Bibiana Bielekova

Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: Results of a phase II clinical trial with an altered peptide ligand

Myelin-specific T lymphocytes are considered essential in the pathogenesis of multiple sclerosis. The myelin basic protein peptide (a.a. 83–99) represents one candidate antigen; therefore, it was chosen to design an altered peptide ligand, CGP77116, for specific immunotherapy of multiple sclerosis. A magnetic resonance imaging-controlled phase II clinical trial with this altered peptide ligand documented that it was poorly tolerated at the dose tested, and the trial had therefore to be halted. Improvement or worsening of clinical or magnetic resonance imaging parameters could not be demonstrated in this small group of individuals because of the short treatment duration. Three patients developed exacerbations of multiple sclerosis, and in two this could be linked to altered peptide ligand treatment by immunological studies demonstrating the encephalitogenic potential of the myelin basic protein peptide (a.a. 83–99) in a subgroup of patients. These data raise important considerations for the use of specific immunotherapies in general.

Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy.

Nine cancer patients were treated with adoptive cell therapy using autologous anti-MAGE-A3 T-cell receptors (TCR)-engineered T cells. Five patients experienced clinical regression of their cancers including 2 on-going responders. Beginning 1–2 days postinfusion, 3 patients (#’s 5, 7, and 8) experienced mental status changes, and 2 patients (5 and 8) lapsed into comas and subsequently died. Magnetic resonance imagining analysis of patients 5 and 8 demonstrated periventricular leukomalacia, and examination of their brains at autopsy revealed necrotizing leukoencephalopathy with extensive white matter defects associated with infiltration of CD3+/CD8+ T cells. Patient 7, developed Parkinson-like symptoms, which resolved over 4 weeks and fully recovered. Immunohistochemical staining of patient and normal brain samples demonstrated rare positively staining neurons with an antibody that recognizes multiple MAGE-A family members. The TCR used in this study recognized epitopes in MAGE-A3/A9/A12. Molecular assays of human brain samples using real-time quantitative-polymerase chain reaction, Nanostring quantitation, and deep-sequencing indicated that MAGE-A12 was expressed in human brain (and possibly MAGE-A1, MAGE-A8, and MAGE-A9). This previously unrecognized expression of MAGE-A12 in human brain was possibly the initiating event of a TCR-mediated inflammatory response that resulted in neuronal cell destruction and raises caution for clinical applications targeting MAGE-A family members with highly active immunotherapies.

Identification of candidate T-cell epitopes and molecular mimics in chronic Lyme disease.

Elucidating the cellular immune response to infectious agents is a prerequisite for understanding disease pathogenesis and designing effective vaccines. In the identification of microbial T-cell epitopes, the availability of purified or recombinant bacterial proteins has been a chief limiting factor. In chronic infectious diseases such as Lyme disease, immune-mediated damage may add to the effects of direct infection by means of molecular mimicry to tissue autoantigens. Here, we describe a new method to effectively identify both microbial epitopes and candidate autoantigens. The approach combines data acquisition by positional scanning peptide combinatorial libraries and biometric data analysis by generation of scoring matrices. In a patient with chronic neuroborreliosis, we show that this strategy leads to the identification of potentially relevant T-cell targets derived from both Borrelia burgdorferi and the host. We also found that the antigen specificity of a single T-cell clone can be degenerate and yet the clone can preferentially recognize different peptides derived from the same organism, thus demonstrating that flexibility in T-cell recognition does not preclude specificity. This approach has potential applications in the identification of ligands in infectious diseases, tumors and autoimmune diseases.

Complex immunomodulatory effects of interferon-β in multiple sclerosis include the upregulation of T helper 1-associated marker genes

Multiple sclerosis (MS) is considered an autoimmune disease that is mediated by proinflammatory T helper‐1 lymphocytes. The putative mechanism of interferon‐β (IFN‐β), an approved treatment for MS, includes the inhibition of T‐cell proliferation, blocking of blood‐brain‐barrier opening and T‐cell transmigration into the brain via interference with cell adhesion, and the upregulation of anti‐inflammatory (TH2) cytokines. In the present study, a gene expression analysis of IFN‐β‐treated peripheral blood mononuclear cells by cDNA microarray documents the broad effects of IFN‐β that are not purely anti‐inflammatory. Specifically, we addressed the effect of IFN‐β on T helper‐1 differentiation‐ or lineage markers such as the IL‐12 receptor β2 chain and the chemokine receptor CCR5 that have been implicated in the pathogenesis of MS. Both markers were significantly upregulated in vitro and in vivo under IFN‐β therapy, supporting that this cytokine exerts complex effects on the immune system. The combination of cDNA microarray and quantitative polymerase chain reaction will expand our knowledge of the immunological effects of such pleiotropic agents as IFN‐β, may provide a key to why certain patients fail to respond, and eventually influence our view of the disease pathogenesis.

