Human retinoic acid-regulated CD161+ regulatory T cells support wound repair in intestinal mucosa.

Repair of tissue damaged during inflammatory processes is key to the return of local homeostasis and restoration of epithelial integrity. Here we describe CD161+ regulatory T (Treg) cells as a distinct, highly suppressive population of Treg cells that mediate wound healing. These Treg cells were enriched in intestinal lamina propria, particularly in Crohn's disease. CD161+ Treg cells had an all-trans retinoic acid (ATRA)-regulated gene signature, and CD161 expression on Treg cells was induced by ATRA, which directly regulated the CD161 gene. CD161 was co-stimulatory, and ligation with the T cell antigen receptor induced cytokines that accelerated the wound healing of intestinal epithelial cells. We identified a transcription-factor network, including BACH2, RORγt, FOSL2, AP-1 and RUNX1, that controlled expression of the wound-healing program, and found a CD161+ Treg cell signature in Crohn's disease mucosa associated with reduced inflammation. These findings identify CD161+ Treg cells as a population involved in controlling the balance between inflammation and epithelial barrier healing in the gut.

PD-L1 signaling on human memory CD4+ T cells induces a regulatory phenotype.

Programmed cell death protein 1 (PD-1) is expressed on T cells upon T cell receptor (TCR) stimulation. PD-1 ligand 1 (PD-L1) is expressed in most tumor environments, and its binding to PD-1 on T cells drives them to apoptosis or into a regulatory phenotype. The fact that PD-L1 itself is also expressed on T cells upon activation has been largely neglected. Here, we demonstrate that PD-L1 ligation on human CD25-depleted CD4+ T cells, combined with CD3/TCR stimulation, induces their conversion into highly suppressive T cells. Furthermore, this effect was most prominent in memory (CD45RA-CD45RO+) T cells. PD-L1 engagement on T cells resulted in reduced ERK phosphorylation and decreased AKT/mTOR/S6 signaling. Importantly, T cells from rheumatoid arthritis patients exhibited high basal levels of phosphorylated ERK and following PD-L1 cross-linking both ERK signaling and the AKT/mTOR/S6 pathway failed to be down modulated, making them refractory to the acquisition of a regulatory phenotype. Altogether, our results suggest that PD-L1 signaling on memory T cells could play an important role in resolving inflammatory responses; maintaining a tolerogenic environment and its failure could contribute to ongoing autoimmunity.

The Role of Regulatory T Cells and Their Therapeutic Potential in Hypertensive Disease of Pregnancy: A Literature Review.

Hypertensive disorders of pregnancy (HDP), including preeclampsia (PE) and gestational hypertension (GH), are major causes of maternal and foetal morbidity and mortality. This review elucidates the role of regulatory T cells (Tregs) in the immunological aspects of HDP and explores their therapeutic potential. Tregs, which play a critical role in maintaining immune homeostasis, are crucial in pregnancy to prevent immune-mediated rejection of the foetus. The review highlights that Tregs contribute to immunological adaptation in normal pregnancy, ensuring foetal acceptance. In contrast, HDP is associated with Treg dysfunction, which is marked by decreased numbers and impaired regulatory capacity, leading to inadequate immune tolerance and abnormal placental development. This dysfunction is particularly evident in PE, in which Tregs fail to adequately modulate the maternal immune response against foetal antigens, contributing to the pathophysiology of the disorder. Therapeutic interventions aiming to modulate Treg activity represent a promising avenue for HDP management. Studies in animal models and limited clinical trials suggest that enhancing Treg functionality could mitigate HDP symptoms and improve pregnancy outcomes. However, given the multifactorial nature of HDP and the intricate regulatory mechanisms of Tregs, the review explores the complexities of translating in vitro and animal model findings into effective clinical therapies. In conclusion, while the precise role of Tregs in HDP is still being unravelled, their central role in immune regulation during pregnancy is indisputable. Further research is needed to fully understand the mechanisms by which Tregs contribute to HDP and to develop targeted therapies that can safely and effectively harness their regulatory potential for treating hypertensive diseases of pregnancy.

OMIP-090: A 20-parameter flow cytometry panel for rapid analysis of cell diversity and homing capacity in human conventional and regulatory T cells.

