Development of T cell lines sensitive to antigen stimulation.

Immortalized T cells such as T cell hybridomas, transfectomas, and transductants are useful tools to study tri-molecular complexes consisting of peptide, MHC, and T cell receptor (TCR) molecules. These cells have been utilized for antigen discovery studies for decades due to simplicity and rapidness of growing cells. However, responsiveness to antigen stimulation is typically less sensitive compared to primary T cells, resulting in occasional false negative outcomes especially for TCRs having low affinity to a peptide-MHC complex (pMHC). To overcome this obstacle, we genetically engineered T cell hybridomas to express additional CD3 molecules as well as CD4 with two amino acid substitutions that increase affinity to MHC class II molecules. The manipulated T cell hybridomas that were further transduced with retroviral vectors encoding TCRs of interest responded to cognate antigens more robustly than non-manipulated cells without evoking non-antigen specific reactivity. Of importance, the manipulation with CD3 and mutated human CD4 expression was effective in increasing responsiveness of T cell hybridomas to a wide variety of TCR, peptide, and MHC combinations across class II genetic loci (i.e. HLA-DR, HLA-DQ, HLA-DP, and murine H2-IA) and species (i.e. both humans and mice), and thus will be useful to identify antigen specificity of T cells.

Modifying Enzymes Are Elicited by ER Stress, Generating Epitopes That Are Selectively Recognized by CD4+ T Cells in Patients With Type 1 Diabetes.

In spite of tolerance mechanisms, some individuals develop T-cell-mediated autoimmunity. Posttranslational modifications that increase the affinity of epitope presentation and/or recognition represent one means through which self-tolerance mechanisms can be circumvented. We investigated T-cell recognition of peptides that correspond to modified ß-cell antigens in subjects with type 1 diabetes. Modified peptides elicited enhanced proliferation by autoreactive T-cell clones. Endoplasmic reticulum (ER) stress in insulinoma cells increased cytosolic calcium and the activity of tissue transglutaminase 2 (tTG2). Furthermore, stressed human islets and insulinomas elicited effector responses from T cells specific for modified peptides, suggesting that ER stress-derived tTG2 activity generated deamidated neoepitopes that autoreactive T cells recognized. Patients with type 1 diabetes had large numbers of T cells specific for these epitopes in their peripheral blood. T cells with these specificities were also isolated from the pancreatic draining lymph nodes of cadaveric donors with established diabetes. Together, these results suggest that self-antigens are enzymatically modified in ß-cells during ER stress, giving rise to modified epitopes that could serve to initiate autoimmunity or to further broaden the antigenic repertoire, activating potentially pathogenic CD4+ T cells that may not be effectively eliminated by negative selection.

Cystic fibrosis-related diabetes is caused by islet loss and inflammation.

Cystic fibrosis-related (CF-related) diabetes (CFRD) is an increasingly common and devastating comorbidity of CF, affecting approximately 35% of adults with CF. However, the underlying causes of CFRD are unclear. Here, we examined cystic fibrosis transmembrane conductance regulator (CFTR) islet expression and whether the CFTR participates in islet endocrine cell function using murine models of ß cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine ß cells did not affect ß cell function. In human islets, CFTR mRNA was minimally expressed, and CFTR protein and electrical activity were not detected. Isolated CF/CFRD islets demonstrated appropriate insulin and glucagon secretion, with few changes in key islet-regulatory transcripts. Furthermore, approximately 65% of ß cell area was lost in CF donors, compounded by pancreatic remodeling and immune infiltration of the islet. These results indicate that CFRD is caused by ß cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation.

Analysis of self-antigen specificity of islet-infiltrating T cells from human donors with type 1 diabetes.

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood-derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors and isolation and identification of DQ8 and DQ2-DQ8 heterodimer-restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.

Tolerogenic nanoparticles inhibit T cell-mediated autoimmunity through SOCS2.

Type 1 diabetes (T1D) is a T cell-dependent autoimmune disease that is characterized by the destruction of insulin-producing ß cells in the pancreas. The administration to patients of ex vivo-differentiated FoxP3(+) regulatory T (Treg) cells or tolerogenic dendritic cells (DCs) that promote Treg cell differentiation is considered a potential therapy for T1D; however, cell-based therapies cannot be easily translated into clinical practice. We engineered nanoparticles (NPs) to deliver both a tolerogenic molecule, the aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), and the ß cell antigen proinsulin (NPITE+Ins) to induce a tolerogenic phenotype in DCs and promote Treg cell generation in vivo. NPITE+Ins administration to 8-week-old nonobese diabetic mice suppressed autoimmune diabetes. NPITE+Ins induced a tolerogenic phenotype in DCs, which was characterized by a decreased ability to activate inflammatory effector T cells and was concomitant with the increased differentiation of FoxP3(+) Treg cells. The induction of a tolerogenic phenotype in DCs by NPs was mediated by the AhR-dependent induction of Socs2, which resulted in inhibition of nuclear factor κB activation and proinflammatory cytokine production (properties of tolerogenic DCs). Together, these data suggest that NPs constitute a potential tool to reestablish tolerance in T1D and potentially other autoimmune disorders.

Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion.

T cell-mediated destruction of insulin-producing ß cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in ß cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in ß cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in ß cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D.

Stimulation of glucagon-like peptide-1 receptor through exendin-4 preserves myocardial performance and prevents cardiac remodeling in infarcted myocardium.

