Nicholas Schwab

Early detrimental T-cell effects in experimental cerebral ischemia are neither related to adaptive immunity nor thrombus formation

T cells contribute to the pathophysiology of ischemic stroke by yet unknown mechanisms. Mice with transgenic T-cell receptors (TCRs) and mutations in costimulatory molecules were used to define the minimal immunologic requirements for T cell-mediated ischemic brain damage. Stroke was induced in recombination activating gene 1-deficient (RAG1(-/-)) mice devoid of T and B cells, RAG1(-/-) mice reconstituted with B cells or T cells, TCR-transgenic mice bearing 1 single CD8(+) (2C/RAG2, OTI/RAG1 mice) or CD4(+) (OTII/RAG1, 2D2/RAG1 mice) TCR, mice lacking accessory molecules of TCR stimulation (CD28(-/-), PD1(-/-), B7-H1(-/-) mice), or mice deficient in nonclassical T cells (natural killer T [NKT] and gammadelta T cells) by transient middle cerebral artery occlusion (tMCAO). Stroke outcome was assessed at day 1. RAG1(-/-) mice and RAG1(-/-) mice reconstituted with B cells developed significantly smaller brain infarctions compared with controls, but thrombus formation after FeCl(3)-induced vessel injury was unimpaired. In contrast, TCR-transgenic mice and mice lacking costimulatory TCR signals were fully susceptible to tMCAO similar to mice lacking NKT and gammadelta T cells. These findings were corroborated by adoptive transfer experiments. Our data demonstrate that T cells critically contribute to cerebral ischemia, but their detrimental effect neither depends on antigen recognition nor TCR costimulation or thrombus formation.

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.

CD8+ T-cell clones dominate brain infiltrates in Rasmussen encephalitis and persist in the periphery.

Rasmussen encephalitis (RE) is a rare neurological disorder of childhood characterized by uni-hemispheric inflammation, progressive neurological deficits and intractable focal epilepsy. Destruction of neurons and astrocytes by cytotoxic CD8 T cells has been proposed as a pathogenic mechanism underlying this enigmatic disorder. We tested this hypothesis by analysing the clonal composition and T-cell receptor (TCR) repertoire of CD4+ and CD8+ T cells using complementarity determining region 3 (CDR3) spectratyping from peripheral blood and corresponding CNS specimens. Severe perturbations of the TCR repertoire were found in brain infiltrates from all specimens (n = 5). Clonal expansions, as evidenced by peripheral blood analysis (n = 14), belonged to the CD8+ T-cell subset, while CD4+ cells showed normal distributions. Some of those expansions were analysed in the respective CNS specimens by histochemistry. The stainings showed Vbeta specific T cells containing the cytotoxic molecule granzyme B and lying in close appositions to NeuN+ neurons and GFAP+ astrocytes. Analysis of corresponding CNS/blood specimens revealed overlapping but also CNS-restricted expansions of certain TCR clonotypes suggesting expansions of T cells within the target organ itself. Longitudinal analysis of peripheral blood samples (n = 5) demonstrated dominance but also longitudinal persistence of specific CD8 T-cell clones over time. The Vbeta/Jbeta usage, length of the CDR3, and biochemical characteristics of the CDR3 amino acids suggested high similarities putatively related to common driving antigen(s) without shared clones. Taken together, our data strongly support the hypothesis of an antigen-driven MHC class-I restricted, CD8+ T cell-mediated attack against neurons and astrocytes in the CNS dominating the pathogenesis in RE.

Ultraviolet B light attenuates the systemic immune response in central nervous system autoimmunity

Environmental conditions (eg, latitude) play a critical role in the susceptibility and severity of many autoimmune disorders, including multiple sclerosis (MS). Here, we investigated the mechanisms underlying the beneficial effects of immune regulatory processes induced in the skin by moderate ultraviolet B (UVB) radiation on central nervous system (CNS) autoimmunity.

Fatal PML associated with efalizumab therapy: insights into integrin αLβ2 in JC virus control.

Objectives: Progressive multifocal leukoencephalopathy (PML) has become much more common with monoclonal antibody treatment for multiple sclerosis and other immune-mediated disorders. Methods: We report 2 patients with severe psoriasis and fatal PML treated for ≥3 years with efalizumab, a neutralizing antibody to αLβ2-leukointegrin (LFA-1). In one patient, we conducted serial studies of peripheral blood and CSF including analyses of leukocyte phenotypes, migration ex vivo, and CDR3 spectratypes with controls coming from HIV-infected patients with PML. Extensive pathologic and histologic analysis was done on autopsy CNS tissue of both patients. Results: Both patients developed progressive cognitive and motor deficits, and JC virus was identified in CSF. Despite treatment including plasma exchange (PE) and signs of immune reconstitution, both died of PML 2 and 6 months after disease onset. Neuropathologic examination confirmed PML. Efalizumab treatment was associated with reduced transendothelial migration by peripheral T cells in vitro. As expression levels of LFA-1 on peripheral T cells gradually rose after PE, in vitro migration increased. Peripheral and CSF T-cell spectratyping showed CD8+ T-cell clonal expansion but blunted activation, which was restored after PE. Conclusions: From these data we propose that inhibition of peripheral and intrathecal T-cell activation and suppression of CNS effector-phase migration both characterize efalizumab-associated PML. LFA-1 may be a crucial factor in homeostatic JC virus control.

