Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency.

We previously reported two siblings with inherited PD-1 deficiency who died from autoimmune pneumonitis at 3 and 11 years of age after developing other autoimmune manifestations, including type 1 diabetes (T1D). We report here two siblings, aged 10 and 11 years, with neonatal-onset T1D (diagnosed at the ages of 1 day and 7 wk), who are homozygous for a splice-site variant of CD274 (encoding PD-L1). This variant results in the exclusive expression of an alternative, loss-of-function PD-L1 protein isoform in overexpression experiments and in the patients' primary leukocytes. Surprisingly, cytometric immunophenotyping and single-cell RNA sequencing analysis on blood leukocytes showed largely normal development and transcriptional profiles across lymphoid and myeloid subsets in the PD-L1-deficient siblings, contrasting with the extensive dysregulation of both lymphoid and myeloid leukocyte compartments in PD-1 deficiency. Our findings suggest that PD-1 and PD-L1 are essential for preventing early-onset T1D but that, unlike PD-1 deficiency, PD-L1 deficiency does not lead to fatal autoimmunity with extensive leukocytic dysregulation.

Single-cell RNAseq identifies clonally expanded antigen-specific T-cells following intradermal injection of gold nanoparticles loaded with diabetes autoantigen in humans.

Gold nanoparticles (GNPs) have been used in the development of novel therapies as a way of delivery of both stimulatory and tolerogenic peptide cargoes. Here we report that intradermal injection of GNPs loaded with the proinsulin peptide C19-A3, in patients with type 1 diabetes, results in recruitment and retention of immune cells in the skin. These include large numbers of clonally expanded T-cells sharing the same paired T-cell receptors (TCRs) with activated phenotypes, half of which, when the TCRs were re-expressed in a cell-based system, were confirmed to be specific for either GNP or proinsulin. All the identified gold-specific clones were CD8+, whilst proinsulin-specific clones were both CD8+ and CD4+. Proinsulin-specific CD8+ clones had a distinctive cytotoxic phenotype with overexpression of granulysin (GNLY) and KIR receptors. Clonally expanded antigen-specific T cells remained in situ for months to years, with a spectrum of tissue resident memory and effector memory phenotypes. As the T-cell response is divided between targeting the gold core and the antigenic cargo, this offers a route to improving resident memory T-cells formation in response to vaccines. In addition, our scRNAseq data indicate that focusing on clonally expanded skin infiltrating T-cells recruited to intradermally injected antigen is a highly efficient method to enrich and identify antigen-specific cells. This approach has the potential to be used to monitor the intradermal delivery of antigens and nanoparticles for immune modulation in humans.

Monitoring islet specific immune responses in type 1 diabetes clinical immunotherapy trials.

The number of immunotherapeutic clinical trials in type 1 diabetes currently being conducted is expanding, and thus there is a need for robust immune-monitoring assays which are capable of detecting and characterizing islet specific immune responses in peripheral blood. Islet- specific T cells can serve as biomarkers and as such can guide drug selection, dosing regimens and immunological efficacy. Furthermore, these biomarkers can be utilized in patient stratification which can then benchmark suitability for participation in future clinical trials. This review focusses on the commonly used immune-monitoring techniques including multimer and antigen induced marker assays and the potential to combine these with single cell transcriptional profiling which may provide a greater understanding of the mechanisms underlying immuno-intervention. Although challenges remain around some key areas such as the need for harmonizing assays, technological advances mean that multiparametric information derived from a single sample can be used in coordinated efforts to harmonize biomarker discovery and validation. Moreover, the technologies discussed here have the potential to provide a unique insight on the effect of therapies on key players in the pathogenesis of T1D that cannot be obtained using antigen agnostic approaches.

Low-dose IL-2 reduces IL-21+ T cell frequency and induces anti-inflammatory gene expression in type 1 diabetes.

