Human Norovirus Triggers Primary B Cell Immune Activation In Vitro.

Human norovirus (HNoV) is a global health and socioeconomic burden, estimated to infect every individual at least five times during their lifetime. The underlying mechanism for the potential lack of long-term immune protection from HNoV infections is not understood and prompted us to investigate HNoV susceptibility of primary human B cells and its functional impact. Primary B cells isolated from whole blood were infected with HNoV-positive stool samples and harvested at 3 days postinfection (dpi) to assess the viral RNA yield by reverse transcriptase quantitative PCR (RT-qPCR). A 3- to 18-fold increase in the HNoV RNA yield was observed in 50 to 60% of donors. Infection was further confirmed in B cells derived from splenic and lymph node biopsy specimens. Next, we characterized infection of whole-blood-derived B cells by flow cytometry in specific functional B cell subsets (naive CD27- IgD+, memory-switched CD27+ IgD-, memory-unswitched CD27+ IgD+, and double-negative CD27- IgD- cells). While the susceptibilities of the subsets were similar, changes in the B cell subset distribution upon infection were observed, which were also noted after treatment with HNoV virus-like particles and the predicted recombinant NS1 protein. Importantly, primary B cell stimulation with the predicted recombinant NS1 protein triggered B cell activation and induced metabolic changes. These data demonstrate that primary B cells are susceptible to HNoV infection and suggest that the NS1 protein can alter B cell activation and metabolism in vitro, which could have implications for viral pathogenesis and immune responses in vivo. IMPORTANCE Human norovirus (HNoV) is the most prevalent causative agent of gastroenteritis worldwide. Infection results in a self-limiting disease that can become chronic and severe in the immunocompromised, the elderly, and infants. There are currently no approved therapeutic and preventative strategies to limit the health and socioeconomic burdens associated with HNoV infections. Moreover, HNoV does not elicit lifelong immunity as repeat infections are common, presenting a challenge for vaccine development. Given the importance of B cells for humoral immunity, we investigated the susceptibility and impact of HNoV infection on human B cells. We found that HNoV replicates in human primary B cells derived from blood, spleen, and lymph node specimens, while the nonstructural protein NS1 can activate B cells. Because of the secreted nature of NS1, we put forward the hypothesis that HNoV infection can modulate bystander B cell function with potential impacts on systemic immune responses.

Insulin Receptor-Expressing T Cells Appear in Individuals at Risk for Type 1 Diabetes and Can Move into the Pancreas in C57BL/6 Transgenic Mice.

Insulin receptor (IR) expression on the T cell surface can indicate an activated state; however, the IR is also chemotactic, enabling T cells with high IR expression to physically move toward insulin. In humans with type 1 diabetes (T1D) and the NOD mouse model, a T cell-mediated autoimmune destruction of insulin-producing pancreatic ß cells occurs. In previous work, when purified IR+ and IR- T cells were sorted from diabetic NOD mice and transferred into irradiated nondiabetic NOD mice, only those that received IR+ T cells developed insulitis and diabetes. In this study, peripheral blood samples from individuals with T1D (new onset to 14 y of duration), relatives at high-risk for T1D, defined by positivity for islet autoantibodies, and healthy controls were examined for frequency of IR+ T cells. High-risk individuals had significantly higher numbers of IR+ T cells as compared with those with T1D (p < 0.01) and controls (p < 0.001); however, the percentage of IR+ T cells in circulation did not differ significantly between T1D and control subjects. With the hypothesis that IR+ T cells traffic to the pancreas in T1D, we developed a (to our knowledge) novel mouse model exhibiting a FLAG-tagged mouse IR on T cells on the C57BL/6 background, which is not susceptible to developing T1D. Interestingly, these C57BL/6-CD3FLAGmIR/mfm mice showed evidence of increased IR+ T cell trafficking into the islets compared with C57BL/6 controls (p < 0.001). This transgenic animal model provides a (to our knowledge) novel platform for investigating the influence of IR expression on T cell trafficking and the development of insulitis.

Characterization and Activation of Fas Ligand-Producing Mouse B Cells and Their Killer Exosomes.

