. This protein has potential for use as a novel immunoregulatory therapeutic for the treatment of inflammatory disease. ​

The linear model of Th cell lineage commitment is being revised due to reports that mature Th cells can trans-differentiate into alternate lineages. This ability of Th cells to reprogram is thought to be regulated by epigenetic mechanisms that control expression of transcription factors characteristic of opposing lineages. It is unclear, however, to what extent this new model of Th cell plasticity holds true in human Th cell subsets that develop under physiological conditions in vivo. We isolated in vivo-differentiated human Th1 and Th17 cells, as well as intermediate Th1/17 cells, and identified distinct epigenetic signatures at cytokine (IFNG and IL17A) and transcription factor (TBX21, RORC, and RORA) loci. We also examined the phenotypic and epigenetic stability of human Th17 cells exposed to Th1-polarizing conditions and found that although they could upregulate TBX21 and IFN-γ, this occurred without loss of IL-17 or RORC expression, and resulted in cells with a Th1/17 phenotype. Similarly, Th1 cells could upregulate IL-17 upon enforced expression of RORC2, but did not lose expression of IFN-γ or TBX21. Despite alterations in expression of these signature genes, epigenetic modifications were remarkably stable aside from the acquisition of active histone methylation marks at cytokine gene promoters. The limited capacity of human Th17 and Th1 cells to undergo complete lineage conversion suggests that the bipotent Th1/17 cells may arise from Th1 and/or Th17 cells. These data also question the broad applicability of the new model of Th cell lineage plasticity to in vivo-polarized human Th cell subsets.

The authors of this article, all women who have been deeply committed to the Federation of Clinical Immunology Societies (FOCIS), performed a retrospective analysis of gender equality practices of FOCIS to identify areas for improvement and make recommendations accordingly. Gender data were obtained and analyzed for the period from January 2010 to July 2021. Outcome measures included numbers of men and women across the following categories: membership enrollment, meeting and course faculty and attendees, committee and leadership composition. FOCIS’ past and present leaders, steering committee members, FCE directors, individual members, as well as education, annual meeting scientific program and FCE committee members and management staff of FOCIS were surveyed by email questionnaire for feedback on FOCIS policies and practice with respect to gender equality and inclusion. Although women represent 50% of the membership, they have been underrepresented in all leadership, educational, and committee roles within the FOCIS organization. Surveying FOCIS leadership and membership revealed a growing recognition of disparities in female leadership across all FOCIS missions, leading to significant improvement in multiple areas since 2016. We highlight these changes and propose a number of recommendations that can be used by FOCIS to improve gender equality.

Regulatory T cells (Tregs) have a critical role in maintaining self-tolerance and immune homeostasis. There is much interest in using Tregs as a cell therapy to re-establish tolerance in conditions such as inflammatory bowel disease and type 1 diabetes, with many ongoing clinical studies testing the safety and efficacy of this approach. Manufacturing of Tregs for therapy typically involves ex vivo expansion to obtain sufficient cell numbers for infusion and comes with the risk of altering the activity of key biological processes. However, this process also offers an opportunity to tailor Treg function to maximize in vivo activity. In this review, we focus on the roles of antigen-presenting cells (APCs) in the generation and function of Tregs in humans. In addition to stimulating the development of Tregs, APCs activate Tregs and provide signals that induce specialized functional and homing marker expression. Cross talk between Tregs and APCs is a critical, often under-appreciated, aspect of Treg biology, with APCs mediating the key properties of infectious tolerance and bystander suppression. Understanding the biology of human Treg-APC interactions will reveal new ways to optimize Treg-based therapeutic approaches.

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.