Despite early clinical successes, the mechanisms of action of low-dose interleukin-2 (LD-IL-2) immunotherapy remain only partly understood. Here we examine the effects of interval administration of low-dose recombinant IL-2 (iLD-IL-2) in type 1 diabetes using high-resolution single-cell multiomics and flow cytometry on longitudinally-collected peripheral blood samples. Our results confirm that iLD-IL-2 selectively expands thymic-derived FOXP3+HELIOS+ regulatory T cells and CD56bright NK cells, and show that the treatment reduces the frequency of IL-21-producing CD4+ T cells and of two innate-like mucosal-associated invariant T and Vγ9Vδ2 CD8+ T cell subsets. The cellular changes induced by iLD-IL-2 associate with an anti-inflammatory gene expression signature, which remains detectable in all T and NK cell subsets analysed one month after treatment. These findings warrant investigations into the potential longer-term clinical benefits of iLD-IL-2 in immunotherapy.

Guidelines for standardizing T-cell cytometry assays to link biomarkers, mechanisms, and disease outcomes in type 1 diabetes.

Cytometric immunophenotyping is a powerful tool to discover and implement T-cell biomarkers of type 1 diabetes (T1D) progression and response to clinical therapy. Although many discovery-based T-cell biomarkers have been described, to date, no such markers have been widely adopted in standard practice. The heterogeneous nature of T1D and lack of standardized assays and experimental design across studies is a major barrier to the broader adoption of T-cell immunophenotyping assays. There is an unmet need to harmonize the design of immunophenotyping assays, including those that measure antigen-agnostic cell populations, such that data collected from different clinical trial sites and T1D cohorts are comparable, yet account for cohort-specific features and different drug mechanisms of action. In these Guidelines, we aim to provide expert advice on how to unify aspects of study design and practice. We provide recommendations for defining cohorts, method implementation, as well as tools for data analysis and reporting by highlighting and building on selected successes. Harmonization of cytometry-based T-cell assays will allow researchers to better integrate findings across trials, ultimately enabling the identification and validation of biomarkers of disease progression and treatment response in T1D.

A phase 1b open-label dose-finding study of ustekinumab in young adults with type 1 diabetes.

OBJECTIVES: We assessed the safety of ustekinumab (a monoclonal antibody used in psoriasis to target the IL-12 and IL-23 pathways) in a small cohort of recent-onset (<100 days of diagnosis) adults with type 1 diabetes (T1D) by conducting a pilot open-label dose-finding and mechanistic study (NCT02117765) at the University of British Columbia. METHODS: We sequentially enrolled 20 participants into four subcutaneous dosing cohorts: (i) 45 mg loading weeks 0/4/16, (ii) 45 mg maintenance weeks 0/4/16/28/40, (iii) 90 mg loading weeks 0/4/16, and (iv) 90 mg maintenance weeks 0/4/16/28/40. The primary endpoint was safety as assessed by an independent data and safety monitoring board (DSMB) but we also measured mixed meal tolerance test C-peptide, insulin use/kg, and HbA1c. Immunophenotyping was performed to assess immune cell subsets and islet antigen-specific T cell responses. RESULTS: Although several adverse events were reported, only two (bacterial vaginosis and hallucinations) were thought to be possibly related to drug administration by the study investigators. At 1 year, the 90 mg maintenance dosing cohort had the smallest mean decline in C-peptide area under the curve (AUC) (0.1 pmol/ml). Immunophenotyping showed that ustekinumab reduced the percentage of circulating Th17, Th1, and Th17.1 cells and proinsulin-specific T cells that secreted IFN-γ and IL-17A. CONCLUSION: Ustekinumab was deemed safe to progress to efficacy studies by the DSMB at doses used to treat psoriasis in adults with T1D. A 90 mg maintenance dosing schedule reduced proinsulin-specific IFN-γ and IL-17A-producing T cells. Further studies are warranted to determine if ustekinumab can prevent C-peptide AUC decline and induce a clinical response.

The effect of methotrexate and targeted immunosuppression on humoral and cellular immune responses to the COVID-19 vaccine BNT162b2: a cohort study.