The panel was developed and optimized for monitoring changes in homing capacity and functional diversity of human CD4+ conventional and regulatory T cell subsets. The analysis was based on expression of only surface markers in freshly isolated peripheral blood mononuclear cells (PBMCs) to reduce at minimum any alteration due to permeabilization or freezing/thawing procedures. We included markers to assess the distribution of naïve and memory populations based on the expression of CD45RA, CCR7, CD25, CD28 and CD95 in both conventional and regulatory T cells. The identification of major functional subsets was performed using CCR4, CCR6, CCR10, CXCR3 and CXCR5. Homing capacity of these subsets to skin, airway tract, gut and inflammatory lesions could finally be assessed with the markers CLA, CCR3, CCR5 and integrin ß7. The panel was tested on freshly isolated PBMCs from healthy donors and patients with allergic rhinitis or autoimmune disorders.

Treg cell therapy: How cell heterogeneity can make the difference.

CD4+ CD25high CD127low/- FOXP3+ T regulatory cells are responsible for maintaining immune tolerance and controlling excessive immune responses. Treg cell use in pre-clinical animal models showed the huge therapeutic potential of these cells in immune-mediated diseases and laid the foundations for their applications in therapy in humans. Currently, there are several clinical trials utilizing the adoptive transfer of Treg cells to reduce the morbidity in autoimmune disorders, allogeneic HSC transplantation, and solid organ transplantation. However, a large part of them utilizes total Treg cells without distinction of their biological variability. Many studies on the heterogeneity of Treg cell population revealed distinct subsets with different functions in the control of the immune response and induction of peripheral tolerance. Some of these subsets also showed a role in controlling the general homeostasis of non-lymphoid tissues. All these Treg cell subsets and their peculiar properties can be therefore exploited to develop novel therapeutic approaches. This review describes these functionally distinct subsets, their phenotype, homing properties and functions in lymphoid and non-lymphoid tissues. In addition, we also discuss the limitations in using Treg cells as a cellular therapy and the strategies to enhance their efficacy.

Isolation and expansion of thymus-derived regulatory T cells for use in pediatric heart transplant patients.

Regulatory T-cells (Tregs) are a subset of T cells generated in the thymus with intrinsic immunosuppressive properties. Phase I clinical trials have shown safety and feasibility of Treg infusion to promote immune tolerance and new studies are ongoing to evaluate their efficacy. During heart transplantation, thymic tissue is routinely discarded providing an attractive source of Tregs. In this study, we developed a GMP-compatible protocol for expanding sorted thymus-derived CD3+ CD4+ CD25+ CD127- (Tregs) as well as CD3+ CD4+ CD25+ CD127- CD45RA+ (RA+ Tregs) cells. We aimed to understand whether thymic RA+ Tregs can be isolated and expanded offering an advantage in terms of stability as it has been previously shown for circulating adult CD45RA+ Tregs. We show that both Tregs and RA+ Tregs could be expanded in large numbers and the presence of rapamycin is essential to inhibit the growth of IFN-γ producing cells. High levels of FOXP3, CTLA4, and CD25 expression, demethylation of the FOXP3 promoter, and high suppressive ability were found with no differences between Tregs and RA+ Tregs. After freezing and thawing, all Treg preparations maintained their suppressive ability, stability, as well as CD25 and FOXP3 expression. The number of thymic Tregs that could be isolated with our protocol, their fold expansion, and functional characteristics allow the clinical application of this cell population to promote tolerance in pediatric heart transplant patients.

Chimeric antigen receptor-modified human regulatory T cells that constitutively express IL-10 maintain their phenotype and are potently suppressive.

Clinical trials of Treg therapy in transplantation are currently entering phases IIa and IIb, with the majority of these employing polyclonal Treg populations that harbor a broad specificity. Enhancing Treg specificity is possible with the use of chimeric antigen receptors (CARs), which can be customized to respond to a specific human leukocyte antigen (HLA). In this study, we build on our previous work in the development of HLA-A2 CAR-Tregs by further equipping cells with the constitutive expression of interleukin 10 (IL-10) and an imaging reporter as additional payloads. Cells were engineered to express combinations of these domains and assessed for phenotype and function. Cells expressing the full construct maintained a stable phenotype after transduction, were specifically activated by HLA-A2, and suppressed alloresponses potently. The addition of IL-10 provided an additional advantage to suppressive capacity. This study therefore provides an important proof-of-principle for this cell engineering approach for next-generation Treg therapy in transplantation.

