Extraislet expression of islet antigen boosts T cell exhaustion to partially prevent autoimmune diabetes.

Persistent antigen exposure results in the differentiation of functionally impaired, also termed exhausted, T cells which are maintained by a distinct population of precursors of exhausted T (TPEX) cells. T cell exhaustion is well studied in the context of chronic viral infections and cancer, but it is unclear whether and how antigen-driven T cell exhaustion controls progression of autoimmune diabetes and whether this process can be harnessed to prevent diabetes. Using nonobese diabetic (NOD) mice, we show that some CD8+ T cells specific for the islet antigen, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) displayed terminal exhaustion characteristics within pancreatic islets but were maintained in the TPEX cell state in peripheral lymphoid organs (PLO). More IGRP-specific T cells resided in the PLO than in islets. To examine the impact of extraislet antigen exposure on T cell exhaustion in diabetes, we generated transgenic NOD mice with inducible IGRP expression in peripheral antigen-presenting cells. Antigen exposure in the extraislet environment induced severely exhausted IGRP-specific T cells with reduced ability to produce interferon (IFN)γ, which protected these mice from diabetes. Our data demonstrate that T cell exhaustion induced by delivery of antigen can be harnessed to prevent autoimmune diabetes.

Imaging for assessment of cancer treatment response to immune checkpoint inhibitors can be complementary in identifying hypophysitis.

Introduction: Hypophysitis is reported in 8.5%-14% of patients receiving combination immune checkpoint inhibition (cICI) but can be a diagnostic challenge. This study aimed to assess the role of routine diagnostic imaging performed during therapeutic monitoring of combination anti-CTLA-4/anti-PD-1 treatment in the identification of hypophysitis and the relationship of imaging findings to clinical diagnostic criteria. Methods: This retrospective cohort study identified patients treated with cICI between January 2016 and January 2019 at a quaternary melanoma service. Medical records were reviewed to identify patients with a documented diagnosis of hypophysitis based on clinical criteria. Available structural brain imaging with magnetic resonance imaging (MRI) or computed tomography (CT) of the brain and 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography with computed tomography (FDG-PET/CT) were assessed retrospectively. The main radiological outcome measures were a relative change in pituitary size or FDG uptake temporally attributed to cICI. Results: There were 162 patients (median age 60 years, 30% female) included. A total of 100 and 134 had serial CT/MRI of the brain and FDG-PET/CT, respectively. There were 31 patients who had a documented diagnosis of hypophysitis and an additional 20 who had isolated pituitary imaging findings. The pituitary gland enlargement was mild, and the largest absolute gland size was 13 mm, with a relative increase of 7 mm from baseline. There were no cases of optic chiasm compression. Pituitary enlargement and increased FDG uptake were universally transient. High-dose glucocorticoid treatment for concurrent irAEs prevented assessment of the pituitary-adrenal axis in 90% of patients with isolated imaging findings. Conclusion: Careful review of changes in pituitary characteristics on imaging performed for assessment of therapeutic response to iICI may lead to increased identification and more prompt management of cICI-induced hypophysitis.

Increased Thyroidal Activity on Routine FDG-PET/CT after Combination Immune Checkpoint Inhibition: Temporal Associations with Clinical and Biochemical Thyroiditis.

BACKGROUND: FDG-PET/CT used for immune checkpoint inhibitor (ICI) response assessment can incidentally identify immune-related adverse events (irAEs), including thyroiditis. This study aimed to correlate the time course of FDG-PET/CT evidence of thyroiditis with clinical and biochemical evolution of thyroid dysfunction. METHODS: A retrospective review was performed by two independent blinded nuclear medicine physicians (NMPs) of thyroidal FDG uptake in 127 patients who underwent PET/CT between January 2016 and January 2019 at baseline and during treatment monitoring of combination ICI therapy for advanced melanoma. Interobserver agreement was assessed and FDG-PET/CT performance defined by a receiver-operating characteristic (ROC) curve using thyroid function tests (TFTs) as the standard of truth. Thyroid maximum standardized uptake value (SUVmax) and its temporal changes with respect to the longitudinal biochemistry were serially recorded. RESULTS: At a median of 3 weeks after commencing ICI, 43/127 (34%) had a diagnosis of thyroiditis established by abnormal TFTs. FDG-PET/CT was performed at baseline and at a median of 11 weeks (range 3-32) following the start of therapy. ROC analysis showed an area under the curve of 0.87 (95% CI 0.80, 0.94) for FDG-PET/CT for detection of thyroiditis with a positive predictive value of 93%. Among patients with biochemical evidence of thyroiditis, those with a positive FDG-PET/CT were more likely to develop overt hypothyroidism (77% versus 35%, p < 0.01). In the evaluation of the index test, there was an almost perfect interobserver agreement between NMPs of 93.7% (95% CI 89.4-98.0), kappa 0.83. CONCLUSION: Increased metabolic activity of the thyroid on routine FDG-PET/CT performed for tumoral response of patients undergoing ICI therapy is generally detected well after routine biochemical diagnosis. Elevation of FDG uptake in the thyroid is predictive of overt clinical hypothyroidism and suggests that an ongoing robust inflammatory response beyond the initial thyrotoxic phase may be indicative of thyroid destruction.

Diabetes induced by checkpoint inhibition in nonobese diabetic mice can be prevented or reversed by a JAK1/JAK2 inhibitor.

Objectives: Immune checkpoint inhibitors have achieved clinical success in cancer treatment, but this treatment causes immune-related adverse events, including type 1 diabetes (T1D). Our aim was to test whether a JAK1/JAK2 inhibitor, effective at treating spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice, can prevent diabetes secondary to PD-L1 blockade. Methods: Anti-PD-L1 antibody was injected into NOD mice to induce diabetes, and JAK1/JAK2 inhibitor LN3103801 was administered by oral gavage to prevent diabetes. Flow cytometry was used to study T cells and beta cells. Mesothelioma cells were inoculated into BALB/c mice to induce a transplantable tumour model. Results: Anti-PD-L1-induced diabetes was associated with increased immune cell infiltration in the islets and upregulated MHC class I on islet cells. Anti-PD-L1 administration significantly increased islet T cell proliferation and islet-specific CD8+ T cell numbers in peripheral lymphoid organs. JAK1/JAK2 inhibitor treatment blocked IFNγ-mediated MHC class I upregulation on beta cells and T cell proliferation mediated by cytokines that use the common γ chain receptor. As a result, anti-PD-L1-induced diabetes was prevented by JAK1/JAK2 inhibitor administered before or after checkpoint inhibitor therapy. Diabetes was also reversed when the JAK1/JAK2 inhibitor was administered after the onset of anti-PD-L1-induced hyperglycaemia. Furthermore, JAK1/JAK2 inhibitor intervention after checkpoint inhibitors did not reverse or abrogate the antitumour effects in a transplantable tumour model. Conclusion: A JAK1/JAK2 inhibitor can prevent and reverse anti-PD-L1-induced diabetes by blocking IFNγ and γc cytokine activities. Our study provides preclinical validation of JAK1/JAK2 inhibitor use in checkpoint inhibitor-induced diabetes.

Massive hypertriglyceridemia associated with paclitaxel; a case report.

This report describes a patient who developed massive hypertriglyceridemia (12,488 mg/dL or 141 mmol/L) during paclitaxel and carboplatin adjuvant chemotherapy for high grade serous fallopian tube carcinoma. Paclitaxel was thought to be the causative agent and she had normal triglyceride levels following a change to carboplatin and gemcitabine. To our knowledge, this is the highest reported triglyceride level associated with paclitaxel. Measurement of serum lipids should be considered in individuals receiving taxane chemotherapy, especially in those with type 2 diabetes mellitus or a history of dyslipidemia.

Harnessing CD8+ T-cell exhaustion to treat type 1 diabetes.

Although immune interventions have shown great promise in type 1 diabetes mellitus (T1D) clinical trials, none are yet in routine clinical use or able to achieve insulin independence in patients. In addition to this, the principles of T1D treatment remain essentially unchanged since the isolation of insulin, almost a century ago. T1D is characterized by insulin deficiency as a result of destruction of insulin-producing beta cells mediated by autoreactive T cells. Therapies that target beta-cell antigen-specific T cells are needed to prevent T1D. CD8+ T-cell exhaustion is an emerging area of research in chronic infection, cancer immunotherapy, and more recently, autoimmunity. Recent data suggest that exhausted T-cell populations are associated with improved markers of T1D. T-cell exhaustion is both characterized and mediated by inhibitory receptors. This review aims to identify which inhibitory receptors may prove useful to induce T-cell exhaustion to treat T1D and identify limitations and gaps in the current literature.

NF-κB is weakly activated in the NOD mouse model of type 1 diabetes.

Type 1 diabetes is an autoimmune disease characterised by selective destruction of pancreatic beta cells by the immune system. The transcription factor nuclear factor-kappa B (NF-κB) regulates innate and adaptive immune responses. Using gene targeting and in vitro analysis of pancreatic islets and immune cells, NF-κB activation has been implicated in type 1 diabetes development. Here we use a non-obese diabetic (NOD) mouse model that expresses a luciferase reporter of transcriptionally active NF-κB to determine its activation in vivo during development of diabetes. Increased luciferase activity was readily detected upon treatment with Toll-like receptor ligands in vitro and in vivo, indicating activation of NF-κB. However, activated NF-κB was detectable at low levels above background in unmanipulated NOD mice, but did not vary with age, despite the progression of inflammatory infiltration in islets over time. NF-κB was highly activated in an accelerated model of type 1 diabetes that requires CD4+ T cells and inflammatory macrophages. These data shed light on the nature of the inflammatory response in the development of type 1 diabetes.

An intrasellar pituitary adenoma-gangliocytoma presenting as acromegaly.

Most cases of acromegaly are due to growth hormone (GH)-secreting pituitary adenomas arising from somatotroph cells. Mixed pituitary adenoma and gangliocytoma tumours are rare and typically associated with hormonal hypersecretion, most commonly GH excess. Differentiating these mixed tumours from conventional pituitary adenomas can be difficult pre-operatively, and careful histological analysis after surgical resection is key to differentiating the two entities. There is little literature addressing the possible mechanisms for the development of mixed pituitary adenoma-gangliocytomas; however, several hypotheses have been proposed. It still remains unclear if these mixed tumours differ from a clinical perspective to pituitary adenomas; however, the additional neural component of the gangliocytoma does not appear to modify the aggressiveness or risk of recurrence after surgical resection. We report a unique case of acromegaly secondary to a mixed GH-secreting pituitary adenoma, co-existing with an intrasellar gangliocytoma. LEARNING POINTS: Acromegaly due to a mixed GH-secreting pituitary adenoma and intrasellar gangliocytoma is rare.These mixed tumours cannot be distinguished easily from ordinary pituitary adenomas on the basis of clinical, endocrine or neuroradiologic findings, and histological analysis is required for a definitive diagnosis.Surgical resection is usually sufficient to provide cure, without the need for adjuvant therapy.These mixed tumours appear to have a good prognosis although the natural history is not well defined.The pathogenesis of these mixed tumours remains debatable, and ongoing research is required.

BIM Deficiency Protects NOD Mice From Diabetes by Diverting Thymocytes to Regulatory T Cells.

Because regulatory T-cell (Treg) development can be induced by the same agonist self-antigens that induce negative selection, perturbation of apoptosis will affect both negative selection and Treg development. But how the processes of thymocyte deletion versus Treg differentiation bifurcate and their relative importance for tolerance have not been studied in spontaneous organ-specific autoimmune disease. We addressed these questions by removing a critical mediator of thymocyte deletion, BIM, in the NOD mouse model of autoimmune diabetes. Despite substantial defects in the deletion of autoreactive thymocytes, BIM-deficient NOD (NODBim(-/-)) mice developed less insulitis and were protected from diabetes. BIM deficiency did not impair effector T-cell function; however, NODBim(-/-) mice had increased numbers of Tregs, including those specific for proinsulin, in the thymus and peripheral lymphoid tissues. Increased levels of Nur77, CD5, GITR, and phosphorylated IκB-α in thymocytes from NODBim(-/-) mice suggest that autoreactive cells receiving strong T-cell receptor signals that would normally delete them escape apoptosis and are diverted into the Treg pathway. Paradoxically, in the NOD model, reduced thymic deletion ameliorates autoimmune diabetes by increasing Tregs. Thus, modulating apoptosis may be one of the ways to increase antigen-specific Tregs and prevent autoimmune disease.

Mouse pancreatic beta cells express MHC class II and stimulate CD4(+) T cells to proliferate.

Type 1 diabetes results from destruction of pancreatic beta cells by autoreactive T cells. Both CD4(+) and CD8(+) T cells have been shown to mediate beta-cell killing. While CD8(+) T cells can directly recognize MHC class I on beta cells, the interaction between CD4(+) T cells and beta cells remains unclear. Genetic association studies have strongly implicated HLA-DQ alleles in human type 1 diabetes. Here we studied MHC class II expression on beta cells in nonobese diabetic mice that were induced to develop diabetes by diabetogenic CD4(+) T cells with T-cell receptors that recognize beta-cell antigens. Acute infiltration of CD4(+) T cells in islets occurred with rapid onset of diabetes. Beta cells from islets with immune infiltration expressed MHC class II mRNA and protein. Exposure of beta cells to IFN-γ increased MHC class II gene expression, and blocking IFN-γ signaling in beta cells inhibited MHC class II upregulation. IFN-γ also increased HLA-DR expression in human islets. MHC class II(+) beta cells stimulated the proliferation of beta-cell-specific CD4(+) T cells. Our study indicates that MHC class II molecules may play an important role in beta-cell interaction with CD4(+) T cells in the development of type 1 diabetes.

Functional cytotoxic T lymphocytes against IGRP206-214 predict diabetes in the non-obese diabetic mouse.

CD8(+) T cells are prominent in autoimmune diabetes of both humans and non-obese diabetic (NOD) mice. For example, CD8(+) T cells against islet-specific glucose 6-phosphatase catalytic subunit-related protein (IGRP) can be detected readily in older NOD mice. It has been suggested that the enumeration of islet-specific CD8(+) T cells in the peripheral blood may be a predictive biomarker for autoimmune type 1 diabetes (T1D). Here, we determined the natural history of the functional endogenous IGRP(206-214)-specific cytotoxic T lymphocytes (CTLs) in NOD mice with regard to age (3- to 15-week-old pre-diabetic mice and diabetic mice) and sex. We demonstrated that in vivo IGRP(206-214)-specific CTLs significantly increased after 12 weeks of age and in vivo cytotoxicity in female NOD mice was significantly higher than in male NOD mice. To determine the in vivo IGRP(206-214)-specific CTL frequency without killing the mice, we performed splenectomies on a cohort of mice after injecting IGRP(206-214)-coated targets and then followed their diabetes progression. We found that CTL frequency correlated with future of disease onset. Thus, our data support that IGRP(206-214)-specific CTLs may be a potent biomarker for T1D.

Proinflammatory cytokines contribute to development and function of regulatory T cells in type 1 diabetes.

Type 1 diabetes is caused by immune-mediated loss of pancreatic beta cells. It has been proposed that inflammatory cytokines play a role in killing beta cells. Expression of interleukin (IL)-1 and tumor necrosis factor (TNF-α) has been detected in islets from patients with type 1 diabetes, and these cytokines can induce beta cell death in vitro. We produced nonobese diabetic (NOD) mice lacking receptors for these cytokines. Islets from mice lacking IL-1RI or TNFR1 were killed when transplanted into wild-type NOD mice, suggesting that cytokine action on beta cells is not required for killing. Mice lacking TNFR1 did not develop diabetes, and mice lacking IL-1R had delayed onset of diabetes, indicating a role for these cytokines in disease development. TNFR1-deficient mice had an increased number of CD4(+) CD25(+) FoxP3(+) regulatory T cells with enhanced suppressive capacity. IL-1 was produced at higher levels in NOD mice and resulted in dilution of suppressor function of CD4(+) CD25(+) FoxP3(+) regulatory T cells. Our data suggest that blocking inflammatory cytokines may increase the capacity of the immune system to suppress type 1 diabetes through regulatory T cells.

TNF receptor 1 deficiency increases regulatory T cell function in nonobese diabetic mice.

TNF has been implicated in the pathogenesis of type 1 diabetes. When administered early in life, TNF accelerates and increases diabetes in NOD mice. However, when administered late, TNF decreases diabetes incidence and delays onset. TNFR1-deficient NOD mice were fully protected from diabetes and only showed mild peri-insulitis. To further dissect how TNFR1 deficiency affects type 1 diabetes, these mice were crossed to ß cell-specific, highly diabetogenic TCR transgenic I-A(g7)-restricted NOD4.1 mice and Kd-restricted NOD8.3 mice. TNFR1-deficient NOD4.1 and NOD8.3 mice were protected from diabetes and had significantly less insulitis compared with wild type NOD4.1 and NOD8.3 controls. Diabetic NOD4.1 mice rejected TNFR1-deficient islet grafts as efficiently as control islets, confirming that TNFR1 signaling is not directly required for ß cell destruction. Flow cytometric analysis showed a significant increase in the number of CD4(+)CD25(+)Foxp3(+) T regulatory cells in TNFR1-deficient mice. TNFR1-deficient T regulatory cells were functionally better at suppressing effector cells than were wild type T regulatory cells both in vitro and in vivo. This study suggests that blocking TNF signaling may be beneficial in increasing the function of T regulatory cells and suppression of type 1 diabetes.

The pro-apoptotic BH3-only protein Bid is dispensable for development of insulitis and diabetes in the non-obese diabetic mouse.

Type 1 diabetes is caused by death of insulin-producing pancreatic beta cells. Beta-cell apoptosis induced by FasL may be important in type 1 diabetes in humans and in the non-obese diabetic (NOD) mouse model. Deficiency of the pro-apoptotic BH3-only molecule Bid protects beta cells from FasL-induced apoptosis in vitro. We aimed to test the requirement for Bid, and the significance of Bid-dependent FasL-induced beta-cell apoptosis in type 1 diabetes. We backcrossed Bid-deficient mice, produced by homologous recombination and thus without transgene overexpression, onto a NOD genetic background. Genome-wide single nucleotide polymorphism analysis demonstrated that diabetes-related genetic regions were NOD genotype. Transferred beta cell antigen-specific CD8+ T cells proliferated normally in the pancreatic lymph nodes of Bid-deficient mice. Moreover, Bid-deficient NOD mice developed type 1 diabetes and insulitis similarly to wild-type NOD mice. Our data indicate that beta-cell apoptosis in type 1 diabetes can proceed without Fas-induced killing mediated by the BH3-only protein Bid.

Autoreactive cytotoxic T lymphocytes acquire higher expression of cytotoxic effector markers in the islets of NOD mice after priming in pancreatic lymph nodes.

Cytotoxic T lymphocytes (CTLs) that cause type 1 diabetes are activated in draining lymph nodes and become concentrated as fully active CTLs in inflamed pancreatic islets. It is unclear whether CTL function is driven by signals received in the lymph node or also in the inflamed tissue. We studied whether the development of cytotoxicity requires further activation in islets. Autoreactive CTLs found in the islets of diabetes-prone NOD mice had acquired much higher expression of the cytotoxic effector markers granzyme B, interferon γ, and CD107a than had those in the pancreatic lymph node (PLN). Increased expression seemed to result from stimulation in the islet itself. T cells held up from migrating from the PLN by administration of the sphingosine-1-phosphate agonist FTY720 did not increase expression of cytotoxic molecules in the PLN. Stimulation did not require antigen presentation or cytokine secretion by the target ß cells because it was not affected by the absence of class I major histocompatibility complex expression or by the overexpression of suppressor of cytokine signaling-1. Activation of CD40-expressing cells stimulated increased CTL function and ß-cell destruction, suggesting that signals derived from CD40-expressing cells promote the acquisition of cytotoxicity in the islet environment. These data provide in vivo evidence that stimulation of cytotoxic effector molecule expression occurs in inflamed islets and is independent of ß cells.

Reducing or increasing ß-cell apoptosis without inflammation does not affect diabetes initiation in neonatal NOD mice.

The presentation of islet antigens in the pancreatic LNs (PLNs) of mice is a developmentally regulated process. It has been hypothesized that, during physiological tissue remodeling, a wave of neonatal ß-cell apoptosis may initiate diabetes in autoimmune-prone strains of mice. If true, increasing or decreasing physiological ß-cell apoptosis in neonatal NOD mice should alter the time-course of antigen presentation in the PLNs. We used transgenic over-expression of either an anti-apoptotic protein (Bcl-2) or a toxic transgene (rat insulin promoter-Kb) in mouse ß cells to reduce or increase neonatal ß-cell apoptosis, respectively. Neither intervention affected the timing of antigen presentation in the PLNs or the initiation of islet infiltration. This suggests that under physiological conditions and in the absence of inflammation, neonatal ß-cell apoptosis in NOD mice is not the trigger for antigen presentation in the draining LNs.