PD-L1 and PD-1 Are Associated with Clinical Outcomes and Alveolar Immune Cell Activation in ARDS.

The relationship between the Programmed Death-Ligand 1 (PD-L1)/Programmed Death-1 (PD-1) pathway, lung inflammation, and clinical outcomes in acute respiratory distress syndrome (ARDS) is poorly understood. We sought to determine whether PD-L1/PD-1 in the lung or blood is associated with ARDS and associated severity. We measured soluble PD-L1 (sPD-L1) in plasma and lower respiratory tract samples (ARDS1 (n = 59) and ARDS2 (n = 78)) or plasma samples alone (ARDS3 (n = 149)) collected from subjects with ARDS and tested for associations with mortality using multiple regression. We used mass cytometry to measure PD-L1/PD-1 expression and intracellular cytokine staining in cells isolated from bronchoalveolar lavage fluid (BALF) (n = 18) and blood (n = 16) from critically-ill subjects with or without ARDS enrolled from a fourth cohort. Higher plasma levels of sPD-L1 were associated with mortality in ARDS1, ARDS2, and ARDS3. In contrast, higher levels of sPD-L1 in the lung were either not associated with mortality (ARDS2) or were associated with survival (ARDS1). Alveolar PD-1POS T cells had more intracellular cytokine staining compared with PD-1NEG T cells. Subjects without ARDS had a higher ratio of PD-L1POS alveolar macrophages to PD-1POS T cells compared with subjects with ARDS. We conclude that sPD-L1 may have divergent cellular sources and/or functions in the alveolar vs. blood compartments given distinct associations with mortality. Alveolar leukocyte subsets defined by PD-L1/PD-1 cell-surface expression have distinct cytokine secretion profiles, and the relative proportions of these subsets are associated with ARDS.

A first-in-human, open-label Phase 1b and a randomised, double-blind Phase 2a clinical trial in recent-onset type 1 diabetes with AG019 as monotherapy and in combination with teplizumab.

AIMS/HYPOTHESIS: We hypothesised that islet beta cell antigen presentation in the gut along with a tolerising cytokine would lead to antigen-specific tolerance in type 1 diabetes. We evaluated this in a parallel open-label Phase 1b study using oral AG019, food-grade Lactococcus lactis bacteria genetically modified to express human proinsulin and human IL-10, as a monotherapy and in a parallel, randomised, double-blind Phase 2a study using AG019 in combination with teplizumab. METHODS: Adults (18-42 years) and adolescents (12-17 years) with type 1 diabetes diagnosed within 150 days were enrolled, with documented evidence of at least one autoantibody and a stimulated peak C-peptide level >0.2 nmol/l. Participants were allocated to interventions using interactive response technology. We treated 42 people aged 12-42 years with recent-onset type 1 diabetes, 24 with Phase 1b monotherapy (open-label) and 18 with Phase 2a combination therapy. In the Phase 2a study, after treatment of the first two open-label participants, all people involved were blinded to group assignment, except for the Data Safety Monitoring Board members and the unblinded statistician. The primary endpoint was safety and tolerability based on the incidence of treatment-emergent adverse events, collected up to 6 months post treatment initiation. The secondary endpoints were pharmacokinetics, based on AG019 detection in blood and faeces, and pharmacodynamic activity. Metabolic and immune endpoints included stimulated C-peptide levels during a mixed meal tolerance test, HbA1c levels, insulin use, and antigen-specific CD4+ and CD8+ T cell responses using an activation-induced marker assay and pooled tetramers, respectively. RESULTS: Data from 24 Phase 1b participants and 18 Phase 2a participants were analysed. No serious adverse events were reported and none of the participants discontinued AG019 due to treatment-emergent adverse events. No systemic exposure to AG019 bacteria, proinsulin or human IL-10 was demonstrated. In AG019 monotherapy-treated adults, metabolic variables were stabilised up to 6 months (C-peptide, insulin use) or 12 months (HbA1c) post treatment initiation. In participants treated with AG019/teplizumab combination therapy, all measured metabolic variables stabilised or improved up to 12 months and CD8+ T cells with a partially exhausted phenotype were significantly increased at 6 months. Circulating preproinsulin-specific CD4+ and CD8+ T cells were detected before and after treatment, with a reduction in the frequency of preproinsulin-specific CD8+ T cells after treatment with monotherapy or combination therapy. CONCLUSIONS/INTERPRETATION: Oral delivery of AG019 was well tolerated and safe as monotherapy and in combination with teplizumab. AG019 was not shown to interfere with the safety profile of teplizumab and may have additional biological effects, including changes in preproinsulin-specific T cells. These preliminary data support continuing studies with this agent alone and in combination with teplizumab or other systemic immunotherapies in type 1 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT03751007, EudraCT 2017-002871-24 FUNDING: This study was funded by Precigen ActoBio.

