Treg Therapy Research Articles

Innate Control of Tissue-Reparative Human Regulatory T Cells.

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.

Research Highlights

Immune Regulation by Glucocorticoids Can Be Linked to Cell Type–dependent Transcriptional Responses Franco LM, Gadkari M, Howe KN, et al. J Exp Med. 2019;216(2):384–406. Glucocorticoids have been a mainstay for treatment of a range of inflammatory disorders for >60 years.1 While widely used, the biological basis underpinning their immunoregulatory effects is still poorly described. In general, glucocorticoids are known to act by binding to the glucocorticoid receptor (GR) in the cytosol, causing GR dimerization and subsequent translocation to the nucleus, where the ligand-bound GR binds to glucocorticoid response elements on the DNA increasing their transcriptional activity.2 Additionally, glucocorticoids are known to interfere with the activity of other transcription factors such as NF-êB.3 The genomic location of GR binding has been shown to differ greatly among different cell types. To further investigate how glucocorticoids differentially affect different cell types, Franco et al4 interrogated the genome-wide transcriptional response to glucocorticoids in 9 distinct human cell types in vitro and used this data to create a pathway-level map of the effects of glucocorticoids across these cell types. They found that the transcriptional response of different cell types to glucocorticoids is highly varied. The number of genes that responded to glucocorticoid treatment was greater in hematopoietic cells than in nonhematopoietic cells, with neutrophils having the greatest number of responsive genes. The specific glucocorticoid-responsive genes identified were also highly variable in each cell type, even when comparing cells of similar origin. Additionally, they found no correlation between the baseline expression of a gene and the average response of that gene to glucocorticoid treatment—many genes that were expressed at comparable baseline levels in hematopoietic and nonhematopoietic cells only responded to glucocorticoid treatment in one of these 2 groups. The authors also identified specific pathways affected by glucocorticoid stimuli. With a a specific focus on B cells, Franco et al found significant upregulation of IL-10, along with significant downregulation of essential B cell receptor signaling proteins, and a decrease in toll-like receptor expression. Functional studies confirmed that glucocorticoids diminished B cell receptor and toll-like receptor signaling. Glucocorticoids also increased expression of the gene encoding BLIMP-1, which is a central part of terminal differentiation in B cells. This could be a mechanism behind the reduced B cell proliferation seen following glucocorticoid administration. With detrimental effects of long-term glucocorticoid use well described, understanding the mechanistic pathways that underpin the immunoregulatory impact of these drugs may prove critical in finding novel agents that exert a similar response without adverse off-target effects. Mitochondrial Complex III Is Essential for Suppressive Function of Regulatory T Cells Weinberg SE, Singer BD, Steinert EM, et al. Nature. 2019;565(7740):495–499. In recent years, there has been a great interest in the potential of regulatory T cell (Treg) therapy for inducing tolerance after solid organ transplantation and for the treatment of graft-versus-host disease. With many promising trials,1 questions surrounding the perpetuation of the suppressive function of Tregs postinfusion remain. It has been shown that there are defined metabolic profiles associated with Treg differentiation and phenotype. Treg cells are largely dependent on mitochondrial metabolism and have been shown to oxidize pyruvate and lipids preferentially when compared with conventional T cells. This unique metabolic profile has been functionally linked to the suppressive phenotype of Treg cells; indeed, the plasticity of Treg suppressive function may well be linked to their metabolic plasticity. To investigate whether Treg suppression is reliant on the mitochondrial respiratory chain, Weinberg et al2 generated mice with a Treg-specific Rieske iron-sulfur protein (RISP) knockout. RISP is an essential part of the mitochondrial respiratory chain complex III, thus ablating mitochondrial complex III function. These mice displayed substantial inflammation by the age of 3 weeks, with enlarged lymph nodes and spleens, thymic atrophy, and increased CD4+ and CD8+ T cell activation and proliferation in the spleen and lymph nodes. This phenotype is highly reminiscent of the Treg deficient Scurfy mice3; however, the number of Tregs in RISP knockout mice remained unchanged. RISP knockout Tregs demonstrated significantly reduced suppressive capacity both in vitro and in vivo. Interestingly, Foxp3 expression levels did not change, demonstrating that Treg suppressive function is reliant on mitochondrial complex III. To further verify that this phenotype was due to a lack in mitochondrial complex III function, the authors also generated mice with a Treg-specific knockout of QPC, another essential subunit of mitochondrial complex III. A similar pattern of systemic inflammation and overt overproliferation of CD4+ and CD8+ T cells was observed, resulting in death of the mice by 3 weeks. These data show that mitochondrial complex III function is necessary for Treg suppressive function and global immune regulation. Understanding how mitochondrial function can affect Treg suppressive potency may prove important for their use as a cellular therapy posttransplantation, given that such therapies may be rendered nonfunctional in an ischemic microenvironment.