B lymphocytes make several contributions to immune regulation including production of antibodies with regulatory properties, release of immune suppressive cytokines, and expression of death-inducing ligands. A role for Fas ligand (FasL)-expressing "killer" B cells in regulating T helper (TH) cell survival and chronic inflammation has been demonstrated in animal models of schistosome worm and other infections, asthma, autoimmune arthritis, and type 1 diabetes. FasL+ B cells were also capable of inducing immune tolerance in a male-to-female transplantation model. Interestingly, populations of B cells found in the spleen and lungs of naïve mice constitutively expresses FasL and have potent killer function against TH cells that is antigen-specific and FasL-dependent. Epstein-Barr virus-transformed human B cells constitutively express FasL and package it into exosomes that co-express MHC Class II molecules and have killer function against antigen-specific TH cells. FasL+ exosomes with markers of B-cell lineage are abundant in the spleen of naïve mice. Killer B cells therefore represent a novel target for immune modulation in many disease settings. Our laboratory has published methods of characterizing FasL+ B cells and inducing their proliferation in vitro. This updated chapter will describe methods of identifying and expanding killer B cells from mice, detecting FasL expression in B cells, extracting FasL+ exosomes from spleen and culture supernatants, and performing functional killing assays against antigen-specific TH cells.

Lymphocyte subset abnormalities in early diffuse cutaneous systemic sclerosis.

BACKGROUND: Abnormalities in lymphocyte surface markers and functions have been described in systemic sclerosis (SSc), but conflicting results abound, and these studies often examined patients with heterogeneous disease duration, severity, clinical phenotype, and concurrent immunosuppressive agents. We studied a clinically homogeneous group of early diffuse cutaneous SSc patients not exposed to immunosuppressive drugs who were enrolled in a clinical trial and compared their immune parameters to healthy control subjects. METHODS: Lymphocyte subsets were enumerated by multi-parameter flow cytometry of peripheral blood mononuclear cells at baseline visit. Production of the cytokines IL-4 and IL-17 was measured by intracellular flow cytometry following T cell activation. RESULTS: SSc patients had increased percentages of CD4+ T cells but lower percentages of CD8+ T cells versus controls. The CD28-negative population was expanded in SSc, in the CD4 subset. Striking expansion of CD319+ T cells was noted among the CD4+ cells, in which they were barely detectable in healthy subjects. Frequencies of IL-4 producing cells did not differ between SSc and controls, but expansion of IL-17 producing cells was observed in SSc. A higher proportion of CD319+ cells produced cytokines, compared to other CD4+ cells. Numbers of activated T cells, regulatory T cells, and B cells were similar in SSc and control groups. Circulating follicular helper but not peripheral helper T cells were slightly expanded in SSc. CONCLUSION: In this carefully selected group of early diffuse cutaneous SSc patients, analysis of immune cell parameters has identified abnormalities that likely reflect disease pathogenesis and that are candidate biomarkers for sub-classification and targeted treatment. The CD4+CD319+ (SLAM-F7+) cells are cytotoxic and oligoclonal, were recently shown to be a dominant T cell population in perivascular lymphocytic infiltrates in SSc skin, actively secrete cytokines, and are emerging as a target for novel treatments of SSc.

Correlation of Immunological Markers with Disease and Clinical Outcome Measures in Patients with Autoimmune Retinopathy.

Purpose: To determine if immunological markers (1) are significantly different between autoimmune retinopathy (AIR) patients and controls and (2) correlate with disease progression in AIR patients. Methods: We enrolled patients with a possible AIR diagnosis, as well as control participants without eye disease, autoimmunity, or cancer. Immunological markers were tested in all participants. In addition, AIR patients had up to three blood draws for testing over their disease course. For AIR patients, clinical measures, including visual acuity (VA) and Goldmann visual field (GVF) area, were recorded at each draw. We used the Mann-Whitney U test to compare the immunological markers between AIR patients and controls. We used multilevel mixed-effect regression to investigate the correlation between markers and clinical parameters over time in AIR patients. Results: Seventeen patients with AIR and 14 controls were included. AIR patients had a higher percent of monocytes (Z = 3.076, P = 0.002). An increase in immunoglobulin G against recoverin was correlated with a VA decrease (ß = 0.0044, P < 0.0001). An increase in monocyte proportion was correlated with a decrease in GVF area (ß = -7.27, P = 0.0021). Several markers of B-cell depletion were correlated with GVF improvement. Conclusions: Monocytes may play a role in AIR pathophysiology and be a disease activity marker. B-cell depletion markers correlated with clinical parameter improvement, particularly GVF. Translational Relevance: This work elucidates immunologic markers that may improve the accuracy of diagnosis and treatment of AIR.