Impact on in-depth immunophenotyping of delay to peripheral blood processing.

Peripheral blood mononuclear cell (PBMC) immunophenotyping is crucial in tracking activation, disease state, and response to therapy in human subjects. Many studies require the shipping of blood from clinical sites to a laboratory for processing to PBMC, which can lead to delays that impact sample quality. We used an extensive cytometry by time-of-flight (CyTOF) immunophenotyping panel to analyze the impacts of delays to processing and distinct storage conditions on cell composition and quality of PBMC from seven adults across a range of ages, including two with rheumatoid arthritis. Two or more days of delay to processing resulted in extensive red blood cell contamination and increased variability of cell counts. While total memory and naïve B- and T-cell populations were maintained, 4-day delays reduced the frequencies of monocytes. Variation across all immune subsets increased with delays of up to 7 days in processing. Unbiased clustering analysis to define more granular subsets confirmed changes in PBMC composition, including decreases of classical and non-classical monocytes, basophils, plasmacytoid dendritic cells, and follicular helper T cells, with each subset impacted at a distinct time of delay. Expression of activation markers and chemokine receptors changed by Day 2, with differential impacts across subsets and markers. Our data support existing recommendations to process PBMC within 36 h of collection but provide guidance on appropriate immunophenotyping experiments with longer delays.

High proinsulin:C-peptide ratio identifies individuals with stage 2 type 1 diabetes at high risk for progression to clinical diagnosis and responses to teplizumab treatment.

AIMS/HYPOTHESIS: Tractable precision biomarkers to identify immunotherapy responders are lacking in type 1 diabetes. We hypothesised that proinsulin:C-peptide (PI:C) ratios, a readout of beta cell stress, could provide insight into type 1 diabetes progression and responses to immunotherapy. METHODS: In this post hoc analysis, proinsulin and C-peptide levels were determined in baseline serum samples from 63 participants with stage 2 type 1 diabetes in the longitudinal TrialNet Teplizumab Prevention Study (n=41 in the teplizumab arm; n=22 in the placebo arm). In addition, previously tested demographic, C-peptide, glucose and proinsulin data were used for the new data analyses. The ratio of intact (unprocessed) proinsulin to C-peptide was analysed and relationships with progression to stage 3 diabetes were investigated. RESULTS: Elevated baseline PI:C was strongly associated with more rapid progression of diabetes in both the placebo and teplizumab treatment groups, but teplizumab abrogated the impact of high pre-treatment PI:C on type 1 diabetes progression. Differential responses of drug treatment in those with high vs low PI:C ratios were independent of treatment effects of teplizumab on the PI:C ratio or on relevant immune cells. CONCLUSIONS/INTERPRETATION: High pre-treatment PI:C identified individuals with stage 2 type 1 diabetes who were exhibiting rapid progression to stage 3 disease and who displayed benefit from teplizumab treatment. These data suggest that readouts of active disease, such as PI:C ratio, could serve to identify optimal candidates or timing for type 1 diabetes disease-modifying therapies.

Th2 cell clonal expansion at diagnosis in human type 1 diabetes.