CD8+CD122+PD-1+ Tregs Synergize With Costimulatory Blockade of CD40/CD154, but Not B7/CD28, to Prolong Murine Allograft Survival.

A transplanted organ is always rejected in the absence of any immunosuppressive treatment due to vigorous alloimmunity. However, continuously global immunosuppression with a conventional immunosuppressant may result in severe side effects, including nephrotoxicity, tumors and infections. Tregs have been widely used to inhibit allograft rejection, especially in animal models. However, it's well accepted that administration of Tregs alone is not satisfactory in immune-competent wild-type animals. Therefore, it's imperative to promote Treg therapies under the cover of other approaches, including costimulatory blockade. In the present study, we demonstrated that administration of in vitro-expanded CD8+CD122+PD-1+ Tregs synergized with costimulatory blockade of CD40/CD154, but not B7/CD28, to prolong skin allograft survival in wild-type mice and to reduce cellular infiltration in skin allografts as well. Treg treatment and blockade of CD40/CD154, but not B7/CD28, also exhibited an additive effect on suppression of T cell proliferation in vitro and pro-inflammatory cytokine expression in skin allografts. Importantly, blocking B7/CD28, but not CD40/CD154, costimulation decreased the number of transferred CD8+CD122+PD-1+ Tregs and their expression of IL-10 in recipient mice. Furthermore, it's B7/CD28, but not CD40/CD154, costimulatory blockade that dramatically reduced IL-10 production by CD8+CD122+PD-1+ Tregs in vitro, suggesting that B7/CD28, but not CD40/CD154, costimulation is critical for their production of IL-10. Indeed, infusion of IL-10-deficient CD8+CD122+PD-1+ Tregs failed to synergize with anti-CD154 Ab treatment to further prolong allograft survival. Our data may explain why blocking B7/CD28 costimulatory pathway does not boost IL-10-dependent Treg suppression of alloimmunity. Thus, these findings could be implicated in clinical organ transplantation.

Past, Present, and Future of Regulatory T Cell Therapy in Transplantation and Autoimmunity.

Regulatory T cells (Tregs) are important for the induction and maintenance of peripheral tolerance therefore, they are key in preventing excessive immune responses and autoimmunity. In the last decades, several reports have been focussed on understanding the biology of Tregs and their mechanisms of action. Preclinical studies have demonstrated the ability of Tregs to delay/prevent graft rejection and to control autoimmune responses following adoptive transfer in vivo. Due to these promising results, Tregs have been extensively studied as a potential new tool for the prevention of graft rejection and/or the treatment of autoimmune diseases. Currently, solid organ transplantation remains the treatment of choice for end-stage organ failure. However, chronic rejection and the ensuing side effects of immunosuppressants represent the main limiting factors for organ acceptance and patient survival. Autoimmune disorders are chronic diseases caused by the breakdown of tolerance against self-antigens. This is triggered either by a numerical or functional Treg defect, or by the resistance of effector T cells to suppression. In this scenario, patients receiving high doses of immunosuppressant are left susceptible to life-threatening opportunistic infections and have increased risk of malignancies. In the last 10 years, a few phase I clinical trials aiming to investigate safety and feasibility of Treg-based therapy have been completed and published, whilst an increasing numbers of trials are still ongoing. The first results showed safety and feasibility of Treg therapy and phase II clinical trials are already enrolling. In this review, we describe our understanding of Tregs focussing primarily on their ontogenesis, mechanisms of action and methods used in the clinic for isolation and expansion. Furthermore, we will describe the ongoing studies and the results from the first clinical trials with Tregs in the setting of solid organ transplantation and autoimmune disorders. Finally, we will discuss strategies to further improve the success of Treg therapy.

Transient increase of activated regulatory T cells early after kidney transplantation

Regulatory T cells (Tregs) are crucial in controlling allospecific immune responses. However, studies in human kidney recipients regarding the contribution of polyspecific Tregs have provided differing results and studies on alloreactive Tregs are missing completely. In this retrospective study, we specifically analyzed activated CD4+CD25highFOXP3+GARP+ Tregs in 17 patients of a living donor kidney transplantation cohort longitudinally over 24 months by flow cytometry (FOXP3: forkhead box protein 3, GARP: glycoprotein A repetitions predominant). We could demonstrate that Tregs of patients with end-stage renal disease (ESRD) are already pre-activated when compared to healthy controls. Furthermore, even though total CD4+CD25highFOXP3+ Treg numbers decreased in the first three months after transplantation, frequency of activated Tregs increased significantly representing up to 40% of all peripheral Tregs. In a cohort of living donor kidney transplantation recipients with stable graft function, frequencies of activated Tregs did not correlate with the occurrence of acute cellular rejection or chronic graft dysfunction. Our results will be important for clinical trials using adoptive Treg therapy after kidney transplantation. Adoptively transferred Tregs could be important to compensate the Treg loss at month 3, while they have to compete within the Treg niche with a large number of activated Tregs.

