Treg Phenotype Research Articles

An essential role for miR-15/16 in Treg suppression and restriction of proliferation

The miR-15/16 family targets a large network of genes in Tcells to restrict their cell cycle, memory formation, and survival. Upon Tcell activation, miR-15/16 are downregulated, allowing rapid expansion of differentiated effector Tcells to mediate a sustained response. Here, we used conditional deletion of miR-15/16 in regulatory Tcells (Tregs) to identify immune functions of the miR-15/16 family in Tcells. miR-15/16 are indispensable to maintain peripheral tolerance by securing efficient suppression by a limited number of Tregs. miR-15/16 deficiency alters expression of critical Treg proteins and results in accumulation of functionally impaired FOXP3loCD25loCD127hi Tregs. Excessive proliferation in the absence of miR-15/16 shifts Treg fate and produces an effector Treg phenotype. These Tregs fail to control immune activation, leading to spontaneous multi-organ inflammation and increased allergic inflammation in a mouse model of asthma. Together, our results demonstrate that miR-15/16 expression in Tregs is essential to maintain immune tolerance.

Analysis of regulatory T lymphocytes in patients with traumatic brain injury

In recent years, traumatic brain injury (TBI) has been one of the most urgent medical and social problems due to its prevalence, predominantly affecting young people of working age, causing high mortality, disability and economic costs for treatment and subsequent rehabilitation of patients. At present, the role of patients’ immune system in evolving neuroinflammation after traumatic brain injury has been proven. Treg cell populations represent an important factor determining the outcome of the disease due to promoting induction of immunological tolerance, being a significant component of immunoregulation. As a result of our study, we found a decrease in the relative content of Treg within the total lymphocyte pool of peripheral blood, which has the CD3+CD4+CD25bright phenotype in patients of the 3rd and 4th groups, in comparison with the data from control group. Moreover, a decreased relative content of Tregs (CD4+CD25+T cells) was revealed which is due to the degree of brain tissue damage and, as a result, their migration to suppress inflammation due to production of anti-inflammatory cytokines (TGF-â, IL-10). The Treg population is heterogeneous, thus prompting us for analysis of the Treg subpopulations profile based on the expression of CD45R0 and CD62L. When assessing subpopulations of regulatory T lymphocytes within CD45Ro and CD62L, significant changes were found only in patients with brain contusion. The changes were revealed within the pool of “naive” T regulatory lymphocytes with the CD3+CD4+CD25brightCD39+ Treg phenotype, capable of producing a wide range of cytokines specific for Th1, Th2, Th17, in patients with mild, moderate and severe TBI. Meanwhile, the level of highly active CD3+CD4+CD25brightCD73+ Tregs was significantly reduced in patients with moderate and severe TBI. These changes indicate an imbalance in immunoregulatory processes resulting from extensive damage to brain tissues.

Attenuated IL-2 muteins leverage the TCR signal to enhance regulatory T cell homeostasis and response in vivo.

Interleukin-2 (IL-2), along with T-cell receptor (TCR) signaling, are required to control regulatory T cell (Treg) homeostasis and function in vivo. Due to the heightened sensitivity to IL-2, Tregs retain the ability to respond to low-dose or attenuated forms of IL-2, as currently being developed for clinical use to treat inflammatory diseases. While attenuated IL-2 increases Treg selectivity, the question remains as to whether a weakened IL-2 signal sufficiently enhances Treg suppressive function(s) toward disease modification. To understand this question, we characterized the in vivo activity and transcriptomic profiles of two different attenuated IL-2 muteins in comparison with wildtype (WT) IL-2. Our study showed that, in addition to favoring Tregs, the attenuated muteins induced disproportionately robust effects on Treg activation and conversion to effector Treg (eTreg) phenotype. Our data furthermore suggested that Tregs activated by attenuated IL-2 muteins showed reduced dependence on TCR signal, at least in part due to the enhanced ability of IL-2 muteins to amplify the TCR signal in vivo. These results point to a new paradigm wherein IL-2 influences Tregs' sensitivity to antigenic signal, and that the combination effect may be leveraged for therapeutic use of attenuated IL-2 muteins.