Soon after diagnosis with type 1 diabetes (T1D), many patients experience a period of partial remission. A longer partial remission is associated with a better response to treatment, but the mechanism is not known. The frequency of CD4+CD25+CD127hi (127-hi) cells, a cell subset with an anti-inflammatory Th2 bias, correlates positively with length of partial remission. The purpose of this study was to further characterize the nature of the Th2 bias in 127-hi cells. Single cell RNA sequencing paired with TCR sequencing of sorted 127-hi memory cells identifies clonally expanded Th2 clusters in 127-hi cells from T1D, but not from healthy donors. The Th2 clusters express GATA3, GATA3-AS1, PTGDR2, IL17RB, IL4R and IL9R. The existence of 127-hi Th2 cell clonal expansion in T1D suggests that disease factors may induce clonal expansion of 127-hi Th2 cells that prolong partial remission and delay disease progression.

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.

Chemokines, soluble PD-L1, and immune cell hyporesponsiveness are distinct features of SARS-CoV-2 critical illness.

Critically ill patients manifest many of the same immune features seen in coronavirus disease 2019 (COVID-19), including both "cytokine storm" and "immune suppression." However, direct comparisons of molecular and cellular profiles between contemporaneously enrolled critically ill patients with and without severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are limited. We sought to identify immune signatures specifically enriched in critically ill patients with COVID-19 compared with patients without COVID-19. We enrolled a multisite prospective cohort of patients admitted under suspicion for COVID-19, who were then determined to be SARS-CoV-2-positive (n = 204) or -negative (n = 122). SARS-CoV-2-positive patients had higher plasma levels of CXCL10, sPD-L1, IFN-γ, CCL26, C-reactive protein (CRP), and TNF-α relative to SARS-CoV-2-negative patients adjusting for demographics and severity of illness (Bonferroni P value < 0.05). In contrast, the levels of IL-6, IL-8, IL-10, and IL-17A were not significantly different between the two groups. In SARS-CoV-2-positive patients, higher plasma levels of sPD-L1 and TNF-α were associated with fewer ventilator-free days (VFDs) and higher mortality rates (Bonferroni P value < 0.05). Lymphocyte chemoattractants such as CCL17 were associated with more severe respiratory failure in SARS-CoV-2-positive patients, but less severe respiratory failure in SARS-CoV-2-negative patients (P value for interaction < 0.01). Circulating T cells and monocytes from SARS-CoV-2-positive subjects were hyporesponsive to in vitro stimulation compared with SARS-CoV-2-negative subjects. Critically ill SARS-CoV-2-positive patients exhibit an immune signature of high interferon-induced lymphocyte chemoattractants (e.g., CXCL10 and CCL17) and immune cell hyporesponsiveness when directly compared with SARS-CoV-2-negative patients. This suggests a specific role for T-cell migration coupled with an immune-checkpoint regulatory response in COVID-19-related critical illness.

An Anti-CD3 Antibody, Teplizumab, in Relatives at Risk for Type 1 Diabetes.

BACKGROUND: Type 1 diabetes is a chronic autoimmune disease that leads to destruction of insulin-producing beta cells and dependence on exogenous insulin for survival. Some interventions have delayed the loss of insulin production in patients with type 1 diabetes, but interventions that might affect clinical progression before diagnosis are needed. METHODS: We conducted a phase 2, randomized, placebo-controlled, double-blind trial of teplizumab (an Fc receptor-nonbinding anti-CD3 monoclonal antibody) involving relatives of patients with type 1 diabetes who did not have diabetes but were at high risk for development of clinical disease. Patients were randomly assigned to a single 14-day course of teplizumab or placebo, and follow-up for progression to clinical type 1 diabetes was performed with the use of oral glucose-tolerance tests at 6-month intervals. RESULTS: A total of 76 participants (55 [72%] of whom were ≤18 years of age) underwent randomization - 44 to the teplizumab group and 32 to the placebo group. The median time to the diagnosis of type 1 diabetes was 48.4 months in the teplizumab group and 24.4 months in the placebo group; the disease was diagnosed in 19 (43%) of the participants who received teplizumab and in 23 (72%) of those who received placebo. The hazard ratio for the diagnosis of type 1 diabetes (teplizumab vs. placebo) was 0.41 (95% confidence interval, 0.22 to 0.78; P = 0.006 by adjusted Cox proportional-hazards model). The annualized rates of diagnosis of diabetes were 14.9% per year in the teplizumab group and 35.9% per year in the placebo group. There were expected adverse events of rash and transient lymphopenia. KLRG1+TIGIT+CD8+ T cells were more common in the teplizumab group than in the placebo group. Among the participants who were HLA-DR3-negative, HLA-DR4-positive, or anti-zinc transporter 8 antibody-negative, fewer participants in the teplizumab group than in the placebo group had diabetes diagnosed. CONCLUSIONS: Teplizumab delayed progression to clinical type 1 diabetes in high-risk participants. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01030861.).