BACKGROUND: Patients on therapeutic immunosuppressants for immune-mediated inflammatory diseases were excluded from COVID-19 vaccine trials. We therefore aimed to evaluate humoral and cellular immune responses to COVID-19 vaccine BNT162b2 (Pfizer-BioNTech) in patients taking methotrexate and commonly used targeted biological therapies, compared with healthy controls. Given the roll-out of extended interval vaccination programmes to maximise population coverage, we present findings after the first dose. METHODS: In this cohort study, we recruited consecutive patients with a dermatologist-confirmed diagnosis of psoriasis who were receiving methotrexate or targeted biological monotherapy (tumour necrosis factor [TNF] inhibitors, interleukin [IL]-17 inhibitors, or IL-23 inhibitors) from a specialist psoriasis centre serving London and South East England. Consecutive volunteers without psoriasis and not receiving systemic immunosuppression who presented for vaccination at Guy's and St Thomas' NHS Foundation Trust (London, UK) were included as the healthy control cohort. All participants had to be eligible to receive the BNT162b2 vaccine. Immunogenicity was evaluated immediately before and on day 28 (±2 days) after vaccination. The primary outcomes were humoral immunity to the SARS-CoV-2 spike glycoprotein, defined as neutralising antibody responses to wild-type SARS-CoV-2, and spike-specific T-cell responses (including interferon-γ, IL-2, and IL-21) 28 days after vaccination. FINDINGS: Between Jan 14 and April 4, 2021, 84 patients with psoriasis (17 on methotrexate, 27 on TNF inhibitors, 15 on IL-17 inhibitors, and 25 on IL-23 inhibitors) and 17 healthy controls were included. The study population had a median age of 43 years (IQR 31-52), with 56 (55%) males, 45 (45%) females, and 85 (84%) participants of White ethnicity. Seroconversion rates were lower in patients receiving immunosuppressants (60 [78%; 95% CI 67-87] of 77) than in controls (17 [100%; 80-100] of 17), with the lowest rate in those receiving methotrexate (seven [47%; 21-73] of 15). Neutralising activity against wild-type SARS-CoV-2 was significantly lower in patients receiving methotrexate (median 50% inhibitory dilution 129 [IQR 40-236]) than in controls (317 [213-487], p=0·0032), but was preserved in those receiving targeted biologics (269 [141-418]). Neutralising titres against the B.1.1.7 variant were similarly low in all participants. Cellular immune responses were induced in all groups, and were not attenuated in patients receiving methotrexate or targeted biologics compared with controls. INTERPRETATION: Functional humoral immunity to a single dose of BNT162b2 is impaired by methotrexate but not by targeted biologics, whereas cellular responses are preserved. Seroconversion alone might not adequately reflect vaccine immunogenicity in individuals with immune-mediated inflammatory diseases receiving therapeutic immunosuppression. Real-world pharmacovigilance studies will determine how these findings reflect clinical effectiveness. FUNDING: UK National Institute for Health Research.

Autoreactive T cell profiles are altered following allogeneic islet transplantation with alemtuzumab induction and re-emerging phenotype is associated with graft function.

Islet transplantation is an effective therapy for life-threatening hypoglycemia, but graft function gradually declines over time in many recipients. We characterized islet-specific T cells in recipients within an islet transplant program favoring alemtuzumab (ATZ) lymphodepleting induction and examined associations with graft function. Fifty-eight recipients were studied: 23 pretransplant and 40 posttransplant (including 5 with pretransplant phenotyping). The proportion with islet-specific T cell responses was not significantly different over time (pre-Tx: 59%; 1-6 m posttransplant: 38%; 7-12 m: 44%; 13-24 m: 47%; and >24 m: 45%). However, phenotype shifted significantly, with IFN-γ-dominated response in the pretransplant group replaced by IL-10-dominated response in the 1-6 m posttransplant group, reverting to predominantly IFN-γ-oriented response in the >24 m group. Clustering analysis of posttransplant responses revealed two main agglomerations, characterized by IFN-γ and IL-10 phenotypes, respectively. IL-10-oriented posttransplant response was associated with relatively low graft function. Recipients within the IL-10+ cluster had a significant decline in C-peptide levels in the period preceding the IL-10 response, but stable graft function following the response. In contrast, an IFN-γ response was associated with subsequently decreased C-peptide. Islet transplantation favoring ATZ induction is associated with an initial altered islet-specific T cell phenotype but reversion toward pretransplant profiles over time. Posttransplant autoreactive T cell phenotype may be a predictor of subsequent graft function.

Type 1 diabetes can present before the age of 6 months and is characterised by autoimmunity and rapid loss of beta cells.