Feasibility, long-term safety, and immune monitoring of regulatory T cell therapy in living donor kidney transplant recipients.

Short-term outcomes in kidney transplantation are marred by progressive transplant failure and mortality secondary to immunosuppression toxicity. Immune modulation with autologous polyclonal regulatory T cell (Treg) therapy may facilitate immunosuppression reduction promoting better long-term clinical outcomes. In a Phase I clinical trial, 12 kidney transplant recipients received 1-10 × 106 Treg per kg at Day +5 posttransplantation in lieu of induction immunosuppression (Treg Therapy cohort). Nineteen patients received standard immunosuppression (Reference cohort). Primary outcomes were rejection-free and patient survival. Patient and transplant survival was 100%; acute rejection-free survival was 100% in the Treg Therapy versus 78.9% in the reference cohort at 48 months posttransplant. Treg therapy revealed no excess safety concerns. Four patients in the Treg Therapy cohort had mycophenolate mofetil withdrawn successfully and remain on tacrolimus monotherapy. Treg infusion resulted in a long-lasting dose-dependent increase in peripheral blood Tregs together with an increase in marginal zone B cell numbers. We identified a pretransplantation immune phenotype suggesting a high risk of unsuccessful ex-vivo Treg expansion. Autologous Treg therapy is feasible, safe, and is potentially associated with a lower rejection rate than standard immunosuppression. Treg therapy may provide an exciting opportunity to minimize immunosuppression therapy and improve long-term outcomes.

Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials.

BACKGROUND: Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. METHODS: The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered with ClinicalTrials.gov, NCT01656135, NCT02252055, NCT02085629, NCT02244801, NCT02371434, NCT02129881, and NCT02091232. FINDINGS: The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2-18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. INTERPRETATION: Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression. FUNDING: The 7th EU Framework Programme.

Correction of Defective T-Regulatory Cells From Patients With Crohn's Disease by Ex Vivo Ligation of Retinoic Acid Receptor-α.

BACKGROUND & AIMS: Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by ex vivo expansion in the presence of factors that activate their regulatory activities. METHODS: We measured levels of the integrin α4ß7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded ex vivo and incubated with rapamycin with or without agonists of the retinoic acid receptor-α (RARA), and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. RESULTS: We found that Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. The pathway that regulates the expression of integrin subunit α is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4ß7, as well as CD62L and FOXP3, compared with cells incubated with rapamycin or rapamycin and all-trans retinoic acid. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines, and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labeled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. CONCLUSIONS: Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. Incubation of patients' ex vivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4ß7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. ClinicalTrials.gov, Number: NCT03185000.

Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn's disease.

BACKGROUND AND AIM: Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohn's blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown. METHODS: To define the optimum population for Treg cell therapy in CD, CD4(+)CD25(+)CD127(lo)CD45RA(+) and CD4(+)CD25(+)CD127(lo)CD45RA(-) Treg subsets were isolated from patients' blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. RESULTS: Tregs can be expanded from the blood of patients with CD to potential target dose within 22-24 days. Expanded CD45RA(+) Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA(-) Tregs. CD45RA(+) Tregs highly express α4ß7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA(+) Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA(+) Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa. CONCLUSIONS: CD4(+)CD25(+)CD127(lo)CD45RA(+) Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials.

Impact of immunosuppressive drugs on the therapeutic efficacy of ex vivo expanded human regulatory T cells.