Single Nucleotide Variant in FAS Associates With Organ Failure and Soluble Fas Cell Surface Death Receptor in Critical Illness.

OBJECTIVES: Multiple organ failure in critically ill patients is associated with poor prognosis, but biomarkers contributory to pathogenesis are unknown. Previous studies support a role for Fas cell surface death receptor (Fas)-mediated apoptosis in organ dysfunction. Our objectives were to test for associations between soluble Fas and multiple organ failure, identify protein quantitative trait loci, and determine associations between genetic variants and multiple organ failure. DESIGN: Retrospective observational cohort study. SETTING: Four academic ICUs at U.S. hospitals. PATIENTS: Genetic analyses were completed in a discovery (n = 1,589) and validation set (n = 863). Fas gene expression and flow cytometry studies were completed in outpatient research participants (n = 250). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In discovery and validation sets of critically ill patients, we tested for associations between enrollment plasma soluble Fas concentrations and Sequential Organ Failure Assessment score on day 3. We conducted a genome-wide association study of plasma soluble Fas (discovery n = 1,042) and carried forward a single nucleotide variant in the FAS gene, rs982764, for validation (n = 863). We further tested whether the single nucleotide variant in FAS (rs982764) was associated with Sequential Organ Failure Assessment score, FAS transcriptional isoforms, and Fas cell surface expression. Higher plasma soluble Fas was associated with higher day 3 Sequential Organ Failure Assessment scores in both the discovery (ß = 4.07; p < 0.001) and validation (ß = 6.96; p < 0.001) sets. A single nucleotide variant in FAS (rs982764G) was associated with lower plasma soluble Fas concentrations and lower day 3 Sequential Organ Failure Assessment score in meta-analysis (-0.21; p = 0.02). Single nucleotide variant rs982764G was also associated with a lower relative expression of the transcript for soluble as opposed to transmembrane Fas and higher cell surface expression of Fas on CD4+ T cells. CONCLUSIONS: We found that single nucleotide variant rs982764G was associated with lower plasma soluble Fas concentrations in a discovery and validation population, and single nucleotide variant rs982764G was also associated with lower organ dysfunction on day 3. These findings support further study of the Fas pathway as a potential mediator of organ dysfunction in critically ill patients.

Clinical and experimental treatment of type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease resulting in the destruction of the insulin-producing pancreatic beta cells. Disease progression occurs along a trajectory from genetic risk, the development of islet autoantibodies, and autoreactive T cells ultimately progressing to clinical disease. Natural history studies and mechanistic studies linked to clinical trials have provided insight into the role of the immune system in disease pathogenesis. Here, we review our current understanding of the underlying etiology of T1D, focusing on the immune cell types that have been implicated in progression from pre-symptomatic T1D to clinical diagnosis and established disease. This knowledge has been foundational for the development of immunotherapies aimed at the prevention and treatment of T1D.

A simple strategy for sample annotation error detection in cytometry datasets.