Control and prevention of kidney transplant rejection: the role and possibilities for the clinical use of regulatory T-cells in transplantation

This article discusses the need to implement effective methods for monitoring immune status and rehabilitation of patients after kidney transplantation. Induction of immunological tolerance which allows minimizing or even completely canceling supportive immunosuppressive therapy is one of the key tasks in the field of organ transplantation. Regulatory T-cells (TREGs) play an important role in maintaining immunological homeostasis, including limiting kidney transplant rejection, and potentially contribute to the development of immunological tolerance. At the same time, for the introduction of TREG therapy into clinical practice, it is necessary to overcome a number of unsolved problems, such as induction and cultivation of a sufficient number of TREG cells for therapeutic action as well as reducing the risks associated with TREG conversion to effector lymphocytes or an undesirable non-specific immunosuppressive effect. This review examines both the impact of common post-transplant pharmacological immunosuppression approaches on TREGs and the therapeutic potential of TREG cell cultures in prevention of kidney transplant rejection. The questions of ex vivo TREG manufacturing process and possible threats of applying cell technologies in this branch of transplantology were considered.

Profiling T Cell Receptor Repertoires in Phase I/II Clinical Trials of Donor Treg Infusion for the Treatment of Chronic Graft-Versus-Host Disease

IntroductionUnder the auspices of the EC-Horizon 2020 project “TREGeneration”, we are conducting five clinical phase I/II trials investigating the safety and efficacy of donor regulatory T cell (Treg) infusion in patients with chronic Graft-versus-Host Disease (cGvHD), a serious complication following hematopoietic stem cell transplantation (HSCT). Workpackage 4 evaluates T cell receptor (TCR) repertoire changes by high-throughput sequencing following the infusion of donor Tregs.MethodsFor repertoire analysis, four different T cell subsets (Treg, conventional CD4 T cells - Tcon, total CD4 and CD8 T cells) are FACS sorted from the infused product and from the cGvHD patients' peripheral blood before the infusion and at 10 time-points up to 12 months post infusion (Fig. 1A). To monitor TCR repertoire changes over time, we developed a novel quantitative “DeepImmune” technique for TCRβ amplification and sequencing. We amplify all TCRβ chain family transcripts from total RNA using a normalized primer set including molecular identifiers for unbiased and quantitative determination of TCRβ copy numbers. We have normalized the concentrations of our TCRβ primers using synthetic TCRβ gene sequences, thus minimizing amplification biases during RT-PCR to ≤3-fold differences among gene families. The inclusion of molecular identifiers during the RT-PCR step allows us to computationally remove additional biases introduced during the secondary PCR step for Illumina library preparation. Thus, our method results in ultra-deep characterization of immune receptor repertoires for sensitive detection of differences among samples and sample cohorts. Depending on T cell numbers in the samples, we routinely identify 1,000 - 150,000 clonotypes per sample.ResultsAs first quality control for repertoire analysis, we cluster the top 200 TCRβ clonotypes from the different sequenced T cell subsets for each patient, resulting in heatmaps showing prominent repertoire differences among the analyzed T cell subsets (Fig. 1B; infused patient example shown). Next, we track the fate of infused donor Treg cells in the time course samples of the patients after Treg infusion, allowing us to quantify the number and frequency of TCRβ clonotypes that persist or expand in the patients after the infusion. We report that TCRβ clonotypes specific to the infused Treg cells (which were not present in the patients before Treg infusion) can be detected in the patients' blood after the infusion. Infusion-specific Treg clonotypes can reach up to 10-15% of the Treg repertoire of the patients and usually decline over time (Fig. 1C; boxplot shows clonotype overlap between infusion-specific Treg clonotypes and patient Treg clonotypes from five patients of the Lisbon cohort over 12 months). In the Tcon compartment of the patients, infusion-specific Treg clonotypes typically reach only 1-2% of the Tcon repertoire of the patients and also decline over time (Fig. 1D), similarly to what is observed in the CD8 T cell compartment (data not shown). This suggests that infused Tregs are not reverting to Tcons, an important control and promising result. We apply the same type of analysis to track clonotypes that were shared between the infused product and the patient prior to infusion, in addition to those clonotypes that were unique to the patient before infusion and not present in the infused product.ConclusionBy applying our robust and sensitive TCRβ sequencing platform to samples from cGvHD patients infused with donor Treg, we are able to detect unique clonotypes of the infused product up to 1 year post-infusion. It remains to be be determined if there is any correlation between the level of detection of the infused clonotypes and the clinical responses of cGVHD to donor Treg therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Epigenetic Profile of Treg-like Cells Induced By Mesenchymal Stem Cells in Vitro Resembles That of Natural Treg