An Imbalance of Two Functionally and Phenotypically Different Subsets of Plasmacytoid Dendritic Cells Characterizes the Dysfunctional Immune Regulation in Multiple Sclerosis

Plasmacytoid dendritic cells (pDCs) are instrumental in peripheral T cell tolerance and innate immunity. How pDCs control peripheral immunetolerance and local parenchymal immune response and contribute to the altered immunoregulation in autoimmune disorders in humans is poorly understood. Based on their surface markers, cytokine production, and ability to prime naive allogenic T cells, we found that purified BDCA-2+BDCA-4+ pDCs consist of at least two separate populations, which differed in their response to oligodeoxynucleotides and IFNs (IFN-β), and differently induced IL-17– or IL-10–producing T cells. To evaluate the potential immunoregulatory role of these two types of pDCs in multiple sclerosis (MS) and other human autoimmune disorders (myasthenia gravis), we studied the phenotype and regulatory function of pDCs isolated from clinically stable, untreated patients with MS (n = 16). Patients with MS showed a reversed ratio of pDC1/pDC2 in peripheral blood (4.4:1 in healthy controls, 0.69:1 in MS), a phenomenon not observed in the other autoimmune disorders. As a consequence, MS pDCs had an overall propensity to prime IL-17–secreting cells over IL-10–secreting CD4+ T cells. Immunomodulatory therapy with IFN-β induced an increase of the pDC1 population in vivo (n = 5). Our data offer a plausible explanation for the disturbed immune tolerance in MS patients and provide evidence that immunomodulatory therapy acts at the level of reconstituting homeostasis of pDC, thus reconstituting the disturbed balance.

T cell suppression by naturally occurring HLA-G-expressing regulatory CD4+ T cells is IL-10-dependent and reversible

CD4+ T cells constitutively expressing the immune‐tolerogenic HLA‐G have been described recently as a new type of nTreg (HLA‐Gpos Treg) in humans. HLA‐Gpos Treg accumulate at sites of inflammation and are potent suppressors of T cell proliferation in vitro, suggesting their role in immune regulation. We here characterize the mechanism of how CD4+ HLA‐Gpos Treg influence autologous HLA‐Gneg Tresp function. Using a suppression system free of APC, we demonstrate a T–T cell interaction, resulting in suppression of HLA‐Gneg Tresp, which is facilitated by TCR engagement on HLA‐Gpos Treg. Suppression is independent of cell–cell contact and is reversible, as the removal of HLA‐Gpos Treg from the established coculture restored the proliferative capability of responder cells. Further, HLA‐Gpos Treg‐mediated suppression critically depends on the secretion of IL‐10 but not TGF‐β.

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.

Immunological and clinical consequences of treating a patient with natalizumab

Background: Long-term therapy with natalizumab increases the risk of progressive multifocal leukoencephalopathy (PML). Objectives: We present a patient study through therapy, the diagnosis of PML (after 29 infusions), plasma exchange (PE) and development of immune reconstitution inflammatory syndrome (IRIS). Methods: Routine diagnostics, magnetic resonance imaging (MRI), immunological status (flow cytometry, T-cell migration assays and T-cell repertoire analysis), and brain biopsy with immunohistological analysis. Results: CD49d decreased after 12 months of treatment. At PML diagnosis, CD49d expression and migratory capacity of T cells was low and peripheral T-cell receptor (TCR) complexity showed severe perturbations. The distribution of peripheral monocytes changed from CCR5+ to CCR7+. After PE some changes reverted: CD49d increased and overshot earliest levels, migratory capacities of T cells recovered and peripheral TCR complexity increased. With no clinical, routine laboratory or cerebrospinal fluid (CSF) changes, MRI 2 months after PE demonstrated progressive lesion development. Brain histopathology confirmed the presence of infiltrates indicative of IRIS without clinical signs, immunologically accompanied by CCR7/CCR5 recovery of peripheral monocytes. Conclusion: Natalizumab-associated immunological changes accompanying PML were reversible after PE; IRIS can occur very late, remain asymptomatic and be elusive to CSF analysis. Our study may provide insights into the changes under treatment with natalizumab associated with JC virus control.

FOXP3+ T regulatory cells in idiopathic inflammatory myopathies.

FOXP3+ T regulatory cells (Tregs) are considered key players in the maintenance of immune homeostasis. Here we studied the presence and potential role of FOXP3+ Tregs in myositis. CD3 and FOXP3 expression in dermatomyositis, polymyositis and inclusion body myositis was assessed by immunohistochemistry and real-time PCR. FOXP3+ Tregs were found in close proximity to effector cells and their numbers correlated with the degree of inflammation. Despite divergent pathogenetic concepts, we observed no differences in the frequency of FOXP3 immunoreactive cells or FOXP3 mRNA expression between different myositis entities. Functional assays using human myoblasts as targets of CD8+ cells demonstrate that Tregs are capable to inhibit the lytic activity of cytotoxic cells. Our data suggest that FOXP3 Tregs serve to counterbalance muscle destruction by cytotoxic T cells in myositis.