Mislabeling samples or data with the wrong participant information can affect study integrity and lead investigators to draw inaccurate conclusions. Quality control to prevent these types of errors is commonly embedded into the analysis of genomic datasets, but a similar identification strategy is not standard for cytometric data. Here, we present a method for detecting sample identification errors in cytometric data using expression of human leukocyte antigen (HLA) class I alleles. We measured HLA-A*02 and HLA-B*07 expression in three longitudinal samples from 41 participants using a 33-marker CyTOF panel designed to identify major immune cell types. 3/123 samples (2.4%) showed HLA allele expression that did not match their longitudinal pairs. Furthermore, these same three samples' cytometric signature did not match qPCR HLA class I allele data, suggesting that they were accurately identified as mismatches. We conclude that this technique is useful for detecting sample-labeling errors in cytometric analyses of longitudinal data. This technique could also be used in conjunction with another method, like GWAS or PCR, to detect errors in cross-sectional data. We suggest widespread adoption of this or similar techniques will improve the quality of clinical studies that utilize cytometry.

Weekly injection of IL-2 using an injectable hydrogel reduces autoimmune diabetes incidence in NOD mice.

AIMS/HYPOTHESIS: IL-2 injections are a promising therapy for autoimmune type 1 diabetes but the short half-life of this cytokine in vivo limits effective tissue exposure and necessitates frequent injections. Here we have investigated whether an injectable hydrogel could be used to promote prolonged IL-2 release in vivo. METHODS: Capitalising on the IL-2-binding capabilities of heparin, an injectable hydrogel incorporating clinical-grade heparin, collagen and hyaluronan polymers was used to deliver IL-2. The IL-2-release kinetics and in vivo stability of this material were examined. The ability of soluble IL-2 vs hydrogel-mediated IL-2 injections to prevent autoimmune diabetes in the NOD mouse model of type 1 diabetes were compared. RESULTS: We observed in vitro that the hydrogel released IL-2 over a 12-day time frame and that injected hydrogel likewise persisted 12 days in vivo. Notably, heparin binding potentiates the activity of IL-2 and enhances IL-2- and TGFß-mediated expansion of forkhead box P3-positive regulatory T cells (FOXP3+ Tregs). Finally, weekly administration of IL-2-containing hydrogel partially prevented autoimmune diabetes while injections of soluble IL-2 did not. CONCLUSIONS/INTERPRETATION: Hydrogel delivery may reduce the number of injections required in IL-2 treatment protocols for autoimmune diabetes. Graphical abstract.

Efficient CRISPR/Cas9 Disruption of Autoimmune-Associated Genes Reveals Key Signaling Programs in Primary Human T Cells.

Risk of autoimmunity is associated with multiple genetic variants. Genome-wide association studies have linked single-nucleotide polymorphisms in the phosphatases PTPN22 (rs2476601) and PTPN2 (rs1893217) to increased risk for multiple autoimmune diseases. Previous mouse studies of loss of function or risk variants in these genes revealed hyperactive T cell responses, whereas studies of human lymphocytes revealed contrasting phenotypes. To better understand this dichotomy, we established a robust gene editing platform to rapidly address the consequences of loss of function of candidate genes in primary human CD4+ T cells. Using CRISPR/Cas9, we obtained efficient gene disruption (>80%) of target genes encoding proteins involved in Ag and cytokine receptor signaling pathways including PTPN22 and PTPN2 Loss-of-function data in all genes studied correlated with previous data from mouse models. Further analyses of PTPN2 gene-disrupted T cells demonstrated dynamic effects, by which hyperactive IL-2R signaling promoted compensatory transcriptional events, eventually resulting in T cells that were hyporesponsive to IL-2. These results imply that altered phosphatase activity promotes evolving phenotypes based on Ag experience and/or other programming signals. This approach enables the discovery of molecular mechanisms modulating risk of autoimmunity that have been difficult to parse in traditional mouse models or cross-sectional human studies.

Treatment of type 1 diabetes with teplizumab: clinical and immunological follow-up after 7 years from diagnosis.

