Previous efforts to preserve β cell function in individuals with type 1 diabetes (T1D) have focused largely on the use of single immunomodulatory agents administered within 100 days of diagnosis. ...Based on human and preclinical studies, we hypothesized that a combination of low-dose anti-thymocyte globulin (ATG) and pegylated granulocyte CSF (G-CSF) would preserve β cell function in patients with established T1D (duration of T1D >4 months and <2 years).
A randomized, single-blinded, placebo-controlled trial was performed on 25 subjects: 17 subjects received ATG (2.5 mg/kg intravenously) followed by pegylated G-CSF (6 mg subcutaneously every 2 weeks for 6 doses) and 8 subjects received placebo. The primary outcome was the 1-year change in AUC C-peptide following a 2-hour mixed-meal tolerance test (MMTT). At baseline, the age (mean ± SD) was 24.6 ± 10 years; mean BMI was 25.4 ± 5.2 kg/m²; mean A1c was 6.5% ± 1.1%; insulin use was 0.31 ± 0.22 units/kg/d; and length of diagnosis was 1 ± 0.5 years.
Combination ATG/G-CSF treatment tended to preserve β cell function in patients with established T1D. The mean difference in MMTT-stimulated AUC C-peptide between treated and placebo subjects was 0.28 nmol/l/min (95% CI 0.001-0.552, P = 0.050). A1c was lower in ATG/G-CSF-treated subjects at the 6-month study visit. ATG/G-CSF therapy was associated with relative preservation of Tregs.
Patients with established T1D may benefit from combination immunotherapy approaches to preserve β cell function. Further studies are needed to determine whether such approaches may prevent or delay the onset of the disease.
Clinicaltrials.gov NCT01106157.
The Leona M. and Harry B. Helmsley Charitable Trust and Sanofi.
N6.2 releases nano-sized vesicles (NVs) with distinct protein and lipid contents. We hypothesized that these NVs play a central role in the delivery of bioactive molecules that may act as mechanistic ...effectors in immune modulation. In this report, we observed that addition of NVs to the human pancreatic cell line βlox5 reduced cytokine-induced apoptosis. Through RNAseq analyses, increased expression of
, and
genes in the aryl hydrocarbon receptor (AHR) pathways were found to be significantly induced in presence of NVs. AHR nuclear translocation was confirmed by confocal microscopy. The role of NVs on beta cell function was further evaluated using primary human pancreatic islets. It was found that NVs significantly increased insulin secretion in presence of high glucose concentrations. These increases positively correlated with increased
and
and coincided with reduced oxidative stress markers. Furthermore, incubation of NVs with THP-1 macrophages promoted the M2 tolerogenic phenotype through STAT3 activation, expression of AHR-dependent genes and secretion of IL10. Altogether, our findings indicate that bacterial NVs have the potential to modulate glucose homeostasis in the host by directly affecting insulin secretion by islets and through the induction of a tolerogenic immune phenotype.
Most natural history models for type 1 diabetes (T1D) propose that overt hyperglycemia results after a progressive loss of insulin-secreting β-cell mass and/or function. To experimentally address ...this concept, we prospectively determined morning blood glucose measurements every other day in multiple cohorts (total n = 660) of female NOD/ShiLtJ mice starting at 8 weeks of age until diabetes onset or 26 weeks of age. Consistent with this notion, a majority of mice that developed diabetes (354 of 489 72%) displayed a progressive increase in blood glucose with transient excursions >200 mg/dL, followed by acute and persistent hyperglycemia at diabetes onset. However, 135 of the 489 (28%) diabetic animals demonstrated normal glucose values followed by acute (i.e., sudden) hyperglycemia. Interestingly, diabetes onset occurred earlier in mice with acute versus progressive disease onset (15.37 ± 0.3207 vs. 17.44 ± 0.2073 weeks of age, P < 0.0001). Moreover, the pattern of onset (i.e., progressive vs. acute) dramatically influenced the ability to achieve reversal of T1D by immunotherapeutic intervention, with increased effectiveness observed in situations of a progressive deterioration in euglycemia. These studies highlight a novel natural history aspect in this animal model, one that may provide important guidance for the selection of subjects participating in human trials seeking disease reversal.