Regulatory T cells (Treg) are a key population in immune tolerance and their potential use in the treatment of chronic inflammatory diseases has been increasingly investigated. A major hurdle to adoptive Treg therapy in humans is the difficulty in obtaining sufficient numbers of clinical grade Treg.We and others have investigated the potential of mesenchymal stem cells (MSC) to induce and/or expand Treg in vitro. Our goal is to ascertain whether MSC co-culture supports the recruitment and expansion of Treg from a non-Treg starting population, which would allow for clinically relevant numbers to be reached, whilst bypassing the costly isolation of Treg under GMP conditions prior to expansion.We found that co-cultures of human peripheral blood mononuclear cells (PBMC) from healthy donors with allogeneic bone marrow-derived MSC specifically increase the absolute counts and frequency (4 and 6-fold, respectively) of Treg-like cells with a CD4+ CD25high Foxp3+ CD127low phenotype. We further observed that this increase in Treg numbers after MSC co-culture is mainly due to the induction of conventional CD4 T cells (Tcon) to acquire a Treg-like phenotype, rather than to Treg expansion.These data led us to investigate if these Treg-like cells induced by MSC also resemble Treg in terms of suppressive ability and epigenetic profile, which would inform on their functional potential and stability, two key features for their applicability in a clinical setting.We performed suppression assays using FACSorted Treg-like cells after co-culture of purified Tcon with MSC as suppressor cells and autologous fresh Tcon labelled with CFSE as responder cells. We compared the suppressive ability of Treg-like cells to that of Treg co-cultured with MSC, as well as of fresh Treg. We found that induced Treg-like cells isolated from Tcon:MSC co-cultures (P=0.0121), natural Treg isolated from Treg:MSC co-cultures (P=0.0025) and fresh Treg (P=0.0006) all significantly suppress Tcon proliferation (% divided cells at 1:1 Tcon:Treg ratio compared to Tcon alone, Mann Whitney test).We observed significantly higher levels of TGF-β in PBMS:MSC co-culture supernatants compared to that of PBMC cultured alone (P<0.05 on Day 10, P<0.01 on Day 14, two-way ANOVA), indicating a role for TGF-β in the induction of these Treg-like cells. TGF-β-induced Treg have been suggested to be less stable than natural Treg, likely due to epigenetic regulation. In particular, DNA demethylation has been shown to be a key factor in Treg stability. The potential therapeutic application of MSC-induced Treg-like cells would hinge on these cells being epigenetically stable. Hence, we investigated whether MSC-induced Treg-like cells are more similar to the original Tcon that they arose from or to natural Treg in terms of their DNA methylation profile.Most Treg epigenetic studies focus on the Treg-specific demethylated region (TSDR) of the FOXP3 gene and, given that FOXP3 is encoded on the X chromosome, opt to use male donors to bypass artefacts of X-chromosome inactivation. In order to avoid a gender biased analysis, we sought an alternative to TSDR. CAMTA1 (calmodulin-binding transciption activator 1) is a Ca2+-dependent calmodulin-binding transcription factor encoded on chromosome 1q3.6, which has been proposed as a potential molecular marker that distinguishes Treg from Tcon in both male and female donors. We assessed the methylation status of 13 CpG sites within the 470bp CAMTA1 intronic region 3 based on the coding DNA strand in Treg-like cells purified after Tcon:MSC co-cultures, as well as in fresh Treg and Tcon from the same donor. Fresh Tcon cells from one of the donors showed 100% methylation of CpGs 1-13, whereas fresh Treg and Treg-like cells from the same donor showed much lower levels of methylation (33.8% and 0%, respectively), with the highest differences in methylation observed in CpGs 2 and 11 (15% in fresh Treg; 0% in Treg-like cells; 100% in fresh Tcon). These two CpG positions, which have been previously demonstrated to be the ones distinguishing Treg from Tcon, were consistently more demethylated in fresh Treg and Treg-like cells than in fresh Tcon, with an average methylation levels of 5.8% for fresh Treg, 17% for Treg-like cells and 73% for fresh Tcon.Overall, our data demonstrate that MSC induce a subset of Tcon to acquire a Treg-like phenotype, which is accompanied by a suppressive ability and an epigenetic profile that resemble that of natural Treg. DisclosuresNo relevant conflicts of interest to declare.

