Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis.

In autoimmunity, the balance of different helper T (Th) cell subsets can influence the tissue damage caused by autoreactive T cells. Pro-inflammatory Th1 and Th17 T cells are implicated as mediators of several human autoimmune conditions such as multiple sclerosis (MS). Autologous hematopoietic stem cell transplantation (aHSCT) has been tested in phase 2 clinical trials for MS patients with aggressive disease. Abrogation of new clinical relapses and brain lesions can be seen after ablative aHSCT, accompanied by significant reductions in Th17, but not Th1, cell populations and activity. The cause of this selective decrease in Th17 cell responses following ablative aHSCT is not completely understood. We identified an increase in the kinetics of natural killer (NK) cell reconstitution, relative to CD4+ T cells, in MS patients post-aHSCT, resulting in an increased NK cell:CD4+ T cell ratio that correlated with the degree of decrease in Th17 responses. Ex vivo removal of NK cells from post-aHSCT peripheral blood mononuclear cells resulted in higher Th17 cell responses, indicating that NK cells can regulate Th17 activity. NK cells were also found to be cytotoxic to memory Th17 cells, and this toxicity is mediated through NKG2D-dependent necrosis. Surprisingly, NK cells induced memory T cells to secrete more IL-17A. This was preceded by an early rise in T cell expression of RORC and IL17A mRNA, and could be blocked with neutralizing antibodies against CD58, a costimulatory receptor expressed on NK cells. Thus, NK cells provide initial co-stimulation that supports the induction of a Th17 response, followed by NKG2D-dependent cytotoxicity that limits these cells. Together these data suggest that rapid reconstitution of NK cells following aHSCT contribute to the suppression of the re-emergence of Th17 cells. This highlights the importance of NK cells in shaping the reconstituting immune system following aHSCT in MS patients.

Postnatal Neural Stem Cells in Treating Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disabilities worldwide. It affects approximately 1.5 million people each year and is associated with severe post-TBI symptoms such as sensory and motor deficits. Several neuro-therapeutic approaches ranging from cell therapy interventions such as the use of neural stem cells (NSCs) to drug-based therapies have been proposed for TBI management. Successful cell-based therapies are tightly dependent on reproducible preclinical animal models to ensure safety and optimal therapeutic benefits. In this chapter, we describe the isolation of NSCs from neonatal mouse brain using the neurosphere assay in culture. Subsequently, dissociated neurosphere-derived cells are used for transplantation into the ipsilateral cortex of a controlled cortical impact (CCI) TBI model in C57BL/6 mice. Following intra-cardiac perfusion and brain removal, the success of NSC transplantation is then evaluated using immunofluorescence in order to assess neurogenesis along with gliosis in the ipsilateral coronal brain sections. Behavioral tests including rotarod and pole climbing are conducted to evaluate the motor activity post-treatment intervention.

Risk factors for multiple sclerosis and associations with anti-EBV antibody titers.

Multiple sclerosis (MS) is an inflammatory demyelination of the central nervous system. We investigated the prevalence of EBV seropositivity and other known risk factors for MS (age, smoking, low vitamin D) and their effect on anti-EBV antibody titers. We retrospectively studied 249 MS patients receiving care at the American University of Beirut Medical Center and 230 controls, during 2010-2014. EBV seropositivity was higher in MS patients compared to controls for both anti-VCA (99.5%; 97.2%) and anti-EBNA-1 (96.3%; 89.4%), and the titers were significantly higher in MS patients. MS patients had a significantly lower vitamin D level (15.5 ± 8.3 ng/ml) compared to controls (20.4 ± 11.3 ng/ml). The proportion of heavy smokers and overweight individuals was significantly higher in MS patients. Lebanese MS patients have risk factors similar to those in western countries. Older age and female gender were associated with a higher anti-VCA titer and male gender with a higher anti-EBNA-1.

Abnormal B-cell cytokine responses a trigger of T-cell-mediated disease in MS?

OBJECTIVE: To study antibody-independent contributions of B cells to inflammatory disease activity, and the immune consequences of B-cell depletion with rituximab, in patients with multiple sclerosis (MS). METHODS: B-Cell effector-cytokine responses were compared between MS patients and matched controls using a 3-signal model of activation. The effects of B-cell depletion on Th1/Th17 CD4 and CD8 T-cell responses in MS patients were assessed both ex vivo and in vivo, together with pharmacokinetic/pharmacodynamic studies as part of 2 rituximab clinical trials in relapsing-remitting MS. RESULTS: B Cells of MS patients exhibited aberrant proinflammatory cytokine responses, including increased lymphotoxin (LT):interleukin-10 ratios and exaggerated LT and tumor necrosis factor (TNF)-alpha secretion, when activated in the context of the pathogen-associated TLR9-ligand CpG-DNA, or the Th1 cytokine interferon-gamma, respectively. B-Cell depletion, both ex vivo and in vivo, resulted in significantly diminished proinflammatory (Th1 and Th17) responses of both CD4 and CD8 T cells. Soluble products from activated B cells of untreated MS patients reconstituted the diminished T-cell responses observed following in vivo B-cell depletion in the same patients, and this effect appeared to be largely mediated by B-cell LT and TNFalpha. INTERPRETATION: We propose that episodic triggering of abnormal B-cell cytokine responses mediates 'bystander activation' of disease-relevant proinflammatory T cells, resulting in new relapsing MS disease activity. Our findings point to a plausible mechanism for the long-recognized association between infections and new MS relapses, and provide novel insights into B-cell roles in both health and disease, and into mechanisms contributing to therapeutic effects of B-cell depletion in human autoimmune diseases, including MS.

Expression of IL-9 receptor alpha chain on human germinal center B cells modulates IgE secretion.

BACKGROUND: IL-9 has been shown to affect the differentiation pathway of different cell types. However, its potential role in the maturation pathway of antigen-driven B-cell differentiation and its functional effects remain unknown. OBJECTIVE: To characterize IL-9 receptor alpha chain (IL-9R alpha) expression on human tonsillar B cells at different maturational stages, and to assess its effect on IgE production. METHODS: Freshly purified human tonsillar B cells were fractionated into 3 populations: low-density (LD), medium-density, and high-density cells. Expression levels of IL-9R alpha were determined by using immunohistochemistry and flow cytometry. IL-9R alpha(high)-expressing cells were stimulated with IL-9 in the presence or absence of IL-4, and IgE release was measured by ELISA. RESULTS: IL-9R alpha was expressed on human LD tonsillar B cells, with an ability to transduce signals through activation of signal transducer and activator of transcription 3 and 5. Although IL-9 was unable to induce IgE secretion by itself, it potentiated IL-4-mediated IgE production from LD cells. Moreover, increased IgE was paralleled by an upregulation of IL-9R alpha and CD27, with the latter a memory B-cell marker implicated in increased IgE secretion. CONCLUSION: These results highlight a crucial role for IL-9 in modulating T-cell-dependent B-cell differentiation and establish a new paradigm for understanding the synergistic role of T(H)2 cytokines and their modulatory effect on B-cell maturation and IgE production. CLINICAL IMPLICATIONS: IL-9 appears to be involved in memory B-cell differentiation and T(H)2-mediated allergic diseases such as asthma.

Regulation of IL-13 receptor alpha 1 expression and signaling on human tonsillar B-lymphocyte subsets.

BACKGROUND: T(H)2 cytokines play crucial roles in driving human B lymphocytes to produce IgE. However, it is unclear whether IL-4 and IL-13 have parallel or sequential roles in the development of B lymphocytes. OBJECTIVE: We investigated IL-13 receptor (IL-13R) expression and regulation in mature and immature human B cells. METHODS: Purified B cells were isolated from human tonsils. We evaluated IL-13Ralpha1 mRNA expression using real-time PCR and IL-13Ralpha1 and IL-4 receptor (IL-4R) alpha expression using flow cytometry and microscopy. Signal transduction was assessed on the basis of signal transducer and activator of transcription 6 phosphorylation. RESULTS: IL13Ralpha1 mRNA was induced after stimulation with anti-CD40 antibodies, anti-CD40 plus IL-4, or anti-IgM/IgG. Baseline surface IL13Ralpha1 levels were low in unstimulated B cells but increased significantly at 24 hours and were sustained for 5 to 14 days. In contrast, IL4R alpha was constitutively expressed on tonsillar B cells, and levels did not significantly vary after stimulation. B cells activated by CD40 ligation or B-cell receptor cross-linking, but not resting B cells, showed significant increases in signal transducer and activator of transcription 6 phosphorylation in response to IL-13. IL-13Ralpha1 expression was induced on mature and memory B cells, as well as on naive subsets. CONCLUSIONS: There is lower constitutive expression and signaling of IL13Ralpha1 in resting tonsillar B lymphocytes compared with that of IL4R alpha. IL-13 is induced on both immature and mature B lymphocytes. CLINICAL IMPLICATIONS: This implies different roles for IL-4 and IL-13 in B-cell development, which would allow for specific targeting of IL-13 in IgE-mediated diseases.

Leukotriene A4 hydrolase expression in PEL cells is regulated at the transcriptional level and leads to increased leukotriene B4 production.

Primary effusion lymphoma (PEL) is a herpesvirus-8-associated lymphoproliferative disease characterized by migration of tumor cells to serous body cavities. PEL cells originate from postgerminal center B cells and share a remarkable alteration in B cell transcription factor expression and/or activation with classical Hodgkin's disease cells. Comparative analysis of gene expression by cDNA microarray of BCBL-1 cells (PEL), L-428 (classical Hodgkin's disease), and BJAB cells revealed a subset of genes that were differentially expressed in BCBL-1 cells. Among these, four genes involved in cell migration and chemotaxis were strongly up-regulated in PEL cells: leukotriene A4 (LTA4) hydrolase (LTA4H), IL-16, thrombospondin-1 (TSP-1), and selectin-P ligand (PSGL-1). Up-regulation of LTA4H was investigated at the transcriptional level. Full-length LTA4H promoter exhibited 50% higher activity in BCBL-1 cells than in BJAB or L-428 cells. Deletion analysis of the LTA4H promoter revealed a positive cis-regulatory element active only in BCBL-1 cells in the promoter proximal region located between -76 and -40 bp. Formation of a specific DNA-protein complex in this region was confirmed by EMSA. Coculture of ionophore-stimulated primary neutrophils with BCBL-1 cells leads to an increased production of LTB4 compared with coculture with BJAB and L-428 cells as measured by enzyme immunoassay, demonstrating the functional significance of LTA4H up-regulation.