AIMS/HYPOTHESIS: Diabetes diagnosed at <6 months of age is usually monogenic. However, 10-15% of affected infants do not have a pathogenic variant in one of the 26 known neonatal diabetes genes. We characterised infants diagnosed at <6 months of age without a pathogenic variant to assess whether polygenic type 1 diabetes could arise at early ages. METHODS: We studied 166 infants diagnosed with type 1 diabetes at <6 months of age in whom pathogenic variants in all 26 known genes had been excluded and compared them with infants with monogenic neonatal diabetes (n = 164) or children with type 1 diabetes diagnosed at 6-24 months of age (n = 152). We assessed the type 1 diabetes genetic risk score (T1D-GRS), islet autoantibodies, C-peptide and clinical features. RESULTS: We found an excess of infants with high T1D-GRS: 38% (63/166) had a T1D-GRS >95th centile of healthy individuals, whereas 5% (8/166) would be expected if all were monogenic (p < 0.0001). Individuals with a high T1D-GRS had a similar rate of autoantibody positivity to that seen in individuals with type 1 diabetes diagnosed at 6-24 months of age (41% vs 58%, p = 0.2), and had markedly reduced C-peptide levels (median <3 pmol/l within 1 year of diagnosis), reflecting rapid loss of insulin secretion. These individuals also had reduced birthweights (median z score -0.89), which were lowest in those diagnosed with type 1 diabetes at <3 months of age (median z score -1.98). CONCLUSIONS/INTERPRETATION: We provide strong evidence that type 1 diabetes can present before the age of 6 months based on individuals with this extremely early-onset diabetes subtype having the classic features of childhood type 1 diabetes: high genetic risk, autoimmunity and rapid beta cell loss. The early-onset association with reduced birthweight raises the possibility that for some individuals there was reduced insulin secretion in utero. Comprehensive genetic testing for all neonatal diabetes genes remains essential for all individuals diagnosed with diabetes at <6 months of age. Graphical abstract.

Phenotypic Analysis of Human Lymph Nodes in Subjects With New-Onset Type 1 Diabetes and Healthy Individuals by Flow Cytometry.

Background: Ultrasound guided sampling of human lymph node (LN) combined with advanced flow cytometry allows phenotypic analysis of multiple immune cell subsets. These may provide insights into immune processes and responses to immunotherapies not apparent from analysis of the blood. Methods: Ultrasound guided inguinal LN samples were obtained by both fine needle aspiration (FNA) and core needle biopsy in 10 adults within 8 weeks of diagnosis of type 1 diabetes (T1D) and 12 age-matched healthy controls at two study centers. Peripheral blood mononuclear cells (PBMC) were obtained on the same occasion. Samples were transported same day to the central laboratory and analyzed by multicolour flow cytometry. Results: LN sampling was well-tolerated and yielded sufficient cells for analysis in 95% of cases. We confirmed the segregation of CD69+ cells into LN and the predominance of CD8+ Temra cells in blood previously reported. In addition, we demonstrated clear enrichment of CD8+ naïve, FOXP3+ Treg, class-switched B cells, CD56bright NK cells and plasmacytoid dendritic cells (DC) in LNs as well as CD4+ T cells of the Th2 phenotype and those expressing Helios and Ki67. Conventional NK cells were virtually absent from LNs as were Th22 and Th1Th17 cells. Paired correlation analysis of blood and LN in the same individuals indicated that for many cell subsets, especially those associated with activation: such as CD25+ and proliferating (Ki67+) T cells, activated follicular helper T cells and class-switched B cells, levels in the LN compartment could not be predicted by analysis of blood. We also observed an increase in Th1-like Treg and less proliferating (Ki67+) CD4+ T cells in LN from T1D compared to control LNs, changes which were not reflected in the blood. Conclusions: LN sampling in humans is well-tolerated. We provide the first detailed "roadmap" comparing immune subsets in LN vs. blood emphasizing a role for differentiated effector T cells in the blood and T cell regulation, B cell activation and memory in the LN. For many subsets, frequencies in blood, did not correlate with LN, suggesting that LN sampling would be valuable for monitoring immuno-therapies where these subsets may be impacted.

Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner.