SARS-CoV-2 spike-specific regulatory T cells (Treg) expand and develop memory in vaccine recipients suggesting a role for immune regulation in preventing severe symptoms in COVID-19

We enrolled healthy subjects that received 2 to 4 injections of mRNA-based vaccination to prevent COVID-19 months to a year from the last vaccine boost, and we found numerous SARS-CoV-2 spike-specific regulatory T cell (Treg) that developed T cell memory as effector memory T cells (TEM) and central memory T cells (TCM). CD4+ CD25high Treg expressed the chemokine receptor CCR6 in a considerable percentage, suggesting T cell homing to the vascular endothelium, lung and gut epithelial cells and brain. Treg phenotype was different than peripherally-induced Treg (pTreg) that revert from pro-inflammatory T cells under repeated stimulatory conditions, suggesting that SARS-CoV-2 spike-specific Treg differentiated from naïve T cells in tissues where the SARS-CoV-2 spike proteins were synthetized. Twenty two of 22 subjects studied responded to vaccination developing a spike-specific CD4+ T helper (Th)1 response, and 20 of 22 developing a spike-specific CD8+ cytotoxic T cells (CTL) response. However, in vaccine recipients the expansion of spike-specific pro-inflammatory T cells was less significant than the expansion of spike-specific Treg. Effector (TEM) and central memory (TCM) Treg were numerous as early as after two vaccine doses, with no significant differences following additional vaccine boosts. In co-culture experiments under stimulatory conditions, Treg regulated naïve T cell differentiation toward a pro-inflammatory phenotype and suppressed interferon (IFN)γ production by SARS-CoV-2-specific CD4 + Th1 cells.

Hypertension-Driven Regulatory T-Cell Perturbations Accelerate Myocardial Ischemia-Reperfusion Injury.

Patients with a history of hypertension have elevated inflammation and a worse prognosis after acute myocardial infarction (AMI). Regulatory T cells (Tregs) are reported to lose their immunosuppressive capacity under pathological conditions. However, whether hypertension leads to Treg dysfunction, thus accelerating myocardial ischemia-reperfusion injury, is still unknown. Studies were performed in hypertensive rats and mice with myocardial ischemia-reperfusion injury. The frequencies and phenotypes of Tregs were analyzed by flow cytometry and immunohistochemistry. Reconstruction Treg experiments were performed to evaluate the effect of Tregs on ischemia-reperfusion injury. Patients with AMI were enrolled to assess circulating Tregs, inflammatory cytokines, and cardiac function. In this study, we found that hypertension leads to proinflammatory Th1 (T helper 1 cell)-like Treg subsets with compromised suppressive capacity. Reconstruction Treg experiments identified that dysfunctional Tregs induced by hypertension play a pathogenic role in the progression of myocardial ischemia-reperfusion injury. In particular, we identified HDAC6 (histone deacetylase 6) as a central regulator in the perturbed Tregs. Clinical studies revealed that the hypertension-induced reduction in circulating Tregs strongly correlated with the higher occurrence rate of microvascular obstruction in AMI patients with hypertension. Our study provided promising clues to explain the poor prognosis of hypertensive AMI patients due to alterations in Tregs. Targeting disturbed Tregs may be a new strategy to treat AMI patients with hypertension.

Development of Beta-Amyloid-Specific CAR-Tregs for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease that remains uncured. Its pathogenesis is characterized by the formation of β-amyloid (Aβ) plaques. The use of antigen-specific regulatory T cells (Tregs) through adoptive transfer has shown promise for the treatment of many inflammatory diseases, although the effectiveness of polyspecific Tregs is limited. Obtaining a sufficient number of antigen-specific Tregs from patients remains challenging. To address this problem, we used an antibody-like single-chain variable fragment from a phage library and subsequently generated a chimeric antigen receptor (CAR) targeting β-amyloid. The β-amyloid-specific CARs obtained were stimulated by both recombinant and membrane-bound Aβ isolated from the murine brain. The generated CAR-Tregs showed a normal Treg phenotype, were antigen-specific activatable, and had suppressive capacity. This study highlights the potential of CAR technology to generate antigen-specific Tregs and presents novel approaches for developing functional CARs.

Regulatory T-cell dysfunctions are associated with increase in tumor necrosis factor α in autoimmune hemolytic anemia and participate in Th17 polarization.

Warm autoimmune hemolytic anemia (wAIHA) is a rare acquired autoimmune disease mediated by antibodies targeting red blood cells. The involvement of CD4 T-helper cells has been scarcely explored, with most findings extrapolated from animal models. Here, we performed quantification of both effector T lymphocytes (Teff) and regulatory T cells (Treg), associated with functional and transcriptomic analyses of Treg in human wAIHA. We observed a shift of Teff toward a Th17 polarization concordant with an increase in serum interleukin-17 concentration that correlates with red blood cell destruction parameters, namely lactate dehydrogenase and bilirubin levels. A decrease in circulating Treg, notably effector Treg, associated with a functional deficiency, as represented by their decrease capability to inhibit Teff proliferation, were also observed. Treg deficiency was associated with a reduced expression of Foxp3, the master transcription factor known to maintain the Treg phenotype stability and suppressive functions. Transcriptomic profiling of Treg revealed activation of the tumor necrosis facto (TNF)-α pathway, which was linked to increased serum TNF-α concentrations that were twice as high as in controls. Treg transcriptomic profiling also suggested that post-translational mechanisms possibly accounted for Foxp3 downregulation and Treg dysfunctions. Since TNF-α participates in the rupture of immune tolerance during wAIHA, its inhibition could be of interest. To this end, the effects of fostamatinib, a SYK inhibitor, were investigated in vitro, and we showed that besides the inhibition of erythrocyte phagocytosis by monocytes, fostamatinib is also able to dampen TNF-α production, thus appearing as a promising multitargeting therapy in wAIHA (clinicaltrials gov. Identifier: NCT02158195).

IL-35 Stabilizes Treg Phenotype to Protect Cardiac Allografts in Mice.

Interleukin-35 (IL-35), secreted by regulatory T cells (Treg) and B cells, is immunosuppressive under both physiological and pathological conditions. However, the role of IL-35 in all responses has yet to be investigated. Here, we demonstrate that IL-35 protects allografts by stabilizing the Treg phenotype and suppressing CD8 + T-cell activation in a mouse heart transplantation model. The effect of IL-35 on immune cell infiltration in grafts and secondary lymphoid organs was examined using mass cytometry, flow cytometry, and immunofluorescence. Moreover, using quantitative real-time polymerase chain reaction, flow cytometry, and phospho-flow assays, we demonstrated that IL-35 maintains Treg phenotypes to restrain CD8 + T cells via the gp130/signal transducer and activator of transcription 1 pathway. Mass cytometry analysis of intragraft immune cells showed that IL-35 decreased CD8 + T-cell infiltration and increased Foxp3 and IL-35 expressions in Treg. In vitro, we demonstrated that IL-35 directly promoted Treg phenotypic and functional stability and its IL-35 secretion, generating a positive feedback loop. However, Treg are required for IL-35 to exert its suppressive effect on CD8 + T cells in vitro. After depleting Treg in the recipient, IL-35 did not prolong graft survival or decrease CD8 + T-cell infiltration. Mechanistically, we found that IL-35 sustained Treg stability via the gp130/signal transducer and activator of transcription 1 signaling pathway. Our findings highlight that IL-35 stabilizes the Treg phenotype to ameliorate CD8 + T-cell infiltration in the allograft, which has never been described in the transplanted immunological milieu.

Purine metabolic pathway regulates regulatory T cell stability and function

Abstract Regulatory T cell (Treg) lymphatic migration and maintenance of transcription factor Foxp3 expression are required for suppressor function and allograft protection, and migrated Tregs express ectonucleotidase CD39 hi. Purine metabolism is implicated in Treg phenotype but the precise functions of purine metabolites on Treg function and stability have remained unclear. Sorted Foxp3+ Tregs were used to test Treg stability, migration, and cellular viability in a transendothelial migration (TEM) in vitro model. Intracellular metabolome of Tregs was analyzed by mass spectrometry, and differentially expressed metabolites between Tregs and exTregs were identified and tested. Foxp3 hiTregs displayed higher suppressive function and migration across LECs compared to Foxp3 loexTregs, but cellular viability was similar. Tregs showed differential metabolic profiles compared to exTregs, and the top 16 most differentially expressed metabolites involved the tricarboxylic acid cycle, polyamines, and purine metabolism. Ten metabolites including nicotinamide and inosine monophosphate (IMP) were downregulated, while six metabolites including putrescine, cadaverine, and N-acetylglycine were upregulated in Tregs compared to exTregs. A purine ectonucleotidase CD73 inhibitor decreased Treg conversion to exTregs and adenosine increased Treg migration and suppressive function concomitant with decreased exTreg conversion. Polyamine metabolites showed only subtle effects on exTreg conversion and migration. These results suggest that purine metabolism is a potent regulator that maintains Treg Foxp3 expression and suppressive function during Treg TEM and thus, could be a promising target for therapeutic interventions. NIH grant RO1 A1062765

Identifiation of novel effector regulatory T cells through simulaneous single cell epigenetic and transcriptomic profiling

Abstract Regulatory T cells (Tregs) harness a central role in peripheral immune tolerance. Since its discovery, there have been numerous efforts to dissect the role of Tregs in the context of disease condition. Recently, Treg fields are moving from the academia to industry since many evidences have been accumulated for the potential of treatment of inflammatory disease by administering genetically engineered Tregs. However, in the pre-clinical or clinical trials, this rare cell subset needs to be expanded in ex-vivo condition to achieve a sufficient number for the controlling of the disease. Our group previously established ex-vivo Treg expansion condition with 25 mer oligonucleotide to maintain the stability and function of Tregs. However, non-biased investigations on the perturbation of transcriptomic and epigenetic changes during the culture condition has not been explored. Since cutting edge multiomics tools which can simultaneously profile epigenetic and transcript signatures in a single cell resolution has been commercialized, we adopt 10x multiome technique to investigate our frozen ex-vivo expanded Tregs(eTreg) compared to freshly isolated frozen human Treg(fTreg) from the same donor. Compared to fTreg cells, we have identified a new subset that exists only in the eTreg cells and named it eeTreg. We found that the eeTreg resembles the phenotype of both effector Tregs and tumor-infiltrated Tregs. eeTreg is characterized by a significantly increased expression and chromatin accessibility score of FOXP3, STAT5B, ETS1 and RUNX1, which are known to be the essential transcription factors of regulatory T cell function. In summary, we suggest a novel human treg subset which might be manufactured by manipulating ex-vivo culture condition.

Investigating the interactions between regulatory T cells and cancer-associated fibroblasts and their role in lung cancer progression

Abstract The tumor microenvironment (TME) is a highly complex multicellular system and has been shown to play a fundamental role in dictating tumorigenesis. High expression of regulatory surface molecules, cytokines, and chemokines in fibroblast niches within lung tumor stroma, paired with their colocalization with tumor infiltrating lymphocytes (TILs), suggests that cancer-associated fibroblast (CAF) niches may alter the functional response of TILs and influence the anti-tumor immune response. Here, utilizing a transcriptomic approach, we have uncovered a distinct CAF population, which we term “iCAFs,” that is enriched for immunomodulatory gene signatures, T cell ligands, and chemoattractants. This population expresses a unique set of cell surface molecules that permit their identification and isolation from lung tumor tissue. Given the demonstrated importance of Tregs in suppressing anti-tumor immunity, we chose to evaluate how Tregs may be interacting with iCAFs within the TME of lung adenocarcinoma. We uncovered significant colocalization patterns of Foxp3 +Tregs with iCAF—a pattern which was found to be unique for Tregs when compared to conventional CD4 +Foxp3 −T cells. Moreover, the Treg to conventional CD4 +T cell ratio is significantly decreased in tumor tissue relative to normal adjacent tissue and healthy controls. Our analyses demonstrate that Tregs preferentially associate with iCAF populations within the tumor, leading us to hypothesize that interactions between Tregs and iCAFs may, in turn, influence Treg trafficking, phenotype and function within the TME. T32AI106711

Expanded T regulatory cells fail to control germinal center responses in the lung lymph nodes of pulmonary fibrosis patients

Abstract Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung diseases (ILD), which is characterized by progressive scarring of the lung. While immune suppression is a viable therapeutic option for many ILDs, it is detrimental in IPF. Nevertheless, enlargement of lung-draining lymph nodes (LLN) is prevalent and predicts survival outcomes in all ILD patients, including IPF, suggesting that there is also an immune component to IPF pathogenesis. To test this hypothesis, we investigated the cellular composition of the LLN in IPF and non-IPF ILD patients. All ILD patients’ LLN had increased germinal centers (GC), implicating that immune responses are not being resolved. As T regulatory cells (Tregs) are key in the control and resolution of immune responses, we investigated the phenotype of Tregs in the LLN of both IPF and non-IPF ILD patients to reveal their role in lung fibrosis. Tregs were expanded in LLN of all ILD patients compared to controls. Interestingly, there was a predominance of GATA3-expressing Tregs in ILD patients’ LLN. However, Tregs of ILD patients expressed significantly elevated levels of ICOS and CD137 compared to controls, indicating recent activation. Surprisingly, CD25 expression was significantly lower on Tregs from ILD patients’ LLN. Sc-RNAseq confirms similar Treg phenotypes in the lungs of IPF patients. In a mouse model of bleomycin-induced pulmonary fibrosis, Tregs expand and display a similar activated phenotype. Preliminary experiments suggest that GATA3 expression in Tregs is detrimental in this model. Together, these data indicate that there are unresolved GC responses in the LLN of all ILD patients, including IPF, and suggest that Tregs are ineffectual at controlling the GC response. American Heart Association Postdoctoral Fellowship, Award number (835938)

DOCK11 deficiency in patients with X-linked actinopathy and autoimmunity

AbstractDedicator of cytokinesis (DOCK) proteins play a central role in actin cytoskeleton regulation. This is highlighted by the DOCK2 and DOCK8 deficiencies leading to actinopathies and immune deficiencies. DOCK8 and DOCK11 activate CDC42, a Rho–guanosine triphosphate hydrolases involved in actin cytoskeleton dynamics, among many cellular functions. The role of DOCK11 in human immune disease has been long suspected but, to the best of our knowledge, has never been described to date. We studied 8 male patients, from 7 unrelated families, with hemizygous DOCK11 missense variants leading to reduced DOCK11 expression. The patients were presenting with early-onset autoimmunity, including cytopenia, systemic lupus erythematosus, skin, and digestive manifestations. Patients' platelets exhibited abnormal ultrastructural morphology and spreading as well as impaired CDC42 activity. In vitro activated T cells and B-lymphoblastoid cell lines from patients exhibited aberrant protrusions and abnormal migration speed in confined channels concomitant with altered actin polymerization during migration. Knock down of DOCK11 recapitulated these abnormal cellular phenotypes in monocytes-derived dendritic cells and primary activated T cells from healthy controls. Lastly, in line with the patients’ autoimmune manifestations, we also observed abnormal regulatory T-cell (Treg) phenotype with profoundly reduced FOXP3 and IKZF2 expression. Moreover, we found reduced T-cell proliferation and impaired STAT5B phosphorylation upon interleukin-2 stimulation of the patients’ lymphocytes. In conclusion, DOCK11 deficiency is a new X-linked immune-related actinopathy leading to impaired CDC42 activity and STAT5 activation, and is associated with abnormal actin cytoskeleton remodeling as well as Treg phenotype, culminating in immune dysregulation and severe early-onset autoimmunity.

Treg cells require Izumo1R to regulate γδT cell-driven inflammation in the skin.

Izumo1R is a pseudo-folate receptor with an essential role in mediating tight oocyte/spermatozoa contacts during fertilization. Intriguingly, it is also expressed in CD4+ T lymphocytes, in particular Treg cells under the control of Foxp3. To understand Izumo1R function in Treg cells, we analyzed mice with Treg-specific Izumo1r deficiency (Iz1rTrKO). Treg differentiation and homeostasis were largely normal, with no overt autoimmunity and only marginal increases in PD1+ and CD44hi Treg phenotypes. pTreg differentiation was also unaffected. Iz1rTrKO mice proved uniquely susceptible to imiquimod-induced, γδT cell-dependent, skin disease, contrasting with normal responses to several inflammatory or tumor challenges, including other models of skin inflammation. Analysis of Iz1rTrKO skin revealed a subclinical inflammation that presaged IMQ-induced changes, with an imbalance of Rorγ+ γδTcells. Immunostaining of normal mouse skin revealed the expression of Izumo1, the ligand for Izumo1R, electively in dermal γδTcells. We propose that Izumo1R on Tregs enables tight contacts with γδTcells, thereby controlling a particular path of skin inflammation.

Impaired response of memory Treg to high density lipoproteins is associated with intermediate/high cardiovascular disease risk in persons with HIV.

Cardiovascular disease (CVD) is a leading cause of enhanced morbidity and mortality in persons with HIV (PWH) in the era of highly active antiretroviral therapy (AART). However, the underlying mechanisms are not fully understood. Regulatory T cells (Treg), notably the highly suppressive memory subset, have been shown to limit CVD. Importantly, memory Treg cell numbers remain low in many treated PWH. High density lipoproteins (HDL) also protect from CVD, and we previously found that Treg-HDL interactions reduce oxidative stress in these cells. Here, we evaluated Treg-HDL interactions in PWH and whether they were operative in those higher CVD risk. To do that, we recruited a cohort of PWH with intermediate/high CVD risk (median ASCVD risk score of 13.2%, n=15) or low/borderline risk (median ASCVD risk score of 3.6%, n=14), as well as a group of statins treated PWH with intermediate/high CVD risk (median ASCVD risk score of 12.7%, n=14). We evaluated Treg frequency, phenotype and response to HDL. PWH with Int/High CVD risk had a significantly lower number of memory Treg, but memory Treg were more activated and displayed an inflammatory phenotype, versus those with Low/BL CVD risk. In untreated patients, Treg absolute numbers were negatively correlated with ASCVD score. Although HDL decreased oxidative stress in memory Treg in all subjects, memory Treg from PWH with Int/High CVD risk were significantly less responsive to HDL than those from PWH with Low/BL CVD risk. The level of oxidative stress in memory Treg positively correlated with ASCVD scores. In contrast, plasma HDL from PWH, regardless of CVD risk, retained their anti-oxidative properties, suggesting that the defect in memory Treg response to HDL is intrinsic. Statin treatment partially ameliorated the memory Treg defect. In conclusion, the defective HDL-Treg interactions may contribute to the inflammation-induced increased CVD risk observed in many AART-treated PWH.

Human small intestine contains 2 functionally distinct regulatory T-cell subsets

Regulatory T (Treg) CD4 cells in mouse gut are mainly specific for intestinal antigens and play an important role in the suppression of immune responses against harmless dietary antigens and members of the microbiota. However, information about the phenotype and function of Treg cells in the human gut is limited. We performed a detailed characterization of Foxp3+ CD4 Treg cells in human normal small intestine (SI) as well as from transplanted duodenum and celiac disease lesions. Treg cells and conventional CD4 T cells derived from SI were subjected to extensive immunophenotyping and their suppressive activity and ability to produce cytokines assessed. SI Foxp3+ CD4 T cells were CD45RA-CD127-CTLA-4+ and suppressed proliferation of autologous T cells. Approximately 60% of Treg cells expressed the transcription factor Helios. When stimulated, Helios-negative Treg cells produced IL-17, IFN-γ, and IL-10, whereas Helios-positive Treg cells produced very low levels of these cytokines. By sampling mucosal tissue from transplanted human duodenum, we demonstrated that donor Helios-negative Treg cells persisted for at least 1 year after transplantation. In normal SI, Foxp3+ Treg cells constituted only 2% of all CD4 T cells, while in active celiac disease, both Helios-negative and Helios-positive subsets expanded 5- to 10-fold. The SI contains 2 subsets of Treg cells with different phenotypes and functional capacities. Both subsets are scarce in healthy gut but increase dramatically in active celiac disease.

Highlights from Recent Cancer Literature.

Highlights from Recent Cancer Literature.

Treg Therapy for the Induction of Immune Tolerance in Transplantation-Not Lost in Translation?