The autoimmune pathogenesis of type 1 diabetes (T1D) involves cellular infiltration from innate and adaptive immune subsets into the islets of Langerhans within the pancreas; however, the direct ...cytotoxic killing of insulin-producing β-cells is thought to be mediated primarily by antigen-specific CD8
T cells. Despite this direct pathogenic role, key aspects of their receptor specificity and function remain uncharacterized, in part, due to their low precursor frequency in peripheral blood. The concept of engineering human T cell specificity, using T cell receptor (TCR) and chimeric antigen receptor (CAR)-based approaches, has been demonstrated to improve adoptive cell therapies for cancer, but has yet to be extensively employed for modeling and treating autoimmunity. To address this limitation, we sought to combine targeted genome editing of the endogenous TCRα chain gene (
) via CRISPR/Cas9 in combination with lentiviral vector (LV)-mediated TCR gene transfer into primary human CD8
T cells. We observed that knockout (KO) of endogenous
enhanced
TCR pairing, which permitted increased peptide:MHC-dextramer staining. Moreover,
KO and TCR gene transfer increased markers of activation and effector function following activation, including granzyme B and interferon-γ production. Importantly, we observed increased cytotoxicity toward an HLA-A*0201
human β-cell line by HLA-A*02:01 restricted CD8
T cells engineered to recognize islet-specific glucose-6-phosphatase catalytic subunit (IGRP). These data support the notion of altering the specificity of primary human T cells for mechanistic analyses of autoreactive antigen-specific CD8
T cells and are expected to facilitate downstream cellular therapeutics to achieve tolerance induction through the generation of antigen-specific regulatory T cells.
Events defining the progression to human type 1 diabetes (T1D) have remained elusive owing to the complex interaction between genetics, the immune system, and the environment. Type 1 interferons ...(T1-IFN) are known to be a constituent of the autoinflammatory milieu within the pancreas of patients with T1D. However, the capacity of IFNα/β to modulate human activated autoreactive CD8
T-cell (cytotoxic T lymphocyte) responses within the islets of patients with T1D has not been investigated. Here, we engineer human β-cell-specific cytotoxic T lymphocytes and demonstrate that T1-IFN augments cytotoxicity by inducing rapid phosphorylation of STAT4, resulting in direct binding at the granzyme B promoter within 2 h of exposure. The current findings provide novel insights concerning the regulation of effector function by T1-IFN in human antigen-experienced CD8
T cells and provide a mechanism by which the presence of T1-IFN potentiates diabetogenicity within the autoimmune islet.
BackgroundCheckpoint inhibitor immunotherapy is becoming an effective treatment modality for an increasing number of malignancies. As a result, autoinflammatory side-effects are also being observed ...more commonly in the clinic. We are currently unable to predict which patients will develop more severe toxicities associated with these treatment regimens.Case presentationWe present a patient with stage IV melanoma that developed rapid onset autoimmune type 1 diabetes (T1D) in response to combination ipilimumab and nivolumab immunotherapy. At the time of the patient’s presentation with diabetes ketoacidosis, a confirmed anti-GAD antibody seroconversion was noted. Longer-term follow-up of this patient has demonstrated a durable complete response based on PET CT imaging along with a persistently undetectable C-peptide level. Single nucleotide polymorphism gene sequencing and HLA risk allele analysis has revealed the patient to lack any established genetic predisposition to the development of autoimmune T1D.ConclusionsWhile larger studies are necessary to better understand the role of genetic risk factors for the development of autoimmune toxicities in those patients undergoing checkpoint inhibitor immunotherapy, these results suggest that pre-screening patients for known T1D risk alleles may not be indicated. Additional investigation is needed to determine whether an approach such as T cell receptor clonotypic analysis to identify the presence of autoreactive T cell clones may be an effective approach for predicting which patients are at risk for the development of autoinflammatory toxicities while undergoing checkpoint inhibitor immunotherapy.
Oxidants participate in lymphocyte activation and function. We previously demonstrated that eliminating the activity of NADPH oxidase 2 (NOX2) significantly impaired the effectiveness of autoreactive ...CD8+ CTLs. However, the molecular mechanisms impacting CD8+ T cell function remain unknown. In the present study, we examined the role of NOX2 in both NOD mouse and human CD8+ T cell function. Genetic ablation or chemical inhibition of NOX2 in CD8+ T cells significantly suppressed activation-induced expression of the transcription factor T-bet, the master transcription factor of the Tc1 cell lineage, and T-bet target effector genes such as IFN-γ and granzyme B. Inhibition of NOX2 in both human and mouse CD8+ T cells prevented target cell lysis. We identified that superoxide generated by NOX2 must be converted into hydrogen peroxide to transduce the redox signal in CD8+ T cells. Furthermore, we show that NOX2-generated oxidants deactivate the tumor suppressor complex leading to activation of RheB and subsequently mTOR complex 1. These results indicate that NOX2 plays a nonredundant role in TCR-mediated CD8+ T cell effector function.
Low-dose antithymocyte globulin (ATG) (2.5 mg/kg) preserves C-peptide and reduces HbA1c in new-onset stage 3 type 1 diabetes, yet efficacy in delaying progression from stage 2 to stage 3 has not been ...evaluated.
Children (n = 6) aged 5-14 years with stage 2 type 1 diabetes received off-label, low-dose ATG. HbA1c, C-peptide, continuous glucose monitoring, insulin requirements, and side effects were followed for 18-48 months.
Three subjects (50%) remained diabetes free after 1.5, 3, and 4 years of follow-up, while three developed stage 3 within 1-2 months after therapy. Eighteen months posttreatment, even disease progressors demonstrated near-normal HbA1c (5.1% 32 mmol/mol, 5.6% 38 mmol/mol, and 5.3% 34 mmol/mol), time in range (93%, 88%, and 98%), low insulin requirements (0.17, 0.18, and 0.34 units/kg/day), and robust C-peptide 90 min after mixed meal (1.3 ng/dL, 2.3 ng/dL, and 1.4 ng/dL).
These observations support additional prospective studies evaluating ATG in stage 2 type 1 diabetes.
Insulitis involves the infiltration of predominantly CD3+ T cells in islets in a patchy distribution between lobes in a pancreas. We hypothesized that gene expression profiling of insulin containing ...islets (INS+CD3-) vs. insulin containing insulitic islets (INS+CD3+) would show heterogeneity in autoimmune and ßcell dysfunction pathways within the same donor. Pancreatic islets were obtained by laser-capture microdissection from nPOD donors (5 autoantibody positive nondiabetic (AAb+) and 6 T1D) . Transcriptome data was collected and compared in 83 individual islets. INS+CD3+ islets were compared to the average of all INS+CD3- islets in each donor. For each INS+CD3+ islet we created two registries of genes >2-fold up or >2-fold downregulated that were analyzed to detect differences in pathways and ontologies. Islets were analyzed based on five categories: immunity, mitochondrial, metabolism, secretion, and cell signaling. A majority of donors (64%) had all 5 categories represented in either up or downregulated pathways. The most upregulated category in T1D donor islets was immunity (39%) and most downregulated was secretion (33%) . These islets were distinguished by extreme heterogeneity in signaling pathways. In AAb+ donor islets, the most upregulated category was immunity (32%) , with the most downregulated noted as metabolism (21%) . Similarly, AAb+ donor islets had heterogeneity in signaling pathways. In contrast, we observed great intra-donor heterogeneity; each individual islet had 65% unique genes upregulated and 58% downregulated compared to other INS+CD3+ islets within the same donor.
In summary, similar themes of up or downregulated categories were noted in INS+CD3+ compared to averaged INS+CD3- islets. Most strikingly, significant heterogeneity between INS+CD3+ islets within donors at the individual gene levels was observed. These findings indicate substantial complexity of pancreatic islet destruction leading to T1D.
Disclosure
A.M.Dye: None. G.Ashbery: None. N.I.Lenchik: n/a. M.A.Atkinson: None. C.E.Mathews: None. M.Campbell-thompson: Consultant; TRex Bio. I.C.Gerling: None.
Funding
NIH-NIDDK (HIRN-CBDS) UC4 DK104155-01LeBonheur Children’s Foundation Research Institute (CFRI) Fellows Research Grant
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