Siponimod enriches regulatory T and B lymphocytes in secondary progressive multiple sclerosis.

BACKGROUNDSiponimod (BAF312) is a selective sphingosine-1-phosphate receptor 1 and 5 (S1PR1, S1PR5) modulator recently approved for active secondary progressive multiple sclerosis (SPMS). The immunomodulatory effects of siponimod in SPMS have not been previously described.METHODSWe conducted a multicentered, randomized, double-blind, placebo-controlled AMS04 mechanistic study with 36 SPMS participants enrolled in the EXPAND trial. Gene expression profiles were analyzed using RNA derived from whole blood with Affymetrix Human Gene ST 2.1 microarray technology. We performed flow cytometry-based assays to analyze the immune cell composition and microarray gene expression analysis on peripheral blood from siponimod-treated participants with SPMS relative to baseline and placebo during the first-year randomization phase.RESULTSMicroarray analysis showed that immune-associated genes involved in T and B cell activation and receptor signaling were largely decreased by siponimod, which is consistent with the reduction in CD4+ T cells, CD8+ T cells, and B cells. Flow cytometric analysis showed that within the remaining lymphocyte subsets there was a reduction in the frequencies of CD4+ and CD8+ naive T cells and central memory cells, while T effector memory cells, antiinflammatory Th2, and T regulatory cells (Tregs) were enriched. Transitional regulatory B cells (CD24hiCD38hi) and B1 cell subsets (CD43+CD27+) were enriched, shifting the balance in favor of regulatory B cells over memory B cells. The proregulatory shift driven by siponimod treatment included a higher proliferative potential of Tregs compared with non-Tregs, and upregulated expression of PD-1 on Tregs. Additionally, a positive correlation was found between Tregs and regulatory B cells in siponimod-treated participants.CONCLUSIONThe shift toward an antiinflammatory and suppressive homeostatic immune system may contribute to the clinical efficacy of siponimod in SPMS.TRIAL REGISTRATIONNCT02330965.

Immune Cell Infiltration into the Eye Is Controlled by IL-10 in Recoverin-Induced Autoimmune Retinopathy.

Autoimmune retinopathy (AIR) is a treatable condition that manifests in acute and progressive vision loss in patients. It has recently been determined that AIR is associated with an imbalance of TH1 versus regulatory T cell immunity toward the retinal protein, recoverin. This study describes a new murine model to understand the immunopathology of AIR and its association with T cell responses toward recoverin. Immunization of C57BL/6 mice with recoverin resulted in ocular inflammation including infiltration of CD4+ and CD8+ T lymphocytes, B cells, and CD11b+Ly6C+ inflammatory monocytes in the eyes. Production of IFN-γ and IL-17 from T cells was exacerbated in IL-10 knockout (KO) mice and kinetics of disease development was accelerated. Infiltration of T cells and inflammatory monocytes into the eyes dramatically increased in recoverin-immunized IL-10 KO mice. An immunodominant peptide of recoverin, AG-16, was capable of inducing disease in IL-10 KO mice and resulted in expansion of AG-16 tetramer-specific CD4+ T cells in lymphoid organs and eyes. Adoptive transfer of recoverin-stimulated cells into naive mice was sufficient to induce AIR, and immunization of B cell-deficient mice led to a milder form of the disease. This model supports the hypothesis that recoverin-specific T cell responses are major drivers of AIR pathogenesis and that IL-10 is an important factor in protection.

CD19+IgM+ cells demonstrate enhanced therapeutic efficacy in type 1 diabetes mellitus.

We describe a protective effect on autoimmune diabetes and reduced destructive insulitis in NOD.scid recipients following splenocyte injections from diabetic NOD donors and sorted CD19+ cells compared with NOD.scid recipients receiving splenocytes alone. This protective effect was age specific (only CD19+ cells from young NOD donors exerted this effect; P < 0.001). We found that the CD19+IgM+ cell is the primary subpopulation of B cells that delayed transfer of diabetes mediated by diabetogenic T cells from NOD mice (P = 0.002). Removal of IgM+ cells from the CD19+ pool did not result in protection. Notably, protection conferred by CD19+IgM+ cotransfers were not dependent on the presence of Tregs, as their depletion did not affect their ability to delay onset of diabetes. Blockade of IL-10 with neutralizing antibodies at the time of CD19+ cell cotransfers also abrogated the therapeutic effect, suggesting that IL-10 secretion was an important component of protection. These results were strengthened by ex vivo incubation of CD19+ cells with IL-5, resulting in enhanced proliferation and IL-10 production and equivalently delayed diabetes progression (P = 0.0005). The potential to expand CD19+IgM+ cells, especially in response to IL-5 stimulation or by pharmacologic agents, may be a new therapeutic option for type 1 diabetes.

