Residual methylprednisolone suppresses human T-cell responses to spleen, but not islet, extracts from deceased organ donors.

Pancreatic islets, transplanted into recipients with type 1 diabetes, are exposed to allogenic and auto-immune T-cell responses. We set out to develop an assay to measure these responses using PBMC. Our approach was to prepare spleen extract from the islet donors (allo-antigen) and islet extracts (auto-antigen). To our surprise, we found that spleen extracts potently inhibited the proliferation of human T cells driven by antigen (tetanus toxoid) and mitogen (anti-CD3 mAb, OKT3), whereas extracts prepared from pancreatic islets from the same donor did not suppress T-cell proliferation. Suppression mediated by spleen extracts was unaffected by blocking mAbs against the IL-10R, transforming growth factor-ß or CD152 (CTLA-4). It was also unaffected by denaturing the spleen extracts by heating, exposing to reducing agents or protease digestion. Because deceased organ donors are commonly given the immunosuppressive glucocorticoid methylprednisolone prior to death, we hypothesized that suppression was due to residual methylprednisolone in the spleen extracts. Methylprednisolone could be detected by mass spectrometry in spleen extracts at concentrations that suppress T-cell proliferation. Finally, the glucocorticoid receptor antagonist mifepristone completely reversed the suppression caused by the spleen extracts. We conclude that extracts of human spleen, but not islets, from deceased organ donors contain sufficient residual methylprednisolone to suppress the proliferation of T-cells in vitro.

Ambulatory intravenous insulin and islet cell transplantation to treat severe type III insulin hypersensitivity in a patient with type 1 diabetes mellitus.

Allogenic pancreatic islet cell transplantation is an appropriate treatment option to consider in the management of refractory cases of severe hypersensitivity to insulin in patients with type 1 diabetes mellitus.

Unresponsiveness to inhaled antigen is governed by conventional dendritic cells and overridden during infection by monocytes.

The nasal-associated lymphoid tissues (NALTs) are mucosal-associated lymphoid organs embedded in the submucosa of the nasal passage. NALTs represent a known site for the deposition of inhaled antigens, but little is known of the mechanisms involved in the induction of immunity within this lymphoid tissue. We find that during the steady state, conventional dendritic cells (cDCs) within the NALTs suppress T cell responses. These cDCs, which are also prevalent within human NALTs (tonsils/adenoids), express a unique transcriptional profile and inhibit T cell proliferation via contact-independent mechanisms that can be diminished by blocking the actions of reactive oxygen species and prostaglandin E2 Although the prevention of unrestrained immune activation to inhaled antigens appears to be the default function of NALT cDCs, inflammation after localized virus infection recruited monocyte-derived DCs (moDCs) to this region, which diluted out the suppressive DC pool, and permitted local T cell priming. Accommodating for inflammation-induced temporal changes in NALT DC composition and function, we developed an intranasal vaccine delivery system that coupled the recruitment of moDCs with the sustained release of antigen into the NALTs, and we were able to substantially improve T cell responses after intranasal immunization. Thus, homeostasis and immunity to inhaled antigens is tuned by inflammatory signals that regulate the balance between conventional and moDC populations within the NALTs.

Human γδ T-cell receptor repertoire is shaped by influenza viruses, age and tissue compartmentalisation.

BACKGROUND: Although γδ T cells comprise up to 10% of human peripheral blood T cells, questions remain regarding their role in disease states and T-cell receptor (TCR) clonal expansions. We dissected anti-viral functions of human γδ T cells towards influenza viruses and defined influenza-reactive γδ TCRs in the context of γδ-TCRs across the human lifespan. METHODS: We performed 51Cr-killing assay and single-cell time-lapse live video microscopy to define mechanisms underlying γδ T-cell-mediated killing of influenza-infected targets. We assessed cytotoxic profiles of γδ T cells in influenza-infected patients and IFN-γ production towards influenza-infected lung epithelial cells. Using single-cell RT-PCR, we characterised paired TCRγδ clonotypes for influenza-reactive γδ T cells in comparison with TCRs from healthy neonates, adults, elderly donors and tissues. RESULTS: We provide the first visual evidence of γδ T-cell-mediated killing of influenza-infected targets and show distinct features to those reported for CD8+ T cells. γδ T cells displayed poly-cytotoxic profiles in influenza-infected patients and produced IFN-γ towards influenza-infected cells. These IFN-γ-producing γδ T cells were skewed towards the γ9δ2 TCRs, particularly expressing the public GV9-TCRγ, capable of pairing with numerous TCR-δ chains, suggesting their significant role in γδ T-cell immunity. Neonatal γδ T cells displayed extensive non-overlapping TCRγδ repertoires, while adults had enriched γ9δ2-pairings with diverse CDR3γδ regions. Conversely, the elderly showed distinct γδ-pairings characterised by large clonal expansions, a profile also prominent in adult tissues. CONCLUSION: Human TCRγδ repertoire is shaped by age, tissue compartmentalisation and the individual's history of infection, suggesting that these somewhat enigmatic γδ T cells indeed respond to antigen challenge.

Influenza-specific lung-resident memory T cells are proliferative and polyfunctional and maintain diverse TCR profiles.

