TIGIT acts as an immune checkpoint upon inhibition of PD1 signaling in autoimmune diabetes

IntroductionChronic activation of self-reactive T cells with beta cell antigens results in the upregulation of immune checkpoint molecules that keep self-reactive T cells under control and delay beta cell destruction in autoimmune diabetes. Inhibiting PD1/PD-L1 signaling results in autoimmune diabetes in mice and humans with pre-existing autoimmunity against beta cells. However, it is not known if other immune checkpoint molecules, such as TIGIT, can also negatively regulate self-reactive T cells. TIGIT negatively regulates the CD226 costimulatory pathway, T-cell receptor (TCR) signaling, and hence T-cell function.MethodsThe phenotype and function of TIGIT expressing islet infiltrating T cells was studied in non-obese diabetic (NOD) mice using flow cytometry and single cell RNA sequencing. To determine if TIGIT restrains self-reactive T cells, we used a TIGIT blocking antibody alone or in combination with anti-PDL1 antibody.ResultsWe show that TIGIT is highly expressed on activated islet infiltrating T cells in NOD mice. We identified a subset of stem-like memory CD8+ T cells expressing multiple immune checkpoints including TIGIT, PD1 and the transcription factor EOMES, which is linked to dysfunctional CD8+ T cells. A known ligand for TIGIT, CD155 was expressed on beta cells and islet infiltrating dendritic cells. However, despite TIGIT and its ligand being expressed, islet infiltrating PD1+TIGIT+CD8+ T cells were functional. Inhibiting TIGIT in NOD mice did not result in exacerbated autoimmune diabetes while inhibiting PD1-PDL1 resulted in rapid autoimmune diabetes, indicating that TIGIT does not restrain islet infiltrating T cells in autoimmune diabetes to the same degree as PD1. Partial inhibition of PD1-PDL1 in combination with TIGIT inhibition resulted in rapid diabetes in NOD mice. DiscussionThese results suggest that TIGIT and PD1 act in synergy as immune checkpoints when PD1 signaling is partially impaired. Beta cell specific stem-like memory T cells retain their functionality despite expressing multiple immune checkpoints and TIGIT is below PD1 in the hierarchy of immune checkpoints in autoimmune diabetes

New approaches to induce tolerance in autoantigen-specific memory T cells in type 1 diabetes

© 2018 Dr. Claudia SelckAutoimmune disorders like type 1 diabetes (T1D) result from the failure of immune tolerance mechanisms. Antigen-specific therapy constitutes an attractive approach to re-establish a tolerant state but has so far not been successful in clinical settings. Importantly, treatment after the onset of autoimmunity is likely to be less effective due to established autoimmune responses and active inflammation in the islets. Moreover, a major hurdle might be the persistence of antigen-experienced islet-reactive T cells and the induction of tolerance in these memory cells is challenging. Hence, therapies intended to induce antigen-specific tolerance may need additional immunomodulatory treatments to be effective in individuals with established autoimmunity. Here, we aimed to identify a combination of interventions that induces tolerance in antigen-specific memory T cells. For this study, we generated non-obese diabetic (NOD) mice with tetracycline-regulated expression of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) in antigen presenting cells (TII mice) and tracked IGRP-specific CD8+ T cells using tetramer enrichment. Notably, while naïve IGRP-specific T cells are eliminated upon antigen expression, memory T cells are refractory to cell depletion. Interestingly, these memory T cells up-regulated several exhaustion markers in response to antigen exposure but showed no functional impairment. We hypothesized that combining short-term treatment with non-Fc-binding anti-CD3 F(ab’)2 and the co-stimulation blocker abatacept together with antigen expression might induce effective tolerance in TII mice. However, after initial depletion of antigen-experienced IGRP-specific cells by anti-CD3 F(ab’)2, these cells re-expanded and were still functional. Thus, our combination approach has only limited efficacy in established autoimmunity. Combination therapies of antigen-specific therapies with agents that support the induction of complete T cell exhaustion might be more successful and should be tested in future studies

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology <i>in situ</i>

<div><p>Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells <i>in vitro</i>. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding <i>in situ</i>.</p></div

Effect of long-term pancreas tissue slice culture on endocrine beta cell viability and function.

<p>(<i>A</i>) Longitudinal <i>in situ</i> imaging of beta cell viability and specificity in pancreas tissue slices before and after 7 days culture in standard and optimized conditions. Under standard culture conditions MIP-GFP fluorescence (green) of beta cells was lost after 7 days and the numbers of dead nuclei (magenta) dramatically increased, whereas in optimized conditions beta cells were still detectable by GFP fluorescence after 7 days and the number of dead cells inside the islet remained low. <i>(B)</i> Quantification of Draq7 nuclei (dead cells) within islets in freshly prepared pancreas tissue slices and during culture in standard and optimized conditions. Islet volume in tissue slices was determined by backscatter LSM. Values are expressed as mean ± SD Draq7 labeled nuclei per mm³ islet volume (n = 11 for optimized and n = 12 for standard conditions). <i>(C)</i> Basal (3 mmol/L glucose) and stimulated (16.7 mmol/L glucose) insulin release from pancreas tissue slices at the day of preparation and after 4 and 7 days of culture in optimized conditions. Insulin release is expressed as percent of total insulin as mean ± SD from 8 slices per time point (n = 8).</p

Preservation of pancreas tissue slice morphology under optimized organotypic culture conditions.

<p><i>(A)</i> Panels show representative images of pancreas slices immediately after preparation and cultured for 7 days under standard and optimized conditions. Slices cultured in standard conditions exhibit dramatic changes of pancreas morphology whereas slices cultured in optimized conditions preserve the typical lobular structure of dense pancreatic tissue. Scale bars  =  2 mm. <i>(B)</i> Pancreas tissue slice area was maintained significantly better after 4 (88.5±5.3% vs. 59.6±19.2%) and 7 (80.2±6.2% vs. 33.1±15.7%) days when cultured under optimized conditions in comparison to standard conditions. Slice area values are expressed as percent of the area on the day of preparation and represent mean ± SD of n = 12 slices for optimized and n = 64 for standard conditions. (C) Quantification of Draq7 nuclei (dead cells) within exocrine tissue in freshly prepared pancreas tissue slices and during culture in standard and optimized conditions. Exocrine tissue volume was determined by backscatter LSM. Values are expressed as mean ± SD Draq7 labeled nuclei per mm³ exocrine tissue volume (n = 24 for optimized and n = 9 for standard conditions).</p

Acinar cell function during long-term culture of pancreas tissue slices.

<p><i>(A)</i> Amylase release from freshly prepared and cultured pancreas tissue slices after 30 min stimulation with indicated caerulein concentrations. The relative amount of released amylase decreases with culture time. However, amylase release shows a typical bell-shaped curve response to increasing caerulein concentrations at all time points. Amylase release is expressed as percent of total amylase as mean ± SD of 16 slices per time point (n = 16). <i>(B)</i> Traces of Ca²⁺_i/Oregon Green BAPTA-1 fluorescence in acinar cells of pancreas tissue slices at indicated time points after preparation. Stimulation with 10 pmol/L caerulein induced oscillations of Ca²⁺_i/Oregon Green BAPTA-1 fluorescence in acinar cells of slices at all time points. Traces are shown as fluorescence ratio (F/F₀), in which F is fluorescence at any given time and F₀ is pre-stimulatory fluorescence.</p

p53-upregulated-modulator-of-apoptosis (PUMA) deficiency affects food intake but does not impact on body weight or glucose homeostasis in diet-induced obesity.

BCL-2 proteins have been implicated in the control of glucose homeostasis and metabolism in different cell types. Thus, the aim of this study was to determine the role of the pro-apoptotic BH3-only protein, p53-upregulated-modulator-of-apoptosis (PUMA), in metabolic changes mediated by diet-induced obesity, using PUMA deficient mice. At 10 weeks of age, knockout and wild type mice either continued consuming a low fat chow diet (6% fat), or were fed with a high fat diet (23% fat) for 14-17 weeks. We measured body composition, glucose and insulin tolerance, insulin response in peripheral tissues, energy expenditure, oxygen consumption, and respiratory exchange ratio in vivo. All these parameters were indistinguishable between wild type and knockout mice on chow diet and were modified equally by diet-induced obesity. Interestingly, we observed decreased food intake and ambulatory capacity of PUMA knockout mice on high fat diet. This was associated with increased adipocyte size and fasted leptin concentration in the blood. Our findings suggest that although PUMA is dispensable for glucose homeostasis in lean and obese mice, it can affect leptin levels and food intake during obesity.info:eu-repo/semantics/publishe

Perinatal tolerance to proinsulin is sufficient to prevent autoimmune diabetes.

High-affinity self-reactive thymocytes are purged in the thymus, and residual self-reactive T cells, which are detectable in healthy subjects, are controlled by peripheral tolerance mechanisms. Breakdown in these mechanisms results in autoimmune disease, but antigen-specific therapy to augment natural mechanisms can prevent this. We aimed to determine when antigen-specific therapy is most effective. Islet autoantigens, proinsulin (PI), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) were expressed in the antigen-presenting cells (APCs) of autoimmune diabetes-prone nonobese diabetic (NOD) mice in a temporally controlled manner. PI expression from gestation until weaning was sufficient to completely protect NOD mice from diabetes, insulitis, and development of insulin autoantibodies. Insulin-specific T cells were significantly diminished, were naive, and did not express IFN-γ when challenged. This long-lasting effect from a brief period of treatment suggests that autoreactive T cells are not produced subsequently. We tracked IGRP206-214-specific CD8(+) T cells in NOD mice expressing IGRP in APCs. When IGRP was expressed only until weaning, IGRP206-214-specific CD8(+) T cells were not detected later in life. Thus, anti-islet autoimmunity is determined during early life, and autoreactive T cells are not generated in later life. Bolstering tolerance to islet antigens in the perinatal period is sufficient to impart lasting protection from diabetes.info:eu-repo/semantics/publishe