AIMS/HYPOTHESIS: The long-term effects of successful immune therapies for treatment of type 1 diabetes have not been well studied. The Autoimmunity-Blocking Antibody for Tolerance (AbATE) trial evaluated teplizumab, an Fc receptor non-binding humanised anti-CD3 monoclonal antibody in individuals with new-onset type 1 diabetes, and ended in 2011. Clinical drug-treated responders showed an increased frequency of 'partially exhausted' CD8+ T cells. We studied the clinical, immunological and metabolic status of participants after an average follow-up of 7 years. METHODS: Participants with detectable C-peptide at year 2 of AbATE returned for follow-up. C-peptide responses were assessed by 4 h mixed-meal tolerance test. Autoantibodies and HbA1c levels were measured and average daily insulin use was obtained from patient logs. Peripheral blood mononuclear cells were analysed by flow cytometry and cytokine release. RESULTS: Fifty-six per cent of the original participants returned. Three of the original control group who did not return had lost all detectable C-peptide by the end of the 2 year trial. The C-peptide responses to a mixed-meal tolerance test were similar overall in the drug vs control group of participants but were significantly improved, with less loss of C-peptide, in drug-treated responders identified at 1 year. However, the improvements in C-peptide response were not associated with lower HbA1c levels or insulin use. Drug-treated responders showed a significantly increased frequency of programmed cell death protein 1-positive central memory and anergic CD8+ T cells at follow-up. CONCLUSIONS/INTERPRETATION: These findings suggest there is reduced decline in C-peptide and persistent immunological responses up to 7 years after diagnosis of diabetes in individuals who respond to teplizumab. TRIAL REGISTRATION: ClinicalTrials.gov NCT02067923; the protocol is available at www.immunetolerance.org (ITN027AI).

Circulating integrin alpha4/beta7+ lymphocytes targeted by vedolizumab have a pro-inflammatory phenotype.

Integrin alpha4/beta7 on circulating lymphocytes identifies them as gut-tropic, and can be targeted by the humanized antibody vedolizumab to treat inflammatory bowel disease (IBD). We found lymphocytes expressing alpha4/beta7 were significantly more responsive to the pro-inflammatory cytokines IL-6, IL-7, and IL-21, and less responsive to the regulatory T cell (Treg)-supporting cytokine IL-2. Alpha4/beta7 was expressed by a smaller percent of FOXP3 + Helios+ thymically-derived Tregs (tTregs) than FOXP3 + Helios- peripherally-derived Tregs (pTregs) or FOXP3- effector T cells. Integrin alpha4/beta7+ CD4 T cells were also rare among cells expressing the Th2 marker CRTh2, but enriched in cells bearing the circulating T follicular helper cell marker CXCR5. Thus the effect of this anti-integrin therapy on the mucosal immune system may be more qualitative than quantitative, and selectively replace pro-inflammatory effector cells with Tregs and Th2 cells to facilitate immune tolerance in the mucosa without globally depleting lymphocytes from the intestinal mucosa.

Attenuated IL-2R signaling in CD4 memory T cells of T1D subjects is intrinsic and dependent on activation state.

The IL-2/IL-2R pathway is implicated in type 1 diabetes (T1D). While its role in regulatory T cell (Treg) biology is well characterized, mechanisms that influence IL-2 responses in effector T cells (Teff) are less well understood. We compared IL-2 responses in 95 healthy control and 98 T1D subjects. In T1D, low IL-2 responsiveness was most pronounced in memory Teff. Unlike Treg, CD25 expression did not influence the Teff responses. Reduced IL-2 responses in memory Teff were not rescued by resting, remained lower after activation and proliferation, and were absent in type 2 diabetes. Comparing basal IL-2 responses in resting versus activated cells, memory Teff displayed lower, but more sustained, responses to IL-2 overtime. These results suggest that T1D-associated defects in the Teff compartment are due to intrinsic factors related to activation. Evaluation of both Teff and Treg IL-2R signaling defects in T1D subjects may inform selection of therapies.