T Helper 1 Cellular Immunity Toward Recoverin Is Enhanced in Patients With Active Autoimmune Retinopathy.

Autoimmune retinopathy (AIR) causes rapidly progressive vision loss that is treatable but often is confused with other forms of retinal degeneration including retinitis pigmentosa (RP). Measurement of anti-retinal antibodies (ARA) by Western blot is a commonly used laboratory assay that supports the diagnosis yet does not reflect current disease activity. To search for better diagnostic indicators, this study was designed to compare immune biomarkers and responses toward the retinal protein, recoverin, between newly diagnosed AIR patients, slow progressing RP patients and healthy controls. All individuals had measurable anti-recoverin IgG and IgM antibodies by ELISA regardless of disease status or Western blot results. Many AIR patients had elevated anti-recoverin IgG1 levels and a strong cellular response toward recoverin dominated by IFNγ. RP patients and controls responded to recoverin with a lower IFNγ response that was balanced by IL-10 production. Both AIR and RP patients displayed lower levels of total peripheral blood mononuclear cells that were due to reductions of CD4+ TH cells. A comparison of messenger RNA (mRNA) for immune-related genes in whole blood of AIR patients versus RP patients or controls indicated lower expression of ATG5 and PTPN22 and higher expression of several genes involved in TH cell signaling/transcription and adhesion. These data indicate that an immune response toward recoverin is normal in humans, but that in AIR patients the balance shifts dramatically toward higher IFNγ production and cellular activation.

Differential Influence on Regulatory B Cells by TH2 Cytokines Affects Protection in Allergic Airway Disease.

The role of regulatory B cells (Bregs) in modulating immune responses and maintaining tolerance are well established. However, how cytokines present during immune responses affect Breg growth and function are not as well defined. Previously, our laboratory reported IL-5- and mCD40L-expressing fibroblast (mCD40L-Fb) stimulation induced IL-10 production from murine B cells. The current study investigated the phenotype and functional relevance of IL-10- producing B cells from this culture. We found IL-5/mCD40L-Fb stimulation induced IL-10 production exclusively from CD5+ splenic B cells of naive mice. After stimulation, the resulting IL-10+ B cells displayed markers of multiple reported Breg phenotypes. Interestingly, when investigating effects of IL-4 (a critical TH2 cytokine) on IL-5/mCD40L-Fb-induced IL-10 production, we found IL-4 inhibited IL-10 production in a STAT6-dependent manner. Upon adoptive transfer, CD5+ B cells previously stimulated with IL-5/mCD40L-Fb were able to reduce development of OVA-induced allergic airway disease in mice. Using B cells from IL-10 mutant mice differentiated by IL-5/mCD40L-Fb, we found protection from allergic airway disease development was dependent on the IL-10 production from the transferred B cells. Bregs have been shown to play crucial roles in the immune tolerance network, and understanding stimuli that modulate their growth and function may be key in development of future treatments for diseases of immune dysregulation.

Autoimmune Retinopathy: An Immunologic Cellular-Driven Disorder.