Lymphodepletion strategies are used in the setting of transplantation (including bone marrow, hematopoietic cell, and solid organ) to create space or to prevent allograft rejection and graft versus host disease. Following lymphodepletion, there is an excess of IL-7 available, and T cells that escape depletion respond to this cytokine undergoing accelerated proliferation. Moreover, this environment promotes the skew of T cells to a Th1 pro-inflammatory phenotype. Existing immunosuppressive regimens fail to control this homeostatic proliferative (HP) response, and thus the development of strategies to successfully control HP while sparing T cell reconstitution (providing a functioning immune system) represents a significant unmet need in patients requiring lymphodepletion. Multipotent adult progenitor cells (MAPC®) have the capacity to control T cell proliferation and Th1 cytokine production. Herein, this study shows that MAPC cells suppressed anti-thymocyte globulin-induced cytokine production but spared T cell reconstitution in a pre-clinical model of lymphodepletion. Importantly, MAPC cells administered intraperitoneally were efficacious in suppressing interferon-γ production and in promoting the expansion of regulatory T cells in the lymph nodes. MAPC cells administered intraperitoneally accumulated in the omentum but were not present in the spleen suggesting a role for soluble factors. MAPC cells suppressed lymphopenia-induced cytokine production in a prostaglandin E2-dependent manner. This study suggests that MAPC cell therapy may be useful as a novel strategy to target lymphopenia-induced pathogenic T cell responses in lymphodepleted patients.

Regulatory T cell dysfunction in type 1 diabetes: what's broken and how can we fix it?

Type 1 diabetes is an autoimmune disease characterised by the destruction of insulin producing beta cells in the pancreas. Whilst it remains unclear what the original triggering factors for this destruction are, observations from the natural history of human type 1 diabetes, including incidence rates in twins, suggest that the disease results from a combination of genetic and environmental factors. Whilst many different immune cells have been implicated, including members of the innate and adaptive immune systems, a view has emerged over the past 10 years that beta cell damage is mediated by the combined actions of CD4+ and CD8+ T cells with specificity for islet autoantigens. In health, these potentially pathogenic T cells are held in check by multiple regulatory mechanisms, known collectively as 'immunological tolerance'. This raises the question as to whether type 1 diabetes develops, at least in part, as a result of a defect in one or more of these control mechanisms. Immunological tolerance includes both central mechanisms (purging of the T cell repertoire of high-affinity autoreactive T cells in the thymus) and peripheral mechanisms, a major component of which is the action of a specialised subpopulation of T cells, known as regulatory T cells (Tregs). In this review, we highlight the evidence suggesting that a reduction in the functional capacity of different Treg populations contributes to disease development in type 1 diabetes. We also address current controversies regarding the putative causes of this defect and discuss strategies to correct it as a means to reduce or prevent islet destruction in a clinical setting.

Metabolic and immune effects of immunotherapy with proinsulin peptide in human new-onset type 1 diabetes.

Immunotherapy using short immunogenic peptides of disease-related autoantigens restores immune tolerance in preclinical disease models. We studied safety and mechanistic effects of injecting human leukocyte antigen-DR4(DRB1*0401)-restricted immunodominant proinsulin peptide intradermally every 2 or 4 weeks for 6 months in newly diagnosed type 1 diabetes patients. Treatment was well tolerated with no systemic or local hypersensitivity. Placebo subjects showed a significant decline in stimulated C-peptide (measuring insulin reserve) at 3, 6, 9, and 12 months versus baseline, whereas no significant change was seen in the 4-weekly peptide group at these time points or the 2-weekly group at 3, 6, and 9 months. The placebo group's daily insulin use increased by 50% over 12 months but remained unchanged in the intervention groups. C-peptide retention in treated subjects was associated with proinsulin-stimulated interleukin-10 production, increased FoxP3 expression by regulatory T cells, low baseline levels of activated ß cell-specific CD8 T cells, and favorable ß cell stress markers (proinsulin/C-peptide ratio). Thus, proinsulin peptide immunotherapy is safe, does not accelerate decline in ß cell function, and is associated with antigen-specific and nonspecific immune modulation.

IL-2 therapy restores regulatory T-cell dysfunction induced by calcineurin inhibitors.

CD4+CD25+FOXP3+ Tregs constitute a heterogeneous lymphocyte subpopulation essential for curtailing effector T cells and establishing peripheral tolerance. Calcineurin inhibitors (CNIs) are among the most effective agents in controlling effector T-cell responses in humans. However, CNIs also reduce the size of the Treg pool. The functional consequences of this negative effect and the mechanisms responsible remain to be elucidated. We report here that CNIs compromise the overall Treg immunoregulatory capacity to a greater extent than would be predicted by the reduction in the size of the Treg compartment, given that they selectively promote the apoptosis of the resting and activated Treg subsets that are known to display the most powerful suppressive function. These effects are caused by reduced access to IL-2, because Tregs remain capable of translocating NFAT even in the presence of high CNI levels. Exogenous IL-2 restores the phenotypic changes and overall gene-expression effects exerted by CNIs and can even promote Treg expansion by enhancing antiapoptotic Bcl-2 expression. In a skin transplant model, the addition of IL-2 synergizes with CNIs treatment, promoting intragraft accumulation of Tregs and prolonged allograft survival. Hence, the combination of IL-2 and CNIs constitutes an optimal immunomodulatory regimen that enhances the pool of suppressive Treg subsets while effectively controlling cytopathic T cells.

Generation of human islet-specific regulatory T cells by TCR gene transfer.

Based on the success in animal models of type 1 diabetes (T1D), clinical trials of adoptive regulatory T cell (Treg) therapy are underway using ex vivo expanded polyclonal Tregs. However, pre-clinical data also demonstrate that islet-specific Tregs are more potent than polyclonal Tregs at reversing T1D. Translation of this approach into man will require methods to generate large populations of islet-specific Tregs which, to date, has proved to be a major hurdle. Here we demonstrate the feasibility of lentiviral-mediated T cell receptor (TCR) gene transfer to confer antigen specificity on polyclonal human Tregs. Targeting has been achieved using TCRs isolated from human islet-specific and viral-specific CD4+ T cell clones. Engineered T cells demonstrated expression of ectopically-delivered TCRs, resulting in endowment of cognate antigen-specific responses. This enabled antigen-specific suppression at increased potency compared to polyclonal Tregs. However, cells transduced with islet-specific TCRs were less responsive to cognate antigen than viral-specific TCRs, and in some cases, required additional methods to isolate functional antigen-specific Tregs. This study demonstrates the potential of TCR gene transfer to develop islet-specific Treg therapies for effective treatment of T1D, but also highlights that additional optimisation may be required to achieve its full potential.

Suppression of IL-7-dependent Effector T-cell Expansion by Multipotent Adult Progenitor Cells and PGE2.

T-cell depletion therapy is used to prevent acute allograft rejection, treat autoimmunity and create space for bone marrow or hematopoietic cell transplantation. The evolved response to T-cell loss is a transient increase in IL-7 that drives compensatory homeostatic proliferation (HP) of mature T cells. Paradoxically, the exaggerated form of this process that occurs following lymphodepletion expands effector T-cells, often causing loss of immunological tolerance that results in rapid graft rejection, autoimmunity, and exacerbated graft-versus-host disease (GVHD). While standard immune suppression is unable to treat these pathologies, growing evidence suggests that manipulating the incipient process of HP increases allograft survival, prevents autoimmunity, and markedly reduces GVHD. Multipotent adult progenitor cells (MAPC) are a clinical grade immunomodulatory cell therapy known to alter γ-chain cytokine responses in T-cells. Herein, we demonstrate that MAPC regulate HP of human T-cells, prevent the expansion of Th1, Th17, and Th22 effectors, and block the development of pathogenic allograft responses. This occurs via IL-1ß-primed secretion of PGE2 and activates T-cell intrinsic regulatory mechanisms (SOCS2, GADD45A). These data provide proof-of-principle that HP of human T-cells can be targeted by cellular and molecular therapies and lays a basis for the development of novel strategies to prevent immunopathology in lymphodepleted patients.

Natural Variation in Interleukin-2 Sensitivity Influences Regulatory T-Cell Frequency and Function in Individuals With Long-standing Type 1 Diabetes.