Remodeling T cell compartments during anti-CD3 immunotherapy of type 1 diabetes.

The immunological mechanism(s) of action whereby teplizumab preserves C-peptide levels in the progression of patients with recent onset type 1 diabetes (T1D) is still not well understood. In the present study, we evaluated the kinetics of T cell modulation in peripheral blood following two 14-day courses of teplizumab therapy one year apart in recent onset T1D participants in the AbATE clinical trial. Transient rises in PD-1+Foxp3+ Treg and potentially anergic (CD57-KLRG1-PD-1+) cells in the circulating CD4 T cell compartment were paralleled by more profound increases in circulating CD8 T cells with traits of exhaustion (CD57-KLRG1+PD-1+, TIGIT+KLRG1+, and persistent down-modulation of CD127). The observed phenotypic changes across cell types were associated with favorable response to treatment in the subgroup of study participants that did not develop anti-drug antibodies after the first course of therapy. These findings provide new insights on the duration and complexity of T cell modulation with teplizumab therapy in recent onset T1D, and in addition, suggest that coordinated immune mechanisms of tolerance that favor CD4 Treg function and restrain CD4 non-Treg and CD8 T cell activation may contribute to treatment success.

Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients.

Most autoimmune diseases (AID) are linked to an imbalance between autoreactive effector T cells (Teffs) and regulatory T cells (Tregs). While blocking Teffs with immunosuppression has long been the only therapeutic option, activating/expanding Tregs may achieve the same objective without the toxicity of immunosuppression. We showed that low-dose interleukin-2 (ld-IL-2) safely expands/activates Tregs in patients with AID, such HCV-induced vasculitis and Type 1 Diabetes (T1D). Here we analyzed the kinetics and dose-relationship of IL-2 effects on immune responses in T1D patients. Ld-IL-2 therapy induced a dose-dependent increase in CD4(+)Foxp3(+) and CD8(+)Foxp3(+) Treg numbers and proportions, the duration of which was markedly dose-dependent. Tregs expressed enhanced levels of activation markers, including CD25, GITR, CTLA-4 and basal pSTAT5, and retained a 20-fold higher sensitivity to IL-2 than Teff and NK cells. Plasma levels of regulatory cytokines were increased in a dose-dependent manner, while cytokines linked to Teff and Th17 inflammatory cells were mostly unchanged. Global transcriptome analyses showed a dose-dependent decrease in immune response signatures. At the highest dose, Teff responses against beta-cell antigens were suppressed in all 4 patients tested. These results inform of broader changes induced by ld-IL-2 beyond direct effects on Tregs, and relevant for further development of ld-IL-2 for therapy and prevention of T1D, and other autoimmune and inflammatory diseases.

Renegade homeostatic cytokine responses in T1D: drivers of regulatory/effector T cell imbalance.

Homeostatic cytokines contribute to the balance between regulatory and effector T cells (Tregs and Teffs respectively) and are necessary to maintain peripheral tolerance. These cytokines include IL-2 that supports Treg and IL-7 and IL-15 that drive Teff. In overt settings of lost tolerance (i.e. graft rejection), IL-2 Treg signatures are decreased while IL-7 and IL-15 Teff signatures are often enhanced. Similar cytokine profile imbalances also occur in some autoimmune diseases. In type 1 diabetes (T1D), there are underlying defects in the IL-2 pathway and Teff cytokine blockade can prevent and treat diabetes in NOD mice. In this review, we summarize evidence of IL-2, IL-7 and IL-15 genetic and cellular alterations in T1D patients. We then discuss how the combined effect of these cytokine profiles may together contribute to altered Treg/Teff ratios and functions in T1D. Implications for combination therapies and suggestions for integrated cytokine and Treg/Teff biomarker development are then proposed.

ECM components guide IL-10 producing regulatory T-cell (TR1) induction from effector memory T-cell precursors.

We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3- IL-10-producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10-dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4(+)CD62L(-)FoxP3(-), suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation.