The paracaspase MALT1 is pivotal in antigen receptor-mediated lymphocyte activation and lymphomagenesis. MALT1 contains a caspase-like domain, but it is unknown whether this domain is proteolytically active. Here we report that MALT1 had arginine-directed proteolytic activity that was activated after T cell stimulation, and we identify the signaling protein Bcl-10 as a MALT1 substrate. Processing of Bcl-10 after Arg228 was required for T cell receptor-induced cell adhesion to fibronectin. In contrast, MALT1 activity but not Bcl-10 cleavage was essential for optimal activation of transcription factor NF-kappaB and production of interleukin 2. Thus, the proteolytic activity of MALT1 is central to T cell activation, which suggests a possible target for the development of immunomodulatory or anticancer drugs.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood-brain barrier, CD56

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Objective:To evaluate the effect of dimethyl fumarate (DMF; Tecfidera, Biogen, Weston, MA) on CD4+ and CD8+ T cell subsets in patients with multiple sclerosis (MS).Methods:Peripheral lymphocyte subsets, including CD4+ and CD8+ memory cells and T helper (TH) cells TH1, TH2, TH17, and peripheral regulatory T cell (pTreg) subpopulations were analyzed before and 6 months after onset of DMF treatment.Results:CD4+ and CD8+ memory T cells were preferentially decreased compared to naive CD4+ and CD8+ T cell populations. Within the CD4+ memory T cell population, frequencies of TH1 cells were decreased, whereas those of TH2 cells were increased and those of TH17 cells remained unaltered. Accordingly, we observed decreased production of interferon γ, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-22 by CD4+ T cells under DMF treatment, whereas the frequency of IL-4- and IL-17A-producing CD4+ T cells remained unchanged. With regard to regulatory T cells, proportions of pTreg increased following DMF treatment.Conclusion:Our data demonstrate that DMF treatment of patients with MS affects predominantly memory T cells accompanied by a shift in TH cell populations, resulting in a shift toward anti-inflammatory responses. These findings indicate that monitoring of memory subsets might enhance vigilance of impaired antiviral immunity and that patients with TH1-driven disease might preferentially benefit from DMF treatment.Classification of Evidence:This study provides Class IV evidence that DMF might preferentially reduce CD4+ and CD8+ memory T cells in MS.

The cell-based assessment of the percentage of l-selectin-expressing CD4 T cells could provide an urgently needed biomarker for individual PML risk assessment.

ObjectiveTo evaluate the influence of Fingolimod treatment on B-cell subset composition and function in multiple sclerosis patients and its potential clinical relevance.MethodsSubset composition and cytokine production of B cells derived from peripheral blood mononuclear cells from multiple sclerosis patients under Fingolimod treatment, untreated multiple sclerosis patients and healthy controls were analyzed by flow cytometry and ELISA. Migration of lymphocyte subsets across primary human brain microvascular endothelial cells was assessed in an in vitro transmigration assay. Cell numbers and composition of B-cell subsets in cerebrospinal fluid and peripheral blood were determined by flow cytometry. Regulatory B-cell frequencies were correlated with parameters of disease stability.ResultsWithin the peripheral B-cell compartment of Fingolimod-treated patients, the proportion of regulatory B cells (CD38+CD27−CD24+CD5+) was significantly increased as compared to treatment-naïve multiple sclerosis patients and to healthy controls, and significantly more regulatory B cells produced Interleukin-10. Fingolimod treatment enhanced the capacity of regulatory B cells to transmigrate across brain endothelial cells in an in vitro model of the blood-brain-barrier. In line with these findings, the cerebrospinal fluid/blood ratio of total B cells and regulatory B cells was strongly increased by Fingolimod treatment, and patients exhibited increased regulatory B-cell frequencies in the cerebrospinal fluid. Finally, elevated regulatory B-cell percentages in the periphery significantly correlated with clinical and paraclinical disease stability.InterpretationThese data suggest a novel and as yet unrecognized role of Fingolimod in correction of the imbalance between regulatory and effector B-cell functions in multiple sclerosis both by direct effects and indirect partitioning effects on B-cell subpopulations.