The human lung harbors a large population of resident memory T cells (Trm cells). These cells are perfectly positioned to mediate rapid protection against respiratory pathogens such as influenza virus, a highly contagious respiratory pathogen that continues to be a major public health burden. Animal models show that influenza-specific lung CD8+ Trm cells are indispensable for crossprotection against pulmonary infection with different influenza virus strains. However, it is not known whether influenza-specific CD8+ Trm cells present within the human lung have the same critical role in modulating the course of the disease. Here, we showed that human lung contains a population of CD8+ Trm cells that are highly proliferative and have polyfunctional progeny. We observed that different influenza virus-specific CD8+ T cell specificities differentiated into Trm cells with varying efficiencies and that the size of the influenza-specific CD8+ T cell population persisting in the lung directly correlated with the efficiency of differentiation into Trm cells. To our knowledge, we provide the first ex vivo dissection of paired T cell receptor (TCR) repertoires of human influenza-specific CD8+ Trm cells. Our data reveal diverse TCR profiles within the human lung Trm cells and a high degree of clonal sharing with other CD8+ T cell populations, a feature important for effective T cell function and protection against the generation of viral-escape mutants.

Islet Transplantation Provides Superior Glycemic Control With Less Hypoglycemia Compared With Continuous Subcutaneous Insulin Infusion or Multiple Daily Insulin Injections.

BACKGROUND: The aim was to compare efficacy of multiple daily injections (MDI), continuous subcutaneous insulin infusion (CSII) and islet transplantation to reduce hypoglycemia and glycemic variability in type 1 diabetes subjects with severe hypoglycemia. METHODS: This was a within-subject, paired comparison of MDI and CSII and CSII with 12 months postislet transplantation in 10 type 1 diabetes subjects referred with severe hypoglycemia, suitable for islet transplantation. Individuals were assessed with HbA1c, Edmonton Hypoglycemia Score (HYPOscore), continuous glucose monitoring (CGM) and in 8 subjects measurements of glucose variability using standard deviation of glucose (SD glucose) from CGM and continuous overlapping net glycemic action using a 4 hour interval (CONGA4). RESULTS: After changing from MDI to CSII before transplantation, 10 subjects reduced median HYPOscore from 2028 to 1085 (P < 0.05) and hypoglycemia events from 24 to 8 per patient-year (P < 0.05). While HbA1c, mean glucose and median percent time hypoglycemic on CGM were unchanged with CSII, SD glucose and CONGA4 reduced significantly (P < 0.05). At 12 months posttransplant 9 of 10 were C-peptide positive, (5 insulin independent). Twelve months postislet transplantation, there were significant reductions in all baseline parameters versus CSII, respectively, HbA1c (6.4% cf 8.2%), median HYPOscore (0 cf 1085), mean glucose (7.1 cf 8.6 mmol L), SD glucose (1.7 cf 3.2 mmol/L), and CONGA4 (1.6 cf 3.0). CONCLUSIONS: In subjects with severe hypoglycemia suitable for islet transplantation, CSII decreased hypoglycemia frequency and glycemic variability compared with MDI whereas islet transplantation resolved hypoglycemia and further improved glycemic variability regardless of insulin independence.

Nuclear factor κB-inducing kinase activation as a mechanism of pancreatic ß cell failure in obesity.

The nuclear factor κB (NF-κB) pathway is a master regulator of inflammatory processes and is implicated in insulin resistance and pancreatic ß cell dysfunction in the metabolic syndrome. Whereas canonical NF-κB signaling is well studied, there is little information on the divergent noncanonical NF-κB pathway in the context of pancreatic islet dysfunction. Here, we demonstrate that pharmacological activation of the noncanonical NF-κB-inducing kinase (NIK) disrupts glucose homeostasis in zebrafish in vivo. We identify NIK as a critical negative regulator of ß cell function, as pharmacological NIK activation results in impaired glucose-stimulated insulin secretion in mouse and human islets. NIK levels are elevated in pancreatic islets isolated from diet-induced obese (DIO) mice, which exhibit increased processing of noncanonical NF-κB components p100 to p52, and accumulation of RelB. TNF and receptor activator of NF-κB ligand (RANKL), two ligands associated with diabetes, induce NIK in islets. Mice with constitutive ß cell-intrinsic NIK activation present impaired insulin secretion with DIO. NIK activation triggers the noncanonical NF-κB transcriptional network to induce genes identified in human type 2 diabetes genome-wide association studies linked to ß cell failure. These studies reveal that NIK contributes a central mechanism for ß cell failure in diet-induced obesity.

A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy.

MicroRNAs are now increasingly recognized as biomarkers of disease progression. Several quantitative real-time PCR (qPCR) platforms have been developed to determine the relative levels of microRNAs in biological fluids. We systematically compared the detection of cellular and circulating microRNA using a standard 96-well platform, a high-content microfluidics platform and two ultra-high content platforms. We used extensive analytical tools to compute inter- and intra-run variability and concordance measured using fidelity scoring, coefficient of variation and cluster analysis. We carried out unprejudiced next generation sequencing to identify a microRNA signature for Diabetic Retinopathy (DR) and systematically assessed the validation of this signature on clinical samples using each of the above four qPCR platforms. The results indicate that sensitivity to measure low copy number microRNAs is inversely related to qPCR reaction volume and that the choice of platform for microRNA biomarker validation should be made based on the abundance of miRNAs of interest.