Immunosuppressive drugs in clinical transplantation are necessary to inhibit the immune response to donor antigens. Although they are effective in controlling acute rejection, they do not prevent long-term transplant loss from chronic rejection. In addition, immunosuppressive drugs have adverse side effects, including increased rate of infections and malignancies. Adoptive cell therapy with human Tregs represents a promising strategy for the induction of transplantation tolerance. Phase I/II clinical trials in transplanted patients are already underway, involving the infusion of Tregs alongside concurrent immunosuppressive drugs. However, it remains to be determined whether the presence of immunosuppressive drugs negatively impacts Treg function and stability. We tested in vitro and in vivo the effects of tacrolimus, mycophenolate and methylprednisolone (major ISDs used in transplantation) on ex vivo expanded, rapamycin-treated human Tregs. The in vitro results showed that these drugs had no effect on phenotype, function and stability of Tregs, although tacrolimus affected the expression of chemokine receptors and IL-10 production. However, viability and proliferative capacity were reduced in a dose-dependent manner by all the three drugs. The in vivo experiments using a humanized mouse model confirmed the in vitro results. However, treatment of mice with only rapamycin maintained the viability, function and proliferative ability of adoptively transferred Tregs. Taken together, our results suggest that the key functions of ex vivo expanded Tregs are not affected by a concurrent immunosuppressive therapy. However, the choice of the drug combination and their timing and dosing should be considered as an essential component to induce and maintain tolerance by Treg.

CD161 expression characterizes a subpopulation of human regulatory T cells that produces IL-17 in a STAT3-dependent manner.

Treg cells are critical for the prevention of autoimmune diseases and are thus prime candidates for cell-based clinical therapy. However, human Treg cells are "plastic", and are able to produce IL-17 under inflammatory conditions. Here, we identify and characterize the human Treg subpopulation that can be induced to produce IL-17 and identify its mechanisms. We confirm that a subpopulation of human Treg cells produces IL-17 in vitro when activated in the presence of IL-1ß, but not IL-6. "IL-17 potential" is restricted to population III (CD4(+) CD25(hi) CD127(lo) CD45RA(-) ) Treg cells expressing the natural killer cell marker CD161. We show that these cells are functionally as suppressive and have similar phenotypic/molecular characteristics to other subpopulations of Treg cells and retain their suppressive function following IL-17 induction. Importantly, we find that IL-17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL-17. Finally, we show that CD161(+) population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL-17-producing Treg-cell population at these sites. As IL-17 production from this Treg-cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.

A rapid diagnostic test for human regulatory T-cell function to enable regulatory T-cell therapy.

Regulatory T cells (CD4(+)CD25(hi)CD127(lo)FOXP3(+) T cells [Tregs]) are a population of lymphocytes involved in the maintenance of self-tolerance. Abnormalities in function or number of Tregs are a feature of autoimmune diseases in humans. The ability to expand functional Tregs ex vivo makes them ideal candidates for autologous cell therapy to treat human autoimmune diseases and to induce tolerance to transplants. Current tests of Treg function typically take up to 120 hours, a kinetic disadvantage as clinical trials of Tregs will be critically dependent on the availability of rapid diagnostic tests before infusion into humans. Here we evaluate a 7-hour flow cytometric assay for assessing Treg function, using suppression of the activation markers CD69 and CD154 on responder T cells (CD4(+)CD25(-) [Tresp]), compared with traditional assays involving inhibition of CFSE dilution and cytokine production. In both freshly isolated and ex vivo expanded Tregs, we describe excellent correlation with gold standard suppressor cell assays. We propose that the kinetic advantage of the new assay may place it as the preferred rapid diagnostic test for the evaluation of Treg function in forthcoming clinical trials of cell therapy, enabling the translation of the large body of preclinical data into potentially useful treatments for human diseases.

Differential effects of rapamycin and retinoic acid on expansion, stability and suppressive qualities of human CD4(+)CD25(+)FOXP3(+) T regulatory cell subpopulations.

Adoptive transfer of ex vivo expanded CD4(+)CD25(+)FOXP3(+) regulatory T cells is a successful therapy for autoimmune diseases and transplant rejection in experimental models. In man, equivalent manipulations in bone marrow transplant recipients appear safe, but questions regarding the stability of the transferred regulatory T cells during inflammation remain unresolved. In this study, protocols for the expansion of clinically useful numbers of functionally suppressive and stable human regulatory T cells were investigated. Regulatory T cells were expanded in vitro with rapamycin and/or all-trans retinoic acid and then characterized under inflammatory conditions in vitro and in vivo in a humanized mouse model of graft-versus-host disease. Addition of rapamycin to regulatory T-cell cultures confirms the generation of high numbers of suppressive regulatory T cells. Their stability was demonstrated in vitro and substantiated in vivo. In contrast, all-trans retinoic acid treatment generates regulatory T cells that retain the capacity to secrete IL-17. However, combined use of rapamycin and all-trans retinoic acid abolishes IL-17 production and confers a specific chemokine receptor homing profile upon regulatory T cells. The use of purified regulatory T-cell subpopulations provided direct evidence that rapamycin can confer an early selective advantage to CD45RA(+) regulatory T cells, while all-trans retinoic acid favors CD45RA(-) regulatory T-cell subset. Expansion of regulatory T cells using rapamycin and all-trans retinoic acid drug combinations provides a new and refined approach for large-scale generation of functionally potent and phenotypically stable human regulatory T cells, rendering them safe for clinical use in settings associated with inflammation.

