NADPH Oxidase 2-Derived Reactive Oxygen Species Promote CD8+ T Cell Effector Function.

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.

T cells display mitochondria hyperpolarization in human type 1 diabetes.

T lymphocytes constitute a major effector cell population in autoimmune type 1 diabetes. Despite essential functions of mitochondria in regulating activation, proliferation, and apoptosis of T cells, little is known regarding T cell metabolism in the progression of human type 1 diabetes. In this study, we report, using two independent cohorts, that T cells from patients with type 1 diabetes exhibited mitochondrial inner-membrane hyperpolarization (MHP). Increased MHP was a general phenotype observed in T cell subsets irrespective of prior antigen exposure, and was not correlated with HbA1C levels, subject age, or duration of diabetes. Elevated T cell MHP was not detected in subjects with type 2 diabetes. T cell MHP was associated with increased activation-induced IFNγ production, and activation-induced IFNγ was linked to mitochondria-specific ROS production. T cells from subjects with type 1 diabetes also exhibited lower intracellular ATP levels. In conclusion, intrinsic mitochondrial dysfunction observed in type 1 diabetes alters mitochondrial ATP and IFNγ production; the latter is correlated with ROS generation. These changes impact T cell bioenergetics and function.

Increased IFN-α-producing plasmacytoid dendritic cells (pDCs) in human Th1-mediated type 1 diabetes: pDCs augment Th1 responses through IFN-α production.

Increasing evidence suggests that type 1 IFN (IFN-αß) is associated with pathogenesis of Th1-mediated type 1 diabetes (T1D). A major source of IFN-αß is plasmacytoid dendritic cells (pDCs). In this study, we analyzed peripheral blood pDC numbers and functions in at-risk, new-onset, and established T1D patients and controls. We found that subjects at risk for T1D and new-onset and established T1D subjects possessed significantly increased pDCs but similar number of myeloid DCs when compared with controls. pDC numbers were not affected by age in T1D subjects but declined with increasing age in control subjects. It was demonstrated that IFN-α production by PBMCs stimulated with influenza viruses was significantly higher in T1D subjects than in controls, and IFN-α production was correlated with pDC numbers in PBMCs. Of interest, only T1D-associated Coxsackievirus serotype B4 but not B3 induced majority of T1D PBMCs to produce IFN-α, which was confirmed to be secreted by pDCs. Finally, in vitro studies demonstrated IFN-α produced by pDCs augmented Th1 responses, with significantly greater IFN-γ-producing CD4(+) T cells from T1D subjects. These findings indicate that increased pDCs and their IFN-αß production may be associated with this Th1-mediated autoimmune disease, especially under certain viral infections linked to T1D pathogenesis.

Anti-CD3 antibody treatment induces hypoglycemia and super tolerance to glucose challenge in mice through enhancing glucose consumption by activated lymphocytes.

Anti-CD3 antibody has been employed for various immune-mediated disorders. However, whether anti-CD3 administration leads to rapid metabolic alternation has not been well investigated. In the current study, we studied how anti-CD3 treatment affected blood glucose levels in mice. We found that anti-CD3 treatment induced immediate reduction of blood glucose after administration. Furthermore, a single dose of anti-CD3 treatment corrected hyperglycemia in all nonobese diabetic mice with recently diagnosed diabetes. This glucose-lowering effect was not attributable to major T cell produced cytokines. Of interest, when tested in a normal strain of mice (C57BL/6), the serum levels of C-peptide in anti-CD3 treated animals were significantly lower than control mice. Paradoxically, anti-CD3 treated animals were highly tolerant to exogenous glucose challenge. Additionally, we found that anti-CD3 treatment significantly induced activation of T and B cells in vitro and in vivo. Further studies demonstrated that anti-CD3 treatment lowered the glucose levels in T cell culture media and increased the intracellular transportation of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2 deoxyglucose (2-NBDG) particularly in activated T and B cells. In addition, injection of anti-CD3 antibodies induced enhanced levels of Glut1 expression in spleen cells. This study suggests that anti-CD3 therapy-induced hypoglycemia likely results from increased glucose transportation and consumption by the activated lymphocytes.

Characterization of Bone Marrow-Derived Dendritic Cells Developed in Serum-Free Media and their Ability to Prevent Type 1 Diabetes in Nonobese Diabetic Mice.

Dendritic cells (DC) have been investigated as a cell-based therapy for Type 1 Diabetes (T1D). BM-DC expanded ex vivo with GM-CSF and IL-4 is typically cultured with fetal bovine serum (FBS). The effect of FBS on NOD BM-DC has not been extensively studied. In the present study we compare BM-DC generated in serum-free culture media (X-VIVO20; FBS-) with BM-DC generated in media containing 10% FBS (RPMI1640/10%FBS; FBS+). We show that FBS- BM-DC display a phenotype and cytokine-producing profile distinct from FBS+ BMDC. Additionally, compared to FBS+ BM-DC, we show evidence of an altered Th cell response induced by FBS- BM-DC. Finally, we demonstrate that only FBS- BM-DC prevent the onset of T1D and induce increased levels of CD4+Foxp3+ regulatory T cells as well as a long-lasting ß cell-specific T cell response. This study indicates that serum-free media generates a more tolerogenic BM-DC capable of preventing T1D in the NOD mice.

Anti-thymocyte globulin (ATG) differentially depletes naïve and memory T cells and permits memory-type regulatory T cells in nonobese diabetic mice.

BACKGROUND: ATG has been employed to deplete T cells in several immune-mediated conditions. However, whether ATG administration affects naïve and memory T cell differently is largely unknown. THE CONTEXT AND PURPOSE OF THE STUDY: In this study, we assessed how murine ATG therapy affected T cell subsets in NOD mice, based on their regulatory and naïve or memory phenotype, as well as its influence on antigen-specific immune responses. RESULTS: Peripheral blood CD4+ and CD8+ T cells post-ATG therapy declined to their lowest levels at day 3, while CD4+ T cells returned to normal levels more rapidly than CD8+ T cells. ATG therapy failed to eliminate antigen-primed T cells. CD4+ T cell responses post-ATG therapy skewed to T helper type 2 (Th2) and possibly IL-10-producing T regulatory type 1 (Tr1) cells. Intriguingly, Foxp3+ regulatory T cells (Tregs) were less sensitive to ATG depletion and remained at higher levels following in vivo recovery compared to controls. Of note, the frequency of Foxp3+ Tregs with memory T cell phenotype was significantly increased in ATG-treated animals. CONCLUSION: ATG therapy may modulate antigen-specific immune responses through inducing memory-like regulatory T cells as well as other protective T cells such as Th2 and IL-10-producing Tr1 cells.

Administration of recombinant human thioredoxin-1 significantly delays and prevents autoimmune diabetes in nonobese diabetic mice through modulation of autoimmunity.

BACKGROUND: Thioredoxin as a biological antioxidant plays an important role in regulating the redox system. The administration of recombinant thioredoxin has been demonstrated to be anti-inflammatory. In this study, the effect of recombinant human thioredoxin-1 (rhTrx-1) in preventing type 1 diabetes (T1D) in nonobese diabetic (NOD) mice was evaluated. METHODS: Eight-week-old NOD mice were treated with intravenous injection of rhTrx-1 (5 µg/mouse/day) for 5 weeks (5 days a week), followed by every other day for additional 5 weeks. Diabetes onset was monitored twice a week. Pancreatic histology and ß-cell mass were examined by hematoxylin and eosin (H&E) and insulin immunohistochemistry staining, respectively. Adoptive transfer experiments were executed to assess autoimmune T cells modulated by rhTrx treatment. RESULTS: The intravenous administration of rhTrx-1 significantly delayed and prevented T1D in NOD mice. The histology data showed that rhTrx-1 treatment markedly reduced insulitic lesions and significantly preserved insulin-producing ß cells. Adoptive transfer of spleen cells from rhTrx-1-treated mice into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice significantly reduced the diabetes onset than transfer of those from phosphate-buffered saline-treated mice. Adoptive co-transfer experiments demonstrated that spleen cells from rhTrx-1-treated mice significantly delayed diabetes induced by the co-transferred diabetogenic spleen cells from the new-onset diabetic mice. CONCLUSIONS: Antioxidant rhTrx-1 effectively prevents T1D which may be attributed to its activity to modulate autoimmunity.

Structural analysis of the DNA target site and its interaction with Mbp1.

The solution structure of a 14 base-pair non-self complementary DNA duplex containing the consensus-binding site of the yeast transcription factor Mbp1 has been determined by NMR using a combination of scalar coupling analysis, time-dependent NOEs, residual dipolar couplings and 13C-edited NMR spectroscopy of a duplex prepared with one strand uniformly labeled with 13C-nucleotides. As expected, the free DNA duplex is within the B-family of structures, and within experimental limits is straight. However, there are clear local structural variations associated with the consensus CGCG element in the binding sequence that are important for sequence recognition. In the complex, the DNA bends around the protein, which also undergoes some conformational rearrangement in the C-terminal region. Structural constraints derived from paramagnetic perturbation experiments with spin-labeled DNA, chemical shift perturbation experiments of the DNA, previous cross-saturation, chemical shift perturbation experiments on the protein, information from mutational analysis, and electrostatics calculations have been used to produce a detailed docked structure using the known solution conformation of the free protein and other spectroscopic information about the Mbp1:DNA complex. A Monte Carlo-based docking procedure with restrained MD in a fully solvated system subjected to available experimental constraints produced models that account for the available structural data, and can rationalize the extensive thermodynamic data about the Mbp1:DNA complex. The protein:DNA interface is closely packed and is associated with a small number of specific contacts. The structure shows an extensive positively charged surface that accounts for the high polyelectrolyte contribution to binding.