The next step for cell therapy?

Immunotherapy Considerable advances have been made in engineering T cells for adoptive cell transfer to treat cancer by manipulating the patients' immune system. In a Perspective, Bluestone and Tang discuss the potential for use of regulatory T cells (Tregs) to treat patients with autoimmunity and other diseases that involve pathological inflammation. Preclinical research has provided encouraging results with Treg therapies, and numerous clinical trials have been initiated to test adoptive Treg transfer in patients with, for example, type I diabetes, organ transplant rejection, and amyotrophic lateral sclerosis. Science , this issue p. [154][1] [1]: /lookup/doi/10.1126/science.aau2688

Optimizing regulatory T cells for therapeutic application in human organ transplantation

Initial clinical trials of adoptive regulatory T-cell (Treg) therapy in solid organ transplantation have proven to be both feasible and well tolerated. With Phase 2 trials underway, efforts have been focused on the optimization of the Treg product. With science and our knowledge on the biology of these cells constantly advancing, we have been able to refine our search for a Treg population that would be ideally suited for therapeutic application. This idealized population must be readily isolated, allow for in-vitro expansion, demonstrate potent and specific suppressor function, maintain lineage stability and demonstrate a relevant homing profile. With the advent of innovative cell analysis/isolation techniques and genetic modifications, we are able to choose and design Tregs to fulfil these criteria. By utilizing advances in science and technology, we can optimize Treg therapy in human organ transplantation maximizing their prospects in the arena of transplantation tolerance.

Taking regulatory T-cell therapy one step further.

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. 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. 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.

Adoptive transfer of xenoantigen‑stimulated T cell receptor Vβ‑restricted human regulatory T cells prevents porcine islet xenograft rejection in humanized mice.

Polyclonal expansion of human regulatory T cells (Tregs) prevents xenogeneic rejection by suppressing effector T cell responses in vitro and in vivo. However, a major limitation to using polyclonally expanded Tregs is that they may cause pan-immunosuppressive effects. The present study was conducted to compare the ability of ex vivo expanded human xenoantigen-stimulated Tregs (Xeno-Treg) and polyclonal Tregs (Poly-Treg) to protect islet xenografts from rejection in NOD-SCID interleukin (IL)-2 receptor (IL2r)γ−/− mice. Human cluster of differentiation (CD)4+CD25+CD127lo Tregs, expanded either by stimulating with porcine peripheral blood mononuclear cells (PBMCs) or anti-CD3/CD28 beads, were characterized by immune cell phenotyping, T cell receptor (TCR) Vβ CDR3 spectratyping and performing suppressive activity assays in vitro. The efficiency of adoptively transferred ex vivo human Tregs was evaluated in vivo using neonatal porcine islet cell clusters (NICC) transplanted into NOD-SCID IL-2rγ−/− mice, which received human PBMCs with or without Xeno-Treg or Poly-Treg. Xeno-Treg, which expressed increased levels of human leukocyte antigen-DR and secreted higher levels of IL-10, demonstrated enhanced suppressive capacity in a pig-human mixed lymphocyte reaction. Spectratypes of TCR Vβ4, Vβ10, Vβ18 and Vβ20 in Xeno-Treg showed restriction and expanded clones at sizes of 205, 441, 332 and 196 respectively, compared to those of Poly-Treg. Reconstitution of mice with human PBMCs and Poly-Treg resulted in NICC xenograft rejection at 63 days. Adoptive transfer with human PBMCs and Xeno-Treg prolonged islet xenograft survival beyond 84 days, with grafts containing intact insulin-secreting cells surrounded by a small number of human CD45+ cells. This study demonstrated that adoptive transfer of ex vivo expanded human Xeno-Treg may potently prevent islet xenograft rejection in humanized NOD-SCID IL2rγ−/− mice compared with Poly-Treg. These findings suggested that adoptive Treg therapy may be used for immunomodulation in islet xenotransplantation by minimizing systemic immunosuppression.

CTLA4Ig Improves Murine iTreg Induction via TGFβ and Suppressor Function In Vitro.