CD28 costimulation regulates FOXP3 in a RelA/NF-κB-dependent mechanism.

The molecular mechanisms whereby CD28 alone or associated with TCR can regulate FOXP3 expression are not understood, although the importance of CD28 as a pivotal regulator of CD4(+) CD25(+) FOXP3(+) T cells is well recognized. We previously demonstrated that unique CD28-induced, NF-κB-dependent signals were sufficient to activate FOXP3 transcription in human CD4(+) CD25(-) T cells; however, the exact mechanisms are currently unknown. In this study, we have identified novel κB-binding sites on FOXP3 gene and demonstrated that CD28 signals mediated FOXP3 trans activation by nuclear translocation of RelA/NF-κB and not of c-Rel. The occupancy of FOXP3 κB-binding sites by RelA dimers that correlated with histone acetylation and recruitment of Pol II were required both to initiate FOXP3 transcription and to control the promoter occupancy by NFAT. Interestingly, knockdown of RelA in CD4(+) CD25(-) T cells stimulated through TCR and CD28 significantly affected FOXP3 expression, confirming that also the transcriptional activation of FOXP3 gene by TCR in the presence of CD28-costimulatory signals is RelA-dependent. In conclusion, these data suggest a new mechanism by which FOXP3 is activated and supports the critical role of CD28 in the regulation of peripheral tolerance.

Isolation and freezing of human peripheral blood mononuclear cells from pregnant patients.

To analyze immune cell populations accurately, a large number of Peripheral Blood Mononuclear Cells (PBMCs) must be obtained from blood samples. Traditional manual isolation and SepMateTM isolation of PBMCs consistently yield blood-stained plasma layers and overall low numbers of CD4+ and CD8+ cells. Here, we describe an optimized protocol, using PBS with EDTA to increase PBMC yield from pregnant patients. This protocol enables analysis of CD4+, CD8+, and Regulatory T Cells and is potentially applicable to any immune cell population. For complete details on the use and execution of this protocol, please refer to the SepMateTM website https://www.stemcell.com/products/brands/SepMateTM-pbmc-isolation.html.

Regulatory T Cells in Pregnancy Adverse Outcomes: A Systematic Review and Meta-Analysis.

Background: Several studies report the role of Regulatory T-cells (Tregs) in the pathophysiology of pregnancy adverse outcomes. Objective: The aim of this systematic review and meta-analysis was to determine whether there is an association between regulatory T cell levels and pregnancy adverse outcomes (PAOs), including pre-eclampsia and preterm birth (PTB). Method: Literature searches were conducted in PubMed/MEDLINE, Embase, and Cochrane CENTRAL databases. Inclusion criteria were original articles (clinical trials, case-control studies and cohort studies) comparing Tregs, sampled from the decidua or maternal blood, in healthy pregnant women versus women with pre-eclampsia or PTB. The outcome was standardised mean difference (SMD) in Treg numbers. The tau-squared (Tau²), inconsistency index (I²), and chi-squared (χ²) test quantified heterogeneity among different studies. Analyses were performed in RevMan software V.5.4.0 for Mac using a random-effects model with outcome data reported with 95% confidence intervals (CI). This study was prospectively registered with PROSPERO (CRD42020205469). PRISMA guidelines were followed. Results: From 4,085 unique studies identified, 36 were included in qualitative synthesis, and 34 were included in quantitative synthesis (meta-analysis). In total, there were 1,783 participants in these studies: healthy controls=964, pre-eclampsia=759, PTB=60. Thirty-two studies compared Tregs in healthy pregnant women and women with pre-eclampsia, and 30 of these sampled Tregs from peripheral blood showing significantly higher Treg numbers in healthy pregnancies (SMD; 1.46; 95% CI, 1.03-1.88; I²=92%). Four studies sampled Tregs from the maternal decidua showing higher Tregs in healthy pregnancies (SMD, 0.76; 95% CI, -0.13-1.65; I²=84%). No difference was found in the number of Tregs between early versus late pre-eclampsia (SMD,-1.17; 95% CI, -2.79-0.44; I²=94%). For PTB, two studies compared Tregs sampled from the peripheral blood with a tendency for higher Tregs in healthy pregnancies but this did not reach significance (SMD, 2.18; 95% CI, -1.34-5.70; I²=96%). Subcohort analysis using Treg analysis (flow cytometry vs. qPCR vs. immunofluorescence tissue staining) showed similar associations. Conclusion: Lower Tregs in pregnancy, sampled from the maternal peripheral blood, are associated with pre-eclampsia. There is a need for further studies to confirm a relationship between low Tregs and PTB. As the precise mechanisms by which Tregs may mediate pre-eclampsia and PTB remain unclear, further fundamental research is necessary to elucidate the underlying processes and highlight the causative link. Systematic Review Registration: PROSPERO, identifier CRD42020205469.