The clinical success of solid organ transplantation is still limited by the insufficiency of immunosuppressive regimens to control chronic rejection and late graft loss. Moreover, serious side effects caused by chronic immunosuppressive treatment increase morbidity and mortality in transplant patients. Regulatory T cells (Tregs) have proven to be efficient in the induction of allograft tolerance and prolongation of graft survival in numerous preclinical models, and treatment has now moved to the clinics. The results of the first Treg-based clinical trials seem promising, proving the feasibility and safety of Treg therapy in clinical organ transplantation. However, many questions regarding Treg phenotype, optimum dosage, antigen-specificity, adjunct immunosuppressants and efficacy remain open. This review summarizes the results of the first Treg-based clinical trials for tolerance induction in solid organ transplantation and recapitulates what we have learnt so far and which questions need to be resolved before Treg therapy can become part of daily clinical practice. In addition, we discuss new strategies being developed for induction of donor-specific tolerance in solid organ transplantation with the clinical aims of prolonged graft survival and minimization of immunosuppression.

Regulatory T cell profiles in patients with N-methyl-ᴅ-aspartate receptor-antibody encephalitis.

Purpose Regulatory T cells (Tregs) have been implicated in the pathogenesis of several autoimmune disorders and used in adoptive cell transfer therapies. Neither have been explored in patients with autoimmune encephalitis where treated patient outcomes remain suboptimal with frequent relapses. Here, to identify new treatment strategies for autoimmune encephalitis, we sought to evaluate the proportion of circulating Tregs and Treg subpopulations in peripheral blood of patients with N-methyl-ᴅ-aspartate receptor-antibody encephalitis (NMDAR-Ab-E) and compared this with healthy controls. We compared the phenotype of peripheral blood Tregs in four adult NMDAR-Ab-E patients and four age- and sex-matched healthy controls using an 11-color flow cytometry assay panel for characterization of Tregs (CD4+ CD25+ FoxP3+) cells into naïve (chemokine receptor [CCR] 7+ CD45RA+), central memory (CCR7+ CD45RA-), and effector memory (CCR7- CD45RA-) cells. We also examined and compared the expression of the CCR6 by circulating Tregs and the respective Treg subpopulations between the study groups. The proportion of circulating Tregs was similar between patients with NMDAR-Ab-E and healthy controls but the proportion of naïve Tregs was lower in NMDAR-Ab-E patients (p = 0.0026). Additionally, the frequency of circulating effector memory Tregs was higher, and the proportion of circulating effector memory Tregs expressing CCR6 was lower, in NMDAR-Ab-E patients compared with healthy controls (p = 0.0026). Altered Treg homeostasis may be a feature of patients with NMDAR-Ab-E. Future studies with larger samples are warranted to validate these findings.

WITHDRAWN: Interleukin-35 dampens T helper 22 phenotype shift in CD4+CD25+CD127dim/- regulatory T cells in primary biliary cholangitis

The phenotype shift in regulatory T cells (Tregs) contributes to immunopathogenesis of autoimmune diseases. The current study was aimed to investigate the regulatory function of interleukin-35 (IL-35) to T helper 22 (Th22) cell phenotype shift in Tregs in primary biliary cholangitis (PBC). Fifty-five PBC patients and twenty-four controls were enrolled. CD4+CD25+CD127dim/- Tregs and Th22 cells were investigated by flow cytometry. Forkhead box P3 (FoxP3) and aryl hydrocarbon receptor (AhR) mRNA levels were assessed by real-time polymerase chain reaction. Plasma IL-10 and IL-22 levels were measured by ELISA. Purified Tregs were stimulated with exogenous IL-35, and were co-cultured with autologous CD4+CD25- T cells. Cellular proliferation and cytokine production was measured. Purified Tregs were also cultured into Th22 condition in the presence or absence of exogenous IL-35, and Th22 phenotype were assessed. PBC patients had lower levels of Treg percentage, FoxP3 mRNA, and plasma IL-10, while had higher levels of Th22 proportion, AhR mRNA, and plasma IL-22. Tregs from PBC patients showed reduced immunosuppressive activity, which presented as increased cellular proliferation, interferon-γ production and decreased IL-35/IL-10 secretion in co-culture system. Tregs shifted into Th22 phenotype in PBC patients with elevated CCR4, CCR6, and CCR10 expression as well as increased IL-22 production. IL-35 not only enhanced inhibitory function of Tregs but also suppressed phenotype shift of Tregs into Th22 phenotype in PBC patients. This process was accompanied by elevation of IL-10 and transforming growth factor-β1 secretion by Tregs from PBC patients. The present data suggested that reduced IL-35 might be insufficient to maintain Tregs function and phenotype shift from Tregs into Th22 phenotype in PBC patients.