Blockade of the CD28:CD80/86 costimulatory pathway has been shown to be potent in blocking T cell activation in vitro and in vivo. The costimulation blocker CTLA4Ig has been approved for the treatment of autoimmune diseases and transplant rejection. The therapeutic application of regulatory T cells (Tregs) has recently gained much attention for its potential of improving allograft survival. However, neither costimulation blockade with CTLA4Ig nor Treg therapy induces robust tolerance on its own. Combining CTLA4Ig with Treg therapy would be an attractive approach for minimizing immunosuppression or for possibly achieving tolerance. However, since the CD28 pathway is more complex than initially thought, the question arose whether blocking CD80/86 would inadvertently impact immunological tolerance by interfering with Treg generation and function. We therefore wanted to investigate the compatibility of CTLA4Ig with regulatory T cells by evaluating direct effects of CTLA4Ig on murine Treg generation and function in vitro. For generation of polyclonal-induced Tregs, we utilized an APC-free in vitro system and added titrated doses of CTLA4Ig at different time points. Phenotypical characterization by flow cytometry and functional characterization in suppressor assays did not reveal negative effects by CTLA4Ig. The costimulation blocker CTLA4Ig does not impair but rather improves murine iTreg generation and suppressor function in vitro.

Co-Stimulatory Modulation of Human Regulatory T cells for Enhanced Immunotherapy

Introduction Regulatory T cells (Treg) are a promising therapy in transplantation. However, current Treg expansion protocols lead to a heterogeneous cell product that contains cells with potentially detrimental pro-inflammatory properties. Ligation of CD27 on T cells with CD70 on APCs promotes T cell function and survival, but impairs Th17 activity. We have previously shown that CD27 expression on human Tregs correlates closely with suppressive potency. In the current study, we hypothesised that modulation of the CD27/CD70 pathway may allow for the generation of a purer population of Tregs with enhanced suppressive properties. Materials and Methods CD4+CD127low/-CD25+ Tregs were flow sorted from PBMCs and expanded in vitro with αCD3/αCD28 stimulation and IL-2. Expanded cells were sorted according to their expression of CD27 and CD70 and further expanded for 14 days. Expanded Treg subsets were examined in the presence or absence of αCD27/αCD70 neutralising antibodies. Phenotype was analysed by flow cytometry. Cytokine production was measured intracellularly and extracellularly by flow cytometry and ELISA. Suppressive function was assessed both in vitro and in vivo in immunodeficient mice. Results and Discussion Human Tregs differentially altered their expression of surface CD27 and CD70 after in vitro expansion, resulting in two distinct subpopulations. Functional in vivo and in vitro assays revealed that suppressive capacity was confined to the CD27+CD70- population. Conversely, CD27-CD70+ Tregs lost their ability to inhibit T cell proliferation. The pre-incubation of total Tregs or CD27-CD70+ Tregs with an αCD70 blocking antibody significantly increased their suppressive potency, suggesting that the CD27/CD70 pathway plays a direct role in Treg activity. Moreover, CD27-CD70+ Tregs, but not CD27+CD70- Tregs, produced high levels of pro-inflammatory cytokines and promoted T cell proliferation in the absence of external co-stimulation.Conclusion Selection on CD27+CD70- after expansion allows for the identification of functionally potent human Tregs. Using these markers eliminates a major component of unstable, proinflammatory cells in the expanded Treg product. This additional selection step may easily be incorporated into current GMP production methods to enhance the efficacy and safety of Treg therapy. In addition, there is an opportunity for the direct modulation of the CD27/CD70 pathway to enhance Treg function.

Tailoring the homing capacity of human Tregs for directed migration to sites of Th1-inflammation or intestinal regions.

Cell-based therapy with CD4+ FOXP3+ regulatory T cells (Tregs) is a promising strategy to limit organ rejection and graft-vs-host disease. Ongoing clinical applications have yet to consider how human Tregs could be modified to direct their migration to specific inflammation sites and/or tissues for more targeted immunosuppression. We show here that stable, homing-receptor-tailored human Tregs can be generated from thymic Tregs isolated from pediatric thymus or adult blood. To direct migration to Th1-inflammatory sites, addition of interferon-γ and IL-12 during Treg expansion produced suppressive, epigenetically stable CXCR3+ TBET+ FOXP3+ T helper (Th)1-Tregs. CXCR3 remained expressed after injection in vivo and Th1-Tregs migrated efficiently towards CXCL10 in vitro. To induce tissue-specific migration, addition of retinoic acid (RA) during Treg expansion induced expression of the gut-homing receptors α4β7-integrin and CCR9. FOXP3+ RA-Tregs had elevated expression of the functional markers latency-associated peptide and glycoprotein A repetitions predominant, increased suppressive capacity in vitro and migrated efficiently to healthy and inflamed intestine after injection into mice. Homing-receptor-tailored Tregs were epigenetically stable even after long-term exposure to inflammatory conditions, suppressive in vivo and characterized by Th1- or gut-homing-specific transcriptomes. Tailoring human thymic Treg homing during in vitro expansion offers a new and clinically applicable approach to improving the potency and specificity of Treg therapy.

Prognostic value of tumor infiltration immune cells in pancreatic cancer

Objective: To investigate the prognostic effect of tumour-infiltrating immune cell, including CD8(+) T cell, regulatory T-cell (Treg) and myeloid-derived suppressor cells (MDSC) on pancreatic patients. Methods: This study retrospectively collected the data of 80 patients who were histologically diagnosed of pancreatic cancer and underwent classical R0 surgical resection at Tianjin Medical University Cancer Institute and Hospital from January 2010 to May 2012. All patients survival were followed up until the cut-off date of January 2015. Clinicopathological features including immunohistochemical staining of FOXP3, CD8 and CD33 were reviewed as indice for evaluating the prognosis of pancreatic patients.The prognostic effect of tumour-infiltrating immune cells were analysed by Kaplan-Meier and Log-rank test. Multiple-factor analysis was conducted with the Cox regression model. The correlation between tumour-infiltrating immune cells and clinicopathological features was analysed by χ(2) test. The C57BL/6 mouse model was used to evaluate the efficacy of Treg and MDSC depletion therapy in vivo. Student's t-test was applied to assess the difference of the tumour volume, Ki-67 positive rate and CD8(+) T-cell infiltration proportion between depletion group and control group. Results: Eventually, 80 patients were included and no patient was lost during the follow-up period. The median follow-up time was 33.2 months (7.4-59.9 months). Patients with high level of tumour-infiltrating CD8(+) T cells had longer overall survival (OS) time ((21.6±11.9)months vs. (13.6±7.4)months, χ(2)=4.647, P = 0.031) than those with low level of tumour-infiltrating CD8(+) T cells. Tumor infiltration FOXP3(+) cells were strongly associated with reduced OS((20.9±8.5)months vs.(13.4±8.8)months, χ(2)=10.528, P=0.001), reduced relapse free survival (RFS) ((15.2±9.0)months vs. (9.5±8.8)months, χ(2)=6.288, P=0.012) and larger tumor size(χ(2)=4.073, r=0.226, P=0.044). The high intratumoural MDSC group showed a significantly shorter OS((23.5±11.8)months vs. (13.8±7.6)months, χ(2)=5.724, P=0.017), RFS((17.9±11.3)months vs. (10.2±7.5)months, χ(2)=7.430, P=0.006) and more advanced N stage (χ(2)=4.714, r=0.243, P=0.030) than the low intratumoural MDSC group. Multivariate Cox analysis revealed that pTNM (P=0.008), tumour-infiltrating Treg density (P=0.009) and intratumoural MDSC density (P=0.034) were independent and negative prognostic factors for OS; pTNM(P=0.003) and tumour-infiltrating MDSC level(P=0.018) were independent and negative factors for RFS. The experiment in vivo revealed that Treg and MDSC depletion therapy significantly decreased tumour volume in the C57BL/6 mouse model of subcutaneous tumours((1 396.3±442.5)mm(3) vs. (3 356.9±533.5)mm(3), t=4.986, P=0.018). Tumour Ki-67 positive rate significantly decreased (23%±5% vs. 55%±10%, t=3.130, P=0.011) in Treg and MDSC depletion group, whereas, the proportion of tumour-infiltrating CD8(+) T cells significantly increased in depletion groups (3.25%±0.69% vs. 0.76%±0.25%, t=3.393, P=0.007). Conclusions: Tumour-infiltrating Treg, MDSC level and pTNM stage are independent prognostic factors for patients with pancreatic cancer. Treg and MDSC depletion therapy can significantly retard tumour growth and increase the level of tumour-infiltrating CD8(+) T-cells in the C57BL/6 mouse model of subcutaneous tumours.

Regulatory T Cells and Kidney Transplantation.

The ability of the immune system to differentiate self from nonself is critical in determining the immune response to antigens expressed on transplanted tissue. Even with conventional immunosuppression, acceptance of the allograft is an active process often determined by the presence of regulatory T cells (Tregs). Tregs classically are CD4+ cells that constitutively express high levels of the IL-2 receptor α chain CD25, along with the transcription factor Foxp3. The use of Tregs in the field of solid organ transplantation is related specifically to the objective of achieving tolerance, with the goal of reducing or eliminating immunosuppressive drugs as well as maintaining tissue repair and managing acute rejection. A key issue in clinical use of Tregs is how to effectively expand the number of Tregs, either through increasing numbers of endogenous Tregs or by the direct infusion of exogenously expanded Tregs. In order to realize the benefits of Treg therapy in solid organ transplantation, a number of outstanding challenges need to be overcome, including assuring an effective expansion of Tregs, improving long-term Treg stability and reduction of risk-related to off-target, nonspecific, immunosuppressive effects related specially to cancer.

Foxp3+ T Regulatory Cells: Still Many Unanswered Questions-A Perspective After 20 Years of Study.

T regulatory (Treg) cells were discovered more than 20 years ago and have remained a topic of intense investigation by immunologists. The initial doubts about their existence were dissipated by the discovery in 2003 of the lineage specific transcription factor Foxp3. In this article, I will discuss some of the questions that I believe still need to be answered before we will be able to fully apply Treg therapy to the clinic. The major issue that remains to be resolved is how they mediate their suppressive functions. In order to correct defective suppression in autoimmune disease (assuming it is a causative factor) or to augment suppression in graft versus host disease or during organ transplantation, we still need to fully understand the biochemical nature of suppressor mechanisms. Similarly, in cancer, it is now widely accepted that reversal of Treg suppression would be highly desirable, yet which of the many purported pathways of suppression are operative in different tumors in different anatomic sites. Many of the concepts we have developed are based on in vitro studies, and it remains unclear if these concepts can readily be applied to Treg function in vivo. Our lack of a specific cell surface marker that readily allows us to identify and target Treg in vivo, particularly in man, remains a major stumbling block. Finally, I will review in some detail controversies regarding the origin of Treg, thymus versus periphery, and attempts to reverse Treg suppression by targeting antigens on their cell surface, particularly members of the TNF receptor superfamily. Hopefully, these areas of controversy will be resolved by in depth studies over the next few years and manipulation of Treg function will be placed on a more solid experimental footing.

A Phase I Clinical Trial with Ex Vivo Expanded Recipient Regulatory T cells in Living Donor Kidney Transplants

There is considerable interest in therapeutic transfer of regulatory T cells (Tregs) for controlling aberrant immune responses. Initial clinical trials have shown the safety of Tregs in hematopoietic stem cell transplant recipients and subjects with juvenile diabetes. Our hypothesis is that infusion(s) of Tregs may induce transplant tolerance thus avoiding long-term use of toxic immunosuppressive agents that cause increased morbidity/mortality. Towards testing our hypothesis, we conducted a phase I dose escalation safety trial infusing billions of ex vivo expanded recipient polyclonal Tregs into living donor kidney transplant recipients. Despite variability in recipient’s renal disease, our expansion protocol produced Tregs which met all release criteria, expressing >98% CD4+CD25+ with <1% CD8+ and CD19+ contamination. Our product displayed >80% FOXP3 expression with stable demethylation in the FOXP3 promoter. Functionally, expanded Tregs potently suppressed allogeneic responses and induced the generation of new Tregs in the recipient’s allo-responders in vitro. Within recipients, expanded Tregs amplified circulating Treg levels in a sustained manner. Clinically, all doses of Treg therapy tested were safe with no adverse infusion related side effects, infections or rejection events up to two years post-transplant. This study provides the necessary safety data to advance Treg cell therapy to phase II efficacy trials.

Development of citrullinated-vimentin-specific CAR for targeting Tregs to treat autoimmune rheumatoid arthritis

Abstract Rheumatoid arthritis (RA) is one of the most common chronic autoimmune diseases that is characterized by an aberrant inflammation of the synovial membrane that causes irreversible joint and bone damage. Regulatory T cells (Tregs) are defective in RA patients while adoptive transfer of expanded autologous Tregs efficiently reversed disease in collagen-induced arthritis (CIA), an animal model of RA. These studies strongly suggest that Treg therapy may be effective in the treatment of RA patients by reducing joint inflammation and inducing immune tolerance. However, clinical treatment with polyclonal Tregs may lead to general immunosuppression. Thus, the development of precise RA Ag-specific Tregs would increase specific activity, promote selective migration to the site of the abnormal inflammation and enhance their function. Thus, we engineered chimeric antigen receptor (CAR)-expressing Tregs specifically targeting an antigen present in the joint of RA patients to induce a localized and effective immunosuppressive response. We developed a single chain Fv directed against a posttranslational modified intermediate filament protein, citrullinated vimentin (CV), an antigen found, almost exclusively, in the extracellular matrix of the synovial tissue of the RA patients and implicated in the pathogenesis of the disease. The scFv was grafted into a functional CAR construct, transduced into human Tregs and shown to react with CV expressed in RA patient synovial fluid and CV-expressing cells. Studies are underway to demonstrate the functional activity of these CAR-expressing Tregs in in vivo mouse models a validation step in the development of a promising therapeutic tool to treat RA and potentially other autoimmune disorders.