Cell therapy with autologous donor-specific regulatory T cells (Tregs) is a promising strategy to minimize immunosuppression in transplant recipients. Chimeric antigen receptor (CAR) technology has recently been used successfully to generate donor-specific Tregs and overcome the limitations of enrichment protocols based on repetitive stimulations with alloantigens. However, the ability of CAR-Treg therapy to control alloreactivity in immunocompetent recipients is unknown. We first analyzed the effect of donor-specific CAR Tregs on alloreactivity in naive, immunocompetent mice receiving skin allografts. Tregs expressing an irrelevant or anti-HLA-A2-specific CAR were administered to Bl/6 mice at the time of transplanting an HLA-A2+ Bl/6 skin graft. Donor-specific CAR-Tregs, but not irrelevant-CAR Tregs, significantly delayed skin rejection and diminished donor-specific antibodies (DSAs) and frequencies of DSA-secreting B cells. Donor-specific CAR-Treg-treated mice also had a weaker recall DSA response, but normal responses to an irrelevant antigen, demonstrating antigen-specific suppression. When donor-specific CAR Tregs were tested in HLA-A2-sensitized mice, they were unable to delay allograft rejection or diminish DSAs. The finding that donor-specific CAR-Tregs restrain de novo but not memory alloreactivity has important implications for their use as an adoptive cell therapy in transplantation.

Background & Aims Bacterial flagellin is an important antigen in inflammatory bowel disease, but the role of flagellin-specific CD4+ T cells in disease pathogenesis remains unclear. Also unknown is how changes in intestinal microbiome intersect with those in microbiota-specific CD4+ T cells. We aimed to quantify and characterize flagellin-specific CD4+ T cells in Crohn’s disease (CD) and ulcerative colitis (UC) patients and study their relationship with intestinal microbiome diversity. Methods Blood was collected from 3 cohorts that included CD patients, UC patients, and healthy controls. Flow cytometry analyzed CD4+ T cells specific for Lachnospiraceae-derived A4-Fla2 and Escherichia coli H18 FliC flagellins, or control vaccine antigens. Serum antiflagellin IgG and IgA antibodies were detected by enzyme-linked immunosorbent assay and stool samples were collected and subjected to 16S ribosomal DNA sequencing. Results Compared with healthy controls, CD and UC patients had lower frequencies of vaccine-antigen–specific CD4+ T cells and, as a proportion of vaccine-specific cells, higher frequencies of flagellin-specific CD4+ T cells. The proportion of flagellin-specific CD4+ T cells that were CXCR3negCCR4+CCR6+ Th17 cells was reduced in CD and UC patients, with increased proportions of CD39+, PD-1+, and integrin β7+ cells. Microbiome analysis showed differentially abundant bacterial species in patient groups that correlated with immune responses to flagellin. Conclusions Both CD and UC patients have relative increases in the proportion of circulating Fla2-specific CD4+ T cells, which may be associated with changes in the intestinal microbiome. Evidence that the phenotype of these cells strongly correlate with disease severity provides insight into the potential roles of flagellin-specific CD4+ T cells in inflammatory bowel disease., Graphical abstract

Regulatory T cell (Treg) therapy is under clinical investigation for the treatment of transplant rejection, autoimmune disease, and graft-versus-host disease. With the advent of genome editing, attention has turned to reinforcing Treg function for therapeutic benefit. A hallmark of Tregs is dampened activation of PI3K-AKT signalling, of which PTEN is a major negative regulator. Loss-of-function studies of PTEN, however, have not conclusively shown a requirement for PTEN in upholding Treg function and stability. Using CRISPR-based genome editing in human Tregs, we show that PTEN ablation does not cause a global defect in Treg function and stability; rather, it selectively blocks their ability to suppress antigen-presenting cells. PTEN-KO Tregs exhibit elevated glycolytic activity, upregulate FOXP3, maintain a Treg phenotype, and have no discernable defects in lineage stability. Functionally, PTEN is dispensable for human Treg-mediated inhibition of T cell activity in vitro and in vivo but is required for suppression of costimulatory molecule expression by antigen-presenting cells. These data are the first to define a role for a signalling pathway in controlling a subset of human Treg activity. Moreover, they point to the functional necessity of PTEN-regulated PI3K-AKT activity for optimal human Treg function.

Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity after Hematopoietic Stem Cell Transplantation (HSCT). In large patient populations, we have shown a CD56bright Natural Killer population to strongly associate with a lack of cGvHD and we hypothesize that these cells function to suppress cGvHD. We aimed to isolate and define the characteristics of NKreg cells associated with suppression of cGvHD. Immunophenotypic evaluation of a large pediatric population found the CD56bright NK population associated with a lack of cGvHD to be perforin-, Granzyme B-, and CD335+. Transcriptome analysis of a small patient cohort of CD56bright compared to CD56dim NK cells found the NKreg cells to also overexpress Granzyme K, IL-7R, GPR183, RANK, GM-CSFR, TCF7, and IL23A. Further analysis of this CD56bright NKreg population found a subpopulation that overexpressed IRF1, and TNF. We also found that viable NKreg cells may be isolated by sorting on CD56+ and CD16- NK cells, and this population can suppress allogeneic CD4+ T cells, but not Treg cells or CD8+ T cells through a non-cytolytic, cell-cell contact dependent mechanism. Suppression was not reliant upon the NKp44, NKp46, or GPR183 receptors. Additionally, NKreg cells do not kill leukemic cells. Moreover, this is the first paper to clearly establish that a CD56brightCD3-CD16-perforin- NKreg population associates with a lack of cGvHD and has several unique characteristics, including the suppression of helper T cell function in vitro. With further investigation we may decipher the mechanism of NKreg suppression and operationalize expansion of NKreg cells associated with cGvHD suppression.

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.

Survivors of severe SARS-CoV-2 infections frequently suffer from a range of post-infection sequelae. Whether survivors of mild or asymptomatic infections can expect any long-term health consequences is not yet known. Herein we investigated lasting changes to soluble inflammatory factors and cellular immune phenotype and function in individuals who had recovered from mild SARS-CoV-2 infections (n=22) compared to those that had recovered from other mild respiratory infections (n=11). Individuals who had mild SARS-CoV-2 infections had elevated levels of C-reactive protein 1-3 months after symptom onset, and changes in phenotype and function of circulating T cells that were not apparent in individuals 6-9 months post-symptom onset. Markers of monocyte activation and expression of adherence and chemokine receptors indicative of altered migratory capacity were also higher at 1-3 months post-infection in individuals who had mild SARS-CoV-2, but these were no longer elevated by 6-9 months post-infection. Perhaps most surprisingly, polyclonal activation of T cells was higher in individuals who had recently experienced a mild SARS-CoV-2 infection compared to individuals with other recent respiratory infections. These data are indicative of prolonged immune activation and systemic inflammation that persists for up to three months after mild or asymptomatic SARS-CoV-2 infections.

Regulatory T (Treg) cell-specific deletion of a gene of interest is a procedure widely used to study mechanisms controlling Treg development, homeostasis and function. Accordingly, several transgenic mouse lines have been generated that bear the Cre recombinase under control of the Foxp3 promoter either as a random transgene insertion or knocked into the endogenous Foxp3 locus, with the Foxp3YFP-Cre strain of mice being one of the most widely used. In an attempt to generate Treg cells that lacked expression of the insulin receptor (Insr), we crossed Foxp3YFP-Cre mice with Insrfl/fl mice. Using a conventional two-band PCR genotyping method we found that offspring genotypes did not correspond to the expected Mendelian ratios. We therefore developed a quantitative PCR-based genotyping method to investigate possible ectopic recombination outside the Treg lineage. With this method we found that ~50% of the F1 -generation mice showed evidence of ectopic recombination and that ~10% of the F2 -generation mice had germline Cre recombination activity leading to a high frequency of offspring with global Insr deletion. Use of the quantitative PCR genotyping method enabled accurate selection of mice without ectopic recombination and only the desired Treg cell-specific Insr deletion. Our data highlight the need to use genotyping methods that allow for assessment of possible ectopic recombination driven by the Foxp3YFP-Cre allele, particularly when studying genes that are systemically expressed.

Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Here we report systematic testing of activation reagents, cell culture media, restimulation timing, and cryopreservation to develop a good-manufacturing-practice (GMP)-compatible method to expand and cryopreserve Tregs. By comparing activation reagents, including soluble antibody tetramers, antibody-conjugated beads and artificial antigen-presenting cells (aAPCs), and different media, we found that the combination of Dynabeads Treg Xpander and ImmunoCult-XF medium preserved FOXP3 expression and suppressive function, and resulted in expansion that was comparable to a single stimulation with aAPCs. Cryopreservation tests revealed a critical timing effect: only cells cryopreserved 1–3 days, but not >3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products.

