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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.

Background: Distinct lesion topography in relapsing-remitting multiple sclerosis (RRMS) might be due to different antigen presentation and/or trafficking routes of immune cells into the central nervous system (CNS). Objective: To investigate whether distinct lesion patterns in multiple sclerosis (MS) might be associated with a predominance of distinct circulating T-helper cell subset as well as their innate counterparts. Methods: Flow cytometric analysis of lymphocytes derived from the peripheral blood of patients with exclusively cerebral (n = 20) or predominantly spinal (n = 12) disease manifestation. Results: Patients with exclusively cerebral or preferential spinal lesion manifestation were associated with increased proportions of circulating granulocyte-macrophage colony-stimulating factor (GM-CSF) producing TH1 cells or interleukin (IL)-17-producing TH17 cells, respectively. In contrast, proportions of peripheral IL-17/IL-22-producing lymphoid tissue inducer (LTi), the innate counterpart of TH17 cells, were enhanced in RRMS patients with exclusively cerebral lesion topography. Conclusions: Distinct T-helper and T-helper-like innate lymphoid cell (ILC) subsets are associated with different lesion topography in RRMS. [ABSTRACT FROM AUTHOR]

Background: Natalizumab treatment is associated with progressive multifocal leukoencephalopathy (PML) development. Treatment duration, prior immunosuppressant use, and JCV serostatus are currently used for risk stratification, but PML incidence stays high. Anti-JCV antibody index and L-selectin (CD62L) have been proposed as additional risk stratification parameters. Objective: This study aimed at verifying and integrating both parameters into one algorithm for risk stratification. Methods: Multicentric, international cohorts of natalizumab-treated MS patients were assessed for JCV index (1921 control patients and nine pre-PML patients) and CD62L (1410 control patients and 17 pre-PML patients). Results: CD62L values correlate with JCV serostatus, as well as JCV index values. Low CD62L in natalizumab-treated patients was confirmed and validated as a biomarker for PML risk with the risk factor “CD62L low” increasing a patient’s relative risk 55-fold (p < 0.0001). Validation efforts established 86% sensitivity/91% specificity for CD62L and 100% sensitivity/59% specificity for JCV index as predictors of PML. Using both parameters identified 1.9% of natalizumab-treated patients in the reference center as the risk group. Conclusions: Both JCV index and CD62L have merit for risk stratification and share a potential biological relationship with implications for general PML etiology. A risk algorithm incorporating both biomarkers could strongly reduce PML incidence. [ABSTRACT FROM AUTHOR]

PAG, the phosphoprotein associated with glycosphingolipid-enriched microdomains (GEM), negatively regulates Src family kinases by recruiting C-terminal Src kinase (Csk) to the membrane, where Csk phosphorylates the inhibitory tyrosine of the Src kinases. S-acylation of a CxxC motif juxtaposed to the transmembrane domain within PAG has been proposed to be responsible for targeting PAG to the lipid rafts. Here, we present the characterization of a mutant PAG molecule lacking the palmitoylation motif. We demonstrate that the mutant protein is expressed at the plasma membrane, but does not localize within the GEM. Despite being displaced, the mutant PAG molecule still binds the Src kinase Fyn and the cytoskeletal adaptor ezrin-radixin-moesin-binding phosphoprotein of 50 kDa, becomes tyrosine-phosphorylated, and recruits Csk to the membrane. Functional characterization of the mutant shows that, unlike WT PAG, it does not block proximal TCR signaling, and surprisingly enhances stromal cell-derived factor 1 (CXCL12)-induced migration. The mutant functions by depleting Csk from the GEM fractions, as apparent by changes in the phosphorylation of the inhibitory tyrosines within the Src kinases. Indeed this mechanism is supported by RNA interference of PAG, which results in enhanced migration and Src kinase activity. Our results therefore support a functional role for the compartmentalization of Src kinases within the membrane. [ABSTRACT FROM AUTHOR]

Background: Distinct lesion topography in relapsing-remitting multiple sclerosis (RRMS) might be due to different antigen presentation and/or trafficking routes of immune cells into the central nervous system (CNS)., Objective: To investigate whether distinct lesion patterns in multiple sclerosis (MS) might be associated with a predominance of distinct circulating T-helper cell subset as well as their innate counterparts., Methods: Flow cytometric analysis of lymphocytes derived from the peripheral blood of patients with exclusively cerebral (n = 20) or predominantly spinal (n = 12) disease manifestation., Results: Patients with exclusively cerebral or preferential spinal lesion manifestation were associated with increased proportions of circulating granulocyte-macrophage colony-stimulating factor (GM-CSF) producing T H 1 cells or interleukin (IL)-17-producing T H 17 cells, respectively. In contrast, proportions of peripheral IL-17/IL-22-producing lymphoid tissue inducer (LTi), the innate counterpart of T H 17 cells, were enhanced in RRMS patients with exclusively cerebral lesion topography., Conclusions: Distinct T-helper and T-helper-like innate lymphoid cell (ILC) subsets are associated with different lesion topography in RRMS.

Background: Natalizumab treatment is associated with progressive multifocal leukoencephalopathy (PML) development. Treatment duration, prior immunosuppressant use, and JCV serostatus are currently used for risk stratification, but PML incidence stays high. Anti-JCV antibody index and L-selectin (CD62L) have been proposed as additional risk stratification parameters., Objective: This study aimed at verifying and integrating both parameters into one algorithm for risk stratification., Methods: Multicentric, international cohorts of natalizumab-treated MS patients were assessed for JCV index (1921 control patients and nine pre-PML patients) and CD62L (1410 control patients and 17 pre-PML patients)., Results: CD62L values correlate with JCV serostatus, as well as JCV index values. Low CD62L in natalizumab-treated patients was confirmed and validated as a biomarker for PML risk with the risk factor "CD62L low" increasing a patient's relative risk 55-fold (p < 0.0001). Validation efforts established 86% sensitivity/91% specificity for CD62L and 100% sensitivity/59% specificity for JCV index as predictors of PML. Using both parameters identified 1.9% of natalizumab-treated patients in the reference center as the risk group., Conclusions: Both JCV index and CD62L have merit for risk stratification and share a potential biological relationship with implications for general PML etiology. A risk algorithm incorporating both biomarkers could strongly reduce PML incidence., (© The Author(s), 2015.)

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.