Autoimmune retinopathy (AIR) was often mistaken for retinitis pigmentosa (RP), due to an overlap of clinical findings, but increasingly has been recognized as a unique entity in the last decade. AIR has distinctive features: sudden onset of photopsias and scotomata in patients with no family history of RP, followed by visual field and central vision loss. Initially, retina exams are normal with no sign of pigment deposits or retinal degeneration. A family history of autoimmune diseases (all types) occurs in 60% of patients. One hallmark of AIR has been the presence of anti-retinal autoimmune antibodies (ARAs) in patients' sera, but patients can continue to have ARAs even when the disease has been quiescent for years. The accumulation of ARAs represents a breakdown of retinal immune tolerance with many different immunoreactive bands found at different reference weights in AIR patients. We began investigating cellular immunity using flow cytometry and found abnormal distributions (>2 StDev) of increased memory lymphocytes and NK cells and decreased regulatory B cell subsets in many AIR patients compared to normal controls. Culture of patient lymphocytes with small amounts (25 µg) of recoverin protein for 6 days led to significant elevations of interferon gamma (IFNγ) and in some cases tumor necrosis factor alpha (TNFα) production. We found the IFNγ/IL-10 ratio in response to recoverin was elevated in patients with more active disease (defined by visual field contraction between visits), but in some patients, there also appeared to be independent factors influencing severity, suggesting other autoimmune mechanisms were at play. These cellular immune parameters may provide improved markers for active AIR.

Dimethyl Fumarate Selectively Reduces Memory T Cells and Shifts the Balance between Th1/Th17 and Th2 in Multiple Sclerosis Patients.

Dimethyl fumarate (DMF; trade name Tecfidera) is an oral formulation of the fumaric acid ester that is Food and Drug Administration approved for treatment of relapsing-remitting multiple sclerosis. To better understand the therapeutic effects of Tecfidera and its rare side effect of progressive multifocal leukoencephalopathy, we conducted cross-sectional and longitudinal studies by immunophenotyping cells from peripheral blood (particularly T lymphocytes) derived from untreated and 4-6 and 18-26 mo Tecfidera-treated stable relapsing-remitting multiple sclerosis patients using multiparametric flow cytometry. The absolute numbers of CD4 and CD8 T cells were significantly decreased and the CD4/CD8 ratio was increased with DMF treatment. The proportions of both effector memory T cells and central memory T cells were reduced, whereas naive T cells increased in treated patients. T cell activation was reduced with DMF treatment, especially among effector memory T cells and effector memory RA T cells. Th subsets Th1 (CXCR3+), Th17 (CCR6+), and particularly those expressing both CXCR3 and CD161 were reduced most significantly, whereas the anti-inflammatory Th2 subset (CCR3+) was increased after DMF treatment. A corresponding increase in IL-4 and decrease in IFN-γ and IL-17-expressing CD4+ T cells were observed in DMF-treated patients. DMF in vitro treatment also led to increased T cell apoptosis and decreased activation, proliferation, reactive oxygen species, and CCR7 expression. Our results suggest that DMF acts on specific memory and effector T cell subsets by limiting their survival, proliferation, activation, and cytokine production. Monitoring these subsets could help to evaluate the efficacy and safety of DMF treatment.

Cystoid macular changes on optical coherence tomography in a patient with maternally inherited diabetes and deafness (MIDD)-associated macular dystrophy.

The clinical presentation and optical coherence tomography findings in a patient with maternally inherited diabetes and deafness (MIDD) are presented to highlight the presence of macular cystoid spaces in some patients with this disease. Typically, patients with MIDD demonstrate progression of a pigmentary maculopathy into areas of geographic macular atrophy. At the time of initial visit, the 30-year-old patient had large macular cystoid changes in addition to retinal pigmentary changes in both eyes. The cystoid changes responded to treatment with systemic immunosuppression and a topical carbonic anhydrase inhibitor (CAI), recurred when treated with topical CAI monotherapy, and finally resolved after an intravitreal triamcinolone acetonide injection. Over time, the retinal atrophy continued to progress, but the macular cysts did not recur. The patient received systemic immunosuppression for renal transplantation due to renal failure resulting from focal glomerulosclerosis. There was no evidence of diabetic retinopathy at any time during the five-and-a-half-year follow-up, and the patient retained good visual acuity in both eyes.

IL-2 augments the therapeutic efficacy of adoptively transferred B cells which directly kill tumor cells via the CXCR4/CXCL12 and perforin pathways.

We previously reported that antitumor B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10. In this study, we defined additional mechanisms involved in B cell antitumor immunity. Administration of IL-2 significantly augmented the therapeutic efficacy of adoptively transferred tumor-draining lymph node (TDLN) B cells which express IL- 2R. Culture supernatant of purified B splenocytes harvested from the mice that received adoptive transfer of 4T1 TDLN B cells plus IL-2 administration produced larger amounts of IgG which bound to 4T1, resulting in 4T1 lysis. Furthermore, we detected CXCR4 expression on 4T1 TDLN B cells, and 4T1 tumor cells produced its ligand CXCL12. Transwell experiments demonstrated the chemoattraction of CXCR4-expressing 4T1 TDLN B cells towards CXCL12- producing 4T1 cells. Blockade of CXCR4 using a CXCR4-specific inhibitor, AMD3100, significantly reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Blockade of FasL and CXCR4 concurrently inhibited B cell-mediated direct killing of tumor cells in an additive manner, indicating that both Fas/FasL and CXCL12/CXCR4 pathways are involved in the direct killing of 4T1 cells by 4T1 TDLN B cells. TDLN B cells produced perforin. Additional transwell experiments showed that effector B cells could directly kill tumor cells in cell-cell contact via the Fas/FasL and CXCR4/CXCL12 pathways as well as perforin, while without cell contact, perforin secreted by B cells led to tumor cell cytotoxicity. These findings underscore the diversity of function by which B cells can play an important role in the host immune response to tumor.

Dimethyl fumarate treatment of relapsing-remitting multiple sclerosis influences B-cell subsets.

OBJECTIVE: To test the hypothesis that dimethyl fumarate (Tecfidera, BG-12) affects B-cell subsets in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: Peripheral blood B cells were compared for surface marker expression in patients with RRMS prior to initiation of treatment, after 4-6 months, and at more than 1 year of treatment with BG-12. Production of interleukin (IL)-10 by RRMS patient B cells was also analyzed. RESULTS: Total numbers of peripheral blood B lymphocytes declined after 4-6 months of BG-12 treatment, due to losses in both the CD27+ memory B cells and CD27(neg) B-cell subsets. Some interpatient variability was observed. In contrast, circulating CD24(high)CD38(high) (T2-MZP) B cells increased in percentage in the majority of patients with RRMS after 4-6 months and were present in higher numbers in all of the patients after 12 months of treatment. The CD43+CD27+ B-1 B cells also increased at the later time point in most patients but were unchanged at 4-6 months compared to pretreatment levels. Purified B cells from 7 of the 9 patients with RRMS tested after 4-6 months of treatment were able to produce IL-10 following CD40 ligand stimulation, and the amount corresponded with the combined levels of T2-MZP and B-1 B cells in the sample. None of the patients with RRMS in this study have had a relapse while taking BG-12. CONCLUSIONS: These data suggest that BG-12 differentially affects B-cell subsets in patients with RRMS, resulting in increased numbers of circulating B lymphocytes with regulatory capacity.

Sudden acquired retinal degeneration syndrome (SARDS) - a review and proposed strategies toward a better understanding of pathogenesis, early diagnosis, and therapy.

Sudden acquired retinal degeneration syndrome (SARDS) is one of the leading causes of currently incurable canine vision loss diagnosed by veterinary ophthalmologists. The disease is characterized by acute onset of blindness due to loss of photoreceptor function, extinguished electroretinogram with an initially normal appearing ocular fundus, and mydriatic pupils which are slowly responsive to bright white light, unresponsive to red, but responsive to blue light stimulation. In addition to blindness, the majority of affected dogs also show systemic abnormalities suggestive of hyperadrenocorticism, such as polyphagia with resulting obesity, polyuria, polydipsia, and a subclinical hepatopathy. The pathogenesis of SARDS is unknown, but neuroendocrine and autoimmune mechanisms have been suggested. Therapies that target these disease pathways have been proposed to reverse or prevent further vision loss in SARDS-affected dogs, but these treatments are controversial. In November 2014, the American College of Veterinary Ophthalmologists' Vision for Animals Foundation organized and funded a Think Tank to review the current knowledge and recently proposed ideas about disease mechanisms and treatment of SARDS. These panel discussions resulted in recommendations for future research strategies toward a better understanding of pathogenesis, early diagnosis, and potential therapy for this condition.

Dimethyl Fumarate Protects Neural Stem/Progenitor Cells and Neurons from Oxidative Damage through Nrf2-ERK1/2 MAPK Pathway.

Multiple sclerosis (MS) is the most common multifocal inflammatory demyelinating disease of the central nervous system (CNS). Due to the progressive neurodegenerative nature of MS, developing treatments that exhibit direct neuroprotective effects are needed. Tecfidera™ (BG-12) is an oral formulation of the fumaric acid esters (FAE), containing the active metabolite dimethyl fumarate (DMF). Although BG-12 showed remarkable efficacy in lowering relapse rates in clinical trials, its mechanism of action in MS is not yet well understood. In this study, we reported the potential neuroprotective effects of dimethyl fumarate (DMF) on mouse and rat neural stem/progenitor cells (NPCs) and neurons. We found that DMF increased the frequency of the multipotent neurospheres and the survival of NPCs following oxidative stress with hydrogen peroxide (H2O2) treatment. In addition, utilizing the reactive oxygen species (ROS) assay, we showed that DMF reduced ROS production induced by H2O2. DMF also decreased oxidative stress-induced apoptosis. Using motor neuron survival assay, DMF significantly promoted survival of motor neurons under oxidative stress. We further analyzed the expression of oxidative stress-induced genes in the NPC cultures and showed that DMF increased the expression of transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) at both levels of RNA and protein. Furthermore, we demonstrated the involvement of Nrf2-ERK1/2 MAPK pathway in DMF-mediated neuroprotection. Finally, we utilized SuperArray gene screen technology to identify additional anti-oxidative stress genes (Gstp1, Sod2, Nqo1, Srxn1, Fth1). Our data suggests that analysis of anti-oxidative stress mechanisms may yield further insights into new targets for treatment of multiple sclerosis (MS).

Characterization of Regulatory B Cells in Graves' Disease and Hashimoto's Thyroiditis.

A hallmark of regulatory B cells is IL-10 production, hence their designation as IL-10+ B cells. Little is known about the ability of self-antigens to induce IL-10+ B cells in Graves' disease (GD), Hashimoto's thyroiditis (HT), or other autoimmune disease. Here we pulsed purified B cells from 12 HT patients, 12 GD patients, and 12 healthy donors with the thyroid self-antigen, thyroglobulin (TG) and added the B cells back to the remaining peripheral blood mononuclear cells (PBMCs). This procedure induced IL-10+ B-cell differentiation in GD. A similar tendency was observed in healthy donors, but not in cells from patients with HT. In GD, B cells primed with TG induced IL-10-producing CD4+ T cells. To assess the maximal frequency of inducible IL-10+ B cells in the three donor groups PBMCs were stimulated with PMA/ionomycin. The resulting IL-10+ B-cell frequency was similar in the three groups and correlated with free T3 levels in GD patients. IL-10+ B cells from both patient groups displayed CD25 or TIM-1 more frequently than did those from healthy donors. B-cell expression of two surface marker combinations previously associated with regulatory B-cell functions, CD24hiCD38hi and CD27+CD43+, did not differ between patients and healthy donors. In conclusion, our findings indicate that autoimmune thyroiditis is not associated with reduced frequency of IL-10+ B cells. These results do not rule out regulatory B-cell dysfunction, however. The observed phenotypic differences between IL-10+ B cells from patients and healthy donors are discussed.

Killer B lymphocytes and their fas ligand positive exosomes as inducers of immune tolerance.

Induction of immune tolerance is a key process by which the immune system is educated to modulate reactions against benign stimuli such as self-antigens and commensal microbes. Understanding and harnessing the natural mechanisms of immune tolerance may become an increasingly useful strategy for treating many types of allergic and autoimmune diseases, as well as for improving the acceptance of solid organ transplants. Our laboratory and others have been interested in the natural ability of some B lymphocytes to express the death-inducing molecule Fas ligand (FasL), and their ability to kill T helper (TH) lymphocytes. We have recently shown that experimental transformation of human B cells by a non-replicative variant of Epstein-Barr virus (EBV) consistently resulted in high expression of functional FasL protein. The production and release of FasL(+) exosomes that co-expressed major histocompatibility complex (MHC) class II molecules and had the capacity to kill antigen-specific TH cells was also observed. Several lines of evidence indicate that FasL+ B cells and FasL(+)MHCII(+) exosomes have important roles in natural immune tolerance and have a great deal of therapeutic potential. Taken together, these findings suggest that EBV-immortalized human B lymphoblastoid cell lines could be used as cellular factories for FasL(+) exosomes, which would be employed to therapeutically establish and/or regain immune tolerance toward specific antigens. The goals of this review are to summarize current knowledge of the roles of FasL(+) B cells and exosomes in immune regulation, and to suggest methods of manipulating killer B cells and FasL(+) exosomes for clinical purposes.