Background & Aims. T-regulatory (Treg) cells suppress immune responses to maintain homeostasis. There are two main subsets of Treg cells, FOXP3-positive Treg cells that do not produce high levels of effector cytokines and Type 1 Treg (Tr1) cells that are FOXP3-negative and secrete interleukin 10 (IL10). IL10 is an important anti-inflammatory cytokine, therefore Tr1 cells might be uniquely suited for development as a treatment for inflammatory bowel diseases. We aimed to develop methods to isolate and expand human Tr1 cells and define their functions. Methods. We obtained blood samples and colon biopsies from patients with Crohn’s disease or ulcerative colitis or healthy individuals (controls). CD4+ T cells were isolated from blood samples, stimulated with anti-CD3/CD28 beads and Tr1 cells purified using an IL10 cytokine capture assay and cell sorting. FOXP3-positive Treg cells were sorted as CD4+CD25highCD127low cells from unstimulated cells. Tr1 and FOXP3-positive Treg cells were expanded, and phenotypes and gene expression profiles were compared. T cells in peripheral blood mononuclear cells from healthy donors were stimulated with anti-CD3/CD28 beads and the suppressive abilities of Tr1 and FOXP3-positive Treg cells were measured. Human colonic organoid cultures were established then cultured with Tr1 or FOXP3-positive Treg cell culture supernatants and analyzed by immunofluorescence and flow cytometry. T84 cells (human colonic adenocarcinoma epithelial cells) were incubated with Tr1 or FOXP3-positive Treg cell culture supernatants and trans-epithelial electrical resistance was measured to determine epithelial cell barrier function. Results. Phenotypes of Tr1 cells isolated from healthy subjects or patients with Crohn’s disease or ulcerative colitis did not differ significantly following expansion. Tr1 cells and FOXP3-positive Treg cells suppressed proliferation of effector T cells, but only Tr1 cells suppressed secretion of IL1beta (IL1B) and TNF from myeloid cells. Tr1 cells, but not FOXP3-positive Treg cells, isolated from healthy subjects and IBD patients secreted IL22, which regulated repair of the epithelium and promoted barrier function of human intestinal epithelial cells. Tr1 cell culture supernatants promoted differentiation of mucin-producing goblet cells in intestinal organoid cultures. Conclusion. Human Tr1 cells suppress proliferation of effector T cells (adaptive immune response) and production of IL1B and TNF by myeloid cells (innate immune response). They also secrete IL22 to regulate repair of the epithelium and promote barrier function. They might be developed as a cell-based therapy for intestinal inflammatory disorders.

Summary Regulatory T cell (Treg) therapy has shown promise in early clinical trials for treating graft-versus-host disease, transplant rejection and autoimmune disorders. A challenge has been to isolate sufficiently pure Tregs and expand them to a clinical dose. However, there has been considerable progress in the development and optimization of these methods, resulting in a variety of manufacturing protocols being tested in clinical trials. In this review, we summarize methods that have been used to manufacture Tregs for clinical trials, including the choice of cell source and protocols for cell isolation and expansion. We also discuss alternative culture or genome editing methods for modulating Treg specificity, function or stability that could be applied to future clinical manufacturing protocols to increase the efficacy of Treg therapy.

Regulatory T cell (Treg) therapy is a potential curative approach for a variety of immune-mediated conditions, including autoimmunity and transplantation, in which there is pathological tissue damage. In mice, IL-33R (ST2)–expressing Tregs mediate tissue repair by producing the growth factor amphiregulin, but whether similar tissue-reparative Tregs exist in humans remains unclear. We show that human Tregs in blood and multiple tissue types produced amphiregulin, but this was neither a unique feature of Tregs nor selectively upregulated in tissues. Human Tregs in blood, tonsil, synovial fluid, colon, and lung tissues did not express ST2, so ST2+ Tregs were engineered via lentiviral-mediated overexpression, and their therapeutic potential for cell therapy was examined. Engineered ST2+ Tregs exhibited TCR-independent, IL-33–stimulated amphiregulin expression and a heightened ability to induce M2-like macrophages. The finding that amphiregulin-producing Tregs have a noneffector phenotype and are progressively lost upon TCR-induced proliferation and differentiation suggests that the tissue repair capacity of human Tregs may be an innate function that operates independently from their classical suppressive function.