Anita Posevitz-Fejfar

The proteolytic activity of the paracaspase MALT1 is key in T cell activation.

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-κB 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.

L-Selectin is a possible biomarker for individual PML risk in natalizumab-treated MS patients

Objective: To find biomarkers identifying patients at risk for the development of progressive multifocal leukoencephalopathy (PML) during natalizumab treatment. Methods: Patients were recruited from 10 European and US cohorts. Of 289 patients with multiple sclerosis (MS), 224 had been treated with natalizumab (18–80 months), 21 received other immune-modulatory treatments, and 28 were untreated. We had access to samples from 16 natalizumab PML patients. Eight of these patients had given blood before the diagnosis of PML. We also analyzed non-natalizumab-treated patients who developed PML (n = 10) and age- and sex-matched healthy donors (n = 31). All flow cytometric assessments were done on previously cryopreserved, viable peripheral blood mononuclear cells. Results: The percentage of l-selectin-expressing CD4+ T cells was significantly lower in patients treated long-term with natalizumab (40.2%) when compared with patients not receiving natalizumab treatment (47.2%; p = 0.016) or healthy controls (61.0%; p < 0.0001). An unusually low percentage (9-fold lower; 4.6%) was highly correlated with the risk of developing PML in the patient group with available pre-PML samples when compared with non-PML natalizumab-treated patients (p ≤ 0.0001). Samples were gathered between 4 and 26 months before PML diagnosis. Conclusions: 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.

Evidence for a TCR Affinity Threshold Delimiting Maximal CD8 T Cell Function

Protective adaptive immune responses rely on TCR-mediated recognition of Ag-derived peptides presented by self-MHC molecules. However, self-Ag (tumor)-specific TCRs are often of too low affinity to achieve best functionality. To precisely assess the relationship between TCR–peptide–MHC binding parameters and T cell function, we tested a panel of sequence-optimized HLA-A*0201/NY–ESO-1157–165–specific TCR variants with affinities lying within physiological boundaries to preserve antigenic specificity and avoid cross-reactivity, as well as two outliers (i.e., a very high- and a low-affinity TCR). Primary human CD8 T cells transduced with these TCRs demonstrated robust correlations between binding measurements of TCR affinity and avidity and the biological response of the T cells, such as TCR cell-surface clustering, intracellular signaling, proliferation, and target cell lysis. Strikingly, above a defined TCR–peptide–MHC affinity threshold (KD < ∼5 μM), T cell function could not be further enhanced, revealing a plateau of maximal T cell function, compatible with the notion that multiple TCRs with slightly different affinities participate equally (codominantly) in immune responses. We propose that rational design of improved self-specific TCRs may not need to be optimized beyond a given affinity threshold to achieve both optimal T cell function and avoidance of the unpredictable risk of cross-reactivity.

PML risk stratification using anti-JCV antibody index and L-selectin.

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.

Fingolimod treatment promotes regulatory phenotype and function of B cells

To evaluate the influence of Fingolimod treatment on B‐cell subset composition and function in multiple sclerosis patients and its potential clinical relevance.

Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation

Significance The importance of natural killer (NK) cells in the control of autoimmunity has recently attracted considerable attention. The current study revealed NK cells as additional players in controlling T-cell activity in CNS autoimmunity. NK-mediated control of T-cell activity in multiple sclerosis (MS) is dysregulated and caused by impaired DNAX accessory molecule-1/CD155 interaction between NK cells and CD4+ T cells. Therapeutic immune modulation of the IL-2 receptor with daclizumab, which has just successfully passed a phase III study in relapsing-remitting MS, not only enhances the cytolytic activity of NK cells but also restores defective NK-mediated immune regulation by increasing the proportion of CD155-expressing CD4+ T cells, thus rendering CD4+ T cells most likely more sensitive to NK-mediated lysis. 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, CD56bright NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α4β1 integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4+ T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4+ T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor’s ligand CD155 on CD4+ T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4+ T cells and the cytolytic activity of NK cells.

An assay to quantify species-specific anti-JC virus antibody levels in MS patients.

Background: The StratifyJCV® test is a qualitative assay to classify MS patients as anti-JC virus (JCV) antibody positive or negative. Quantification of anti-JCV antibody levels in serum and cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients might add to the progressive multifocal leukoencephalopathy (PML) risk assessment. Objective: The objective of this study is to test sera of patients in a quantitative anti-JCV antibody assay, and to compare the results with preexisting data from the StratifyJCV® test. Methods: Sera of a total of 175 MS patients and matched non-MS-controls were tested for anti-JCV antibodies using glutathione S-transferase-tagged-VP1 as antigen. Antibody reactivity was quantified in arbitrary units using human immunoglobulin as standard. Results: The comparison of our assay with StratifyJCV® showed good inter-assay agreement (kappa 0.6), and strong correlation for antibody reactivity (r2 = 0.94). Discordant samples had low-reactive positivity, and a higher proportion (13% vs. 4%) tested positive in the StratifyJCV® test only. Conclusions: The method presented is a tool for the reliable quantification of anti-JCV antibodies, which demonstrates good agreement with results from StratifyJCV®. In contrast to StratifyJCV®, we pre-adsorbed all of the sera with BK virus (BKV) VP1 protein to reduce cross-reactivity. This step may account for a higher species-specificity of our assay. As such, our assay might be a promising additional tool for PML risk assessment.