We have demonstrated that GLP-1 improved myocardial functional recovery in acute myocardial ischemic injury. However, whether stimulation of the GLP-1 receptor (GLP-1R) with exendin-4, a selective GLP-1R agonist, could initiate a protective effect in the heart remains to be determined. Mouse myocardial infarction (MI) was created by ligation of the left descending artery. After 48 h of MI, animals were divided into the following groups (n = 5-7/group): 1) sham (animals that underwent thoracotomy without ligation), 2) MI [animals that underwent MI and received a daily dose of intraperitoneal injection (ip) of saline]; and 3) MI + exendin-4 [infarcted mice that received injections of exendin-4 (0.1 mg/kg ip)]. Two weeks later, cardiac function was assessed by echocardiography and an isovolumetrically perfused heart. Compared with control MI hearts, stimulation of GLP-1R improved cardiac function, which was associated with attenuation of myocardial hypertrophy, the mitigation of interstitial fibrosis, and an increase in survival rate in post-MI hearts. Furthermore, H9c2 cardiomyoblasts were preconditioned with exendin-4 at a dose of 100 nmol/l and then subjected to hydrogen peroxide exposure at concentrations of 50 and 100 µmol/l. The exendin-4 treatment decreased lactate dehydrogenase leakage and increased cell survival. Notably, this event was also associated with the reduction of cleaved caspase-3 and caspase-9 and attenuation of reactive oxygen species production. Exendin-4 treatments improved mitochondrial respiration and suppressed the opening of mitochondrial permeability transition pore and protected mitochondria function. Our results indicate that GLP-1R serves as a novel approach to eliciting cardioprotection and mitigating oxidative stress-induced injury.

Specific inhibition of HDAC4 in cardiac progenitor cells enhances myocardial repairs.

We have recently shown that in vivo inhibition of histone deacetylase (HDAC) stimulates endogenous myocardial regeneration in infarcted hearts (Zhang L et al. J Pharmacol Exp Ther 341: 285-293, 2012). Furthermore, our observation demonstrates that HDAC inhibition promotes cardiogenesis, which is associated with HDAC4 reduction. However, it remains unknown as to whether specific inhibition of HDAC4 modulates cardiac stem cells (CSCs) to facilitate myocardial repair and to preserve cardiac performance. c-kit(+) CSCs were isolated from adult mouse hearts and were transfected with HDAC4 siRNA to knockdown HDAC4 of c-kit(+) CSCs. The transfection of HDAC4 siRNA caused a marked reduction of HDAC4 mRNA and proteins in c-kit(+) CSCs. Mouse myocardial infarction (MI) was created to assess the effect of HDAC4 inhibition in c-kit(+) CSCs on myocardial regeneration in vivo when cells were introduced into MI hearts. Transplantation of HDAC4 siRNA-treated c-kit(+) CSCs into MI hearts improved ventricular function, attenuated ventricular remodeling, and promoted CSC-derived regeneration and neovascularization. Furthermore, Ki67 and BrdU positively proliferative myocytes increased in MI hearts receiving HDAC4 siRNA-treated c-kit(+) CSCs compared with MI hearts engrafted with control siRNA-treated c-kit(+) CSCs. In addition, compared with MI hearts engrafted with control adenoviral GFP-infected c-kit(+) CSCs, MI hearts receiving adenoviral HDAC4-infected c-kit(+) CSCs exhibited attenuated cardiac functional recovery, CSC-derived regeneration, and neovascularization, which was accompanied with adverse ventricular remodeling and decrease in Ki67 and BrdU positively proliferative myocytes. HDAC4 inhibition facilitated c-kit(+) CSCs into the differentiation into cardiac lineage commitments in vitro, while HDAC4 overexpression attenuated c-kit(+) CSC-derived cardiogenesis. Our results indicate that HDAC4 inhibition promotes CSC-derived cardiac regeneration and improves the restoration of cardiac function.

HDAC inhibition promotes cardiogenesis and the survival of embryonic stem cells through proteasome-dependent pathway.

Histone deacetylase (HDAC) inhibition plays a crucial role in mediating cardiogenesis and myocardial protection, whereas HDAC degradation has recently attracted attention in mediating the biological function of HDACs. However, it remains unknown whether HDAC inhibition modulates cardiogenesis and embryonic stem cell (ESC) survival through the proteasome pathway. Using the well-established mouse ESC culture, we demonstrated that HDAC inhibitors, both trichostatin A (TSA,50 nmol/L) and sodium butyrate (NaB, 200 µmol/L) that causes the pronounced reduction of HDAC4 activity, decreased cell death and increased viability of ESCs in response to oxidant stress. HDAC inhibition reduced the cleaved caspases 3, 6, 9, PARP, and TUNEL positive ESCs, which were abrogated with MG132 (0.5 µmol/L), a specific proteasome inhibitor. Furthermore, HDAC inhibition stimulates the growth of embryoid bodies (EB), which are associated with a faster spontaneous rhythmic contraction. HDAC inhibition increases the up-regulation of GATA4, MEF2C, Nkx2.5, cardiac actin, and α-SMA mRNA and protein levels that were abrogated by MG132. TSA and NaB resulted in a significant increase in cardiac lineage commitments that were blocked by the proteasome inhibition. Notably, HDAC inhibitors led to noticeable HDAC4 degradation, which was effectively prevented by MG132. Luciferase assay demonstrates an activation of MEF2 cardiac transcriptional factor by HDAC inhibition, which was repressed by MG132, revealing that the degradation of HDAC4 allows for the activation of MEF2. Taken together, our study is the first to demonstrate that HDAC inhibition through proteasome pathway forms a novel signaling to determine the cardiac lineage commitment and elicits the survival pathway.