Spatiotemporal in vivo tracking of polyclonal human regulatory T cells (Tregs) reveals a role for innate immune cells in Treg transplant recruitment.

Regulatory T cells (Tregs) are emerging as a new cell-based therapy in solid organ transplantation. Adoptive transfer of Tregs has been shown preclinically to protect from graft rejection, and the safety of Treg therapy has been demonstrated in clinical trials. Despite these successes, the in vivo distribution and persistence of adoptively transferred Tregs remained elusive, which hampers clinical translation. Here we isolated human Tregs using a GMP-compatible protocol and lentivirally transduced them with the human sodium iodide symporter to render them traceable in vivo by radionuclide imaging. Engineered human Tregs were characterized for phenotype, survival, suppressive capacity, and reporter function. To study their trafficking behavior, they were subsequently administered to humanized mice with human skin transplants. Traceable Tregs were quantified in skin grafts by non-invasive nano-single-photon emission computed tomography (nanoSPECT)/computed tomography (CT) for up to 40 days, and the results were validated ex vivo. Using this approach, we demonstrated that Treg trafficking to skin grafts was regulated by the presence of recipient Gr-1+ innate immune cells. We demonstrated the utility of radionuclide reporter gene-afforded quantitative Treg in vivo tracking, addressing a fundamental need in Treg therapy development and offering a clinically compatible methodology for future Treg therapy imaging in humans.

FOXP3 induced by CD28/B7 interaction regulates CD25 and anergic phenotype in human CD4+CD25- T lymphocytes.

Among the signals necessary to generate CD4(+)CD25(+)FOXP3(+) T cells from CD4(+)CD25(-)FOXP3(-) T cells, a pivotal role is played by CD28. However, in humans, it is not known whether CD28 signaling independently of TCR promotes forkhead box protein 3 (FOXP3) expression and regulates CD4(+)CD25(+)FOXP3(+) T cell functions. To address this issue, starting from our previous experience, we analyzed the unique signals delivered by CD28 following stimulation by its natural ligand B7. Our results show that, in primary CD4(+)CD25(-) T cells, CD28 signals independent of TCR-mediated stimulatory pathways are sufficient to induce the transcription of FOXP3 in a small number of CD4(+)CD25(-) T cells committed to express FOXP3. These signals are dependent on CD28-derived PI3K/Akt pathways and resistant to cyclosporin A. In addition, we demonstrated that translated FOXP3 was recruited to CD25, Il-2, and Ctla4 target promoters. CD28-mediated FOXP3 expression was transient and correlated with CD25 expression. The presence of FOXP3 in CD28-activated CD4(+)CD25(-) T cells correlated with a transient unresponsiveness to antigenic stimuli. The addition of exogenous IL-2 did not influence either FOXP3 or CD25 expression but rescued CD28-activated T cells from apoptosis. Our results, demonstrating that FOXP3 expression driven solely by the CD28/B7 interaction inhibited T cell activation, support the role of CD28 in the regulation of peripheral tolerance and suggest a new mechanism through which it could occur.