Anti-JC-virus antibody prevalence in a German MS cohort.

Dear Sir, The anti-JC-virus-IgG (anti-JCV) antibody status has been introduced to stratify patients with multiple sclerosis (MS) treated with natalizumab for the risk of developing progressive multifocal leukoencephalopathy (PML). We tested sera of 511 patients (360 females, 151 males) from 9 German MS centres for the anti-JCV antibody status applying the published protocol.1 The samples were either taken for the purpose of treatment decision making (e.g. treatment discontinuation, treatment initiation with natalizumab) or within the prospective German natalizumab pharmacovigilance study after obtaining written informed consent. This study was approved by the local ethical committees at the different sites. The overall seroprevalence in our German cohort was 56%, and there was no significant sex difference (females 55%, males 58%, Figure 1A). The seropositivity rate gradually increased with age, from 37% in patients younger than 20 years to 77% in patients at the age of 60 years or beyond (Figure 1B). Our data independently confirm the published data in other cohorts, applying the same methodology.1, 2 The observed increase in seropositivity with age, fitting a reported annual seroconversion rate for anti-JCVantibody status ranging between 2% and 3%,1 strongly argues for a close clinical and serological follow-up of patients tested negative for anti-JCV antibodies when applying the test to stratify patients for the risk of developing PML in clinical practice. In addition, we studied six patients with PML from our cohort prior to (n = 3) or at the time of PML diagnosis (n = 3). All of these patients tested positive for anti-JCV antibodies. All non-PML patients with detectable JCVDNA in serum (1 of 33 tested) or urine (3 of 18 tested) also tested positive for anti-JCV antibodies, supporting the potential utility of this method: no false-negative results were observed. However, no firm conclusions, such as risk calculations, can be drawn from these data as the number of patients with PML included was too low. Long-term observational studies are on the way to prospectively correlate the JCV antibody positivity and the development of PML during treatment with natalizumab.3 In the meantime the test might already be a useful tool to assist in treatment decisions in a proportion of patients.4 Nevertheless, high clinical and paraclinical vigilance for signs of severe 429955 MSJ18710.1177/1352458511429955Warnke et al.Multiple Sclerosis Journal 2012

Distinct pattern of lesion distribution in multiple sclerosis is associated with different circulating T-helper and helper-like innate lymphoid cell subsets

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.

JCV index and L-selectin for natalizumab-associated PML risk stratification

important biomarker in neuromyelitis optica. Furthermore, autoantibodies to myelin oligodendrocyte glycoprotein (MOG) are detected in pediatric demyelinating disorders. We examined a cohort of adults with AQP4 antibody-negative neuromyelitis optica spectrum disorder (NMOSD) for antibodies to MOG. Methods: We performed a flow cytometry cell-based assay using live human embryonic kidney 293 cells that were lentivirus-transduced cells to express full-length surface MOG. Serum was tested in 23 AQP4 antibody-negative NMOSD patients with bilateral and/or recurrent optic neuritis (BON, n = 11), longitudinally extensive transverse myelitis (LETM, n = 10), and sequential BON and LETM (n= 2). Control cohorts included patients with clinically definite multiple sclerosis (MS, n = 76), as well as age matched healthy and other neurological disease controls (n= 52). Results: MOG antibodies were detected in 9/23 AQP4 antibodynegative NMOSD patients compared to 1/76 MS patients and 0/52 controls (P b 0.001). In all patients, the MOG antibodies were of the IgG rather than the IgM isotype. MOG antibodies were detected in 8/11 BON, 0/10 LETM, and 1/2 sequential BON and LETM patients. 6/9 MOG antibody-positive AQP4 antibody-negative patients had a relapsing course. MOG antibody-positive patients were more often female, of younger age at disease onset, and had a preceding viral prodrome compared to MOG antibody-negative patients (P values not significant). MOG antibody-positive patients had prominent bilateral optic disc swelling at presentation, andweremore likely to have a rapid response to steroid therapy and relapse on steroid cessation than MOG antibody-negative patients (P= 0.034, P = 0.029, respectively). While 8/9 MOG antibody-positive patients had good follow-up visual acuity, one experienced sustained impairments in visual acuity and visual field testing. Furthermore, three patients had retinal nerve fiber layer atrophy on optical coherence tomography at follow-up, and one had residual spinal disability. Conclusions: MOG antibodies have a strong association with BON and may be a useful clinical biomarker. MOG antibody-associated BON is a relapsing disorder that is frequently steroid responsive and often steroid dependent. Failure of early recognition and institution of immunotherapy may be associated with sustained impairment.