Defective immune homeostasis in the balance between FOXP3+ regulatory T cells (Tregs) and effector T cells is a likely contributing factor in the loss of self-tolerance observed in type 1 diabetes (T1D). Given the importance of interleukin-2 (IL-2) signaling in the generation and function of Tregs, observations that polymorphisms in genes in the IL-2 pathway associate with T1D and that some individuals with T1D exhibit reduced IL-2 signaling indicate that impairment of this pathway may play a role in Treg dysfunction and the pathogenesis of T1D. Here, we have examined IL-2 sensitivity in CD4+ T-cell subsets in 70 individuals with long-standing T1D, allowing us to investigate the effect of low IL-2 sensitivity on Treg frequency and function. IL-2 responsiveness, measured by STAT5a phosphorylation, was a very stable phenotype within individuals but exhibited considerable interindividual variation and was influenced by T1D-associated PTPN2 gene polymorphisms. Tregs from individuals with lower IL-2 signaling were reduced in frequency, were less able to maintain expression of FOXP3 under limiting concentrations of IL-2, and displayed reduced suppressor function. These results suggest that reduced IL-2 signaling may be used to identify patients with the highest Treg dysfunction and who may benefit most from IL-2 immunotherapy.

Phenotypic Complexity of the Human Regulatory T Cell Compartment Revealed by Mass Cytometry.

Regulatory T cells (Tregs) are an essential component of the cellular immune response, occupying a key role in maintaining immunological tolerance and present an attractive therapeutic target in a range of immunopathologies. Comprehensive analysis of the human Treg compartment has been restricted due to technical limitations. The advent of mass cytometry enables simultaneous assessment of vastly increased phenotypic parameters at single-cell resolution. In this study, we used mass cytometry to examine the complexity of human Tregs using an extensive panel of surface markers associated with Treg function and phenotype. We applied unsupervised clustering analysis, revealing 22 distinct subpopulations of Tregs, representing previously identified and novel subpopulations. Our data represent the most in-depth phenotypic description of the human Treg compartment at single-cell resolution and show a hitherto unrecognized degree of phenotypic complexity among cells of the regulatory lineage.

Immune modulation in humans: implications for type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is the result of autoimmune destruction of pancreatic ß cells in genetically predisposed individuals with impaired immune regulation. The insufficiency in the modulation of immune attacks on the ß cells might be partly due to genetic causes; indeed, several of the genetic variants that predispose individuals to T1DM have functional features of impaired immune regulation. Whilst defects in immune regulation in patients with T1DM have been identified, many patients seem to have immune regulatory capacities that are indistinguishable from those of healthy individuals. Insight into the regulation of islet autoimmunity might enable us to restore immune imbalances with therapeutic interventions. In this Review, we discuss the current knowledge on immune regulation and dysfunction in humans that is the basis of tissue-specific immune regulation as an alternative to generalized immune suppression.

Clinical-grade multipotent adult progenitor cells durably control pathogenic T cell responses in human models of transplantation and autoimmunity.

A major goal of immunotherapy remains the control of pathogenic T cell responses that drive autoimmunity and allograft rejection. Adherent progenitor cells, including mesenchymal stromal cells (MSCs) and multipotent adult progenitor cells (MAPCs), represent attractive immunomodulatory cell therapy candidates currently active in clinical trials. MAPCs can be distinguished from MSCs on the basis of cellular phenotype, size, transcriptional profile, and expansion capacity. However, despite their ongoing evaluation in autoimmune and allogeneic solid organ transplantation settings, data supporting the immune regulatory potential of clinical-grade MAPCs are limited. In this study, we used allogeneic islet transplantation as a model indication to assess the ability of clinical-grade MAPCs to control T cell responses that drive immunopathology in human autoimmune disease and allograft rejection. MAPCs suppressed T cell proliferation and Th1 and Th17 cytokine production while increasing secretion of IL-10 and were able to suppress effector functions of bona fide autoreactive T cells from individuals with type 1 diabetes mellitus, including killing of human islets. Furthermore, MAPCs favored the proliferation of regulatory T cells during homeostatic expansion driven by γ-chain cytokines and exerted a durable, yet reversible, control of T cell function. MAPC suppression required licensing and proceeded via IDO-mediated tryptophan catabolism. Therefore, the common immune modulatory characteristics of clinical-grade MAPCs shown in this study suggest that they can be regarded as an alternative source of adult progenitor cells with similar clinical usefulness to MSCs. Taken collectively, these findings may guide the successful deployment of both MSCs and MAPCs for the amelioration of human autoimmunity and allograft rejection.