In primary human T cells, anergy induction results in enhanced p59Fyn activity. Because Fyn is the kinase primarily responsible for the phosphorylation of PAG (the phosphoprotein associated with glycosphingolipid-enriched microdomains), which negatively regulates Srckinase activity by recruiting Csk (the Cterminal Src kinase) to the membrane, we investigated whether anergy induction also affects PAG. Analysis of anergic T cells revealed that PAG is hyperphosphorylated at the Csk binding site, leading to IntroductionIn the thymus, most T cells undergo negative selection as they bind with high affinity to the self-peptide/MHC complexes presented. However, thymic selection is not a tight process and some autoreactive T cells escape into the periphery. 1 To protect the body from these potentially destructive cells, there must exist mechanisms of peripheral tolerance that prevent autoreactive T cells from becoming activated; one such mechanism is anergy. 2 T cells receiving a strong signal via the T-cell receptor (TCR) [signal 1] in the absence of costimulation [signal 2] enter into an unresponsive state (ie, anergy) in which the cells are alive but fail to display functional responses, such as proliferation in response to full activation [signal 1 ϩ 2]. 3,4 Although the mechanism responsible for the induction of anergy remains unknown, the characterization of anergic T cells has clearly demonstrated that the lack of proliferation is due to a block in interleukin-2 (IL-2) production and that anergic T cells proliferate after addition of exogenous IL-2. The defect in IL-2 production is attributed to a block in Ras activation, 5-7 a small G-protein that activates the mitogen-activated protein kinase cascade, ultimately resulting in activation of the transcription factor activator protein-1 (AP-1). Indeed, the ionomycininduced anergy model shows that activation of nuclear factor of activated T cells in the absence of AP-1 and nuclear factor of the -enhancer in B cells (NF-B) leads to the transcription of genes that induce anergy. 8 Within the TCR signaling cascade, the earliest alteration detected in anergic T cells is the up-regulation of the Src family kinase (SFK) p59Fyn. 9,10 However the mechanism by which Fyn contributes to anergy is unknown. In addition to the TCR, 11 Fyn also binds the phosphoprotein associated with glycosphingolipidenriched microdomains (PAG) 12 (also known as the Csk binding protein [Cbp]). 13 Fyn is the kinase responsible for PAG phosphorylation. 14 PAG, a transmembrane adaptor that localizes within the lipid rafts, negatively regulates Src kinase activity by recruiting the C-terminal Src kinase (Csk) to the membrane. 15 Csk in turn phosphorylates the inhibitory tyrosines conserved within the Src kinases. 16 It is noteworthy that T cells overexpressing PAG are resistant to activation via the TCR, a phenotype similar to anergy. 12,17 Therefore, we chose to investigate whether anergy induction leads to changes within PAG that might contribute to the unresponsiveness of anergic T cells. Material...

Human biospecimen collection, processing and preservation are rapidly emerging subjects providing essential support to clinical as well as basic researchers. Unlike collection of other biospecimens (e.g. DNA and serum), biobanking of viable immune cells, such as peripheral blood mononuclear cells (PBMC) and/or isolated immune cell subsets is still in its infancy. While certain aspects of processing and freezing conditions have been studied in the past years, little is known about the effect of blood transportation on immune cell survival, phenotype and specific functions. However, especially for multicentric and cooperative projects it is vital to precisely know those effects. In this study we investigated the effect of blood shipping and pre-processing delay on immune cell phenotype and function both on cellular and subcellular levels. Peripheral blood was collected from healthy volunteers (n = 9): at a distal location (shipped overnight) and in the central laboratory (processed immediately). PBMC were processed in the central laboratory and analyzed post-cryopreservation. We analyzed yield, major immune subset distribution, proliferative capacity of T cells, cytokine pattern and T-cell receptor signal transduction. Results show that overnight transportation of blood samples does not globally compromise T- cell subsets as they largely retain their phenotype and proliferative capacity. However, NK and B cell frequencies, the production of certain PBMC-derived cytokines and IL-6 mediated cytokine signaling pathway are altered due to transportation. Various control experiments have been carried out to compare issues related to shipping versus pre-processing delay on site. Our results suggest the implementation of appropriate controls when using multicenter logistics for blood transportation aiming at subsequent isolation of viable immune cells, e.g. in multicenter clinical trials or studies analyzing immune cells/subsets. One important conclusion might be that despite changes due to overnight shipment, highly standardized central processing (and analysis) could be superior to multicentric de-central processing with more difficult standardization.