Expansion and Functional Relevance of High-Avidity Myelin-Specific CD4+ T Cells in Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune disease in which myelin-specific T cells are believed to play a crucial pathogenic role. Nevertheless, so far it has been extremely difficult to demonstrate differences in T cell reactivity to myelin Ag between MS patients and controls. We believe that by using unphysiologically high Ag concentrations previous studies have missed a highly relevant aspect of autoimmune responses, i.e., T cells recognizing Ag with high functional avidity. Therefore, we focused on the characterization of high-avidity myelin-specific CD4+ T cells in a large cohort of MS patients and controls that was matched demographically and with respect to expression of MHC class II alleles. We demonstrated that their frequency is significantly higher in MS patients while the numbers of control T cells specific for influenza hemagglutinin are virtually identical between the two cohorts; that high-avidity T cells are enriched for previously in vivo-activated cells and are significantly skewed toward a proinflammatory phenotype. Moreover, the immunodominant epitopes that were most discriminatory between MS patients and controls differed from those described previously and were clearly biased toward epitopes with lower predicted binding affinities to HLA-DR molecules, pointing at the importance of thymic selection for the generation of the autoimmune T cell repertoire. Correlations between selected immunological parameters and magnetic resonance imaging markers indicate that the specificity and function of these cells influences phenotypic disease expression. These data have important implications for autoimmunity research and should be considered in the development of Ag-specific therapies in MS.

Effect of Anti-CD25 Antibody Daclizumab in the Inhibition of Inflammation and Stabilization of Disease Progression in Multiple Sclerosis

BACKGROUND Several questions arise concerning the use of the anti-CD25 antibody daclizumab to treat multiple sclerosis (MS). OBJECTIVES To answer the following 3 questions related to the efficacy of daclizumab therapy in patients with MS: Is the therapeutic effect of daclizumab dependent on combination with interferon beta? Is a higher dosage of daclizumab more efficacious in patients with persistent disease activity? Can biomarkers predict full vs partial therapeutic response to daclizumab? DESIGN An open-label baseline vs treatment phase II clinical trial of daclizumab in patients having MS with inadequate response to interferon beta. Three months of interferon beta treatment at baseline were followed by 5.5 months of interferon beta-daclizumab combination therapy. If patients experienced more than 75% reduction of contrast-enhancing lesions (CELs) on brain magnetic resonance imaging at month 5.5 compared with baseline, daclizumab was continued as monotherapy for 10 months. Otherwise, the dosage of daclizumab was doubled. SETTING Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland. PATIENTS Fifteen patients with MS receiving standard preparations of interferon beta who experienced more than 1 MS exacerbation or whose clinical disability increased in the preceding 12 months and who had at least 2 CELs on baseline brain magnetic resonance images. INTERVENTION Daclizumab (1 mg/kg) as an intravenous infusion every 4 weeks in combination with interferon beta (months 0-5.5) and as monotherapy (months 6.5-15.5). MAIN OUTCOME MEASURES The primary outcome was the reduction of CELs among interferon beta monotherapy, interferon beta-daclizumab combination therapy, and daclizumab monotherapy. The secondary outcomes included immunologic biomarkers and changes in clinical disability. RESULTS Overall, 5 of 15 patients (33%) experienced adverse effects of therapy. Two patients developed systemic adverse effects, and daclizumab therapy was discontinued. Although daclizumab monotherapy was efficacious in 9 of 13 patients with MS, interferon beta-daclizumab combination therapy was necessary to stabilize disease activity in the other 4 patients. Daclizumab therapy led to 72% inhibition of new CELs and significant improvement in clinical disability. Pilot biomarkers (increase in CD56bright natural killer cells and decrease in CD8+ T cells) were identified that can differentiate between full and partial daclizumab responders. CONCLUSIONS Daclizumab monotherapy is effective in most patients who experienced persistent MS disease activity with interferon beta therapy. Interferon beta-daclizumab combination therapy or higher dosages of daclizumab may be necessary to achieve optimal therapeutic response in all patients. Biomarkers may identify patients with suboptimal response to daclizumab monotherapy. Administration among a large patient sample during a longer period is needed to fully define the safety and long-term efficacy of daclizumab as treatment for high-inflammatory MS. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00001934.

Mechanisms of immunomodulation by glatiramer acetate.

Objective To define the mechanism of action of glatiramer acetate (GA; formerly known as copolymer-1) as an immunomodulatory treatment for MS. Background The proposed mechanisms of action of GA include 1) functional inhibition of myelin-reactive T cells by human leukocyte antigen (HLA) blocking, 2) T-cell receptor (TCR) antagonism, and 3) induction of T helper 2 (Th2) immunomodulatory cells. In this report, the authors examined the effects of GA on the functional activation of human T-cell clones (TCC) specific for myelin basic protein (MBP) and for foreign antigens. Several questions were addressed: Is the inhibitory effect of GA specific for autoantigens? Is it mediated by blocking the interaction between peptide and HLA molecule? Is GA a partial agonist or TCR antagonist, or does it induce anergy? Does it induce Th2 modulatory T cells? Methods The effects of GA on antigen-induced activation of human TCC specific for MBP, influenza virus hemagglutinin, and Borrelia burgdorferi were studied by proliferation and cytokine measurements, TCR downmodulation, and anergy assays. GA-specific TCC were generated in vitro from the peripheral blood of patients and healthy controls by limiting dilution. Results GA more strongly inhibited the proliferation of MBP, as compared with foreign antigen-specific TCC; in some MBP-specific TCC, the production of Th1-type cytokines was preferentially inhibited. In addition to HLA competition, the induction of anergy, but not direct TCR antagonism, was observed. Numerous GA-specific TCC were generated from the peripheral blood of both MS patients and normal controls, and a fraction of these showed a Th2 phenotype. Conclusions This study confirms a preferential inhibitory effect of GA on autoreactive TCC. With respect to cellular mechanisms, although HLA competition appears to play the most important role in functional inhibition in vitro, a direct effect on the TCR may be involved at least in some autoreactive T cells as shown by anergy induction. Although not confirmed at the clonal level, it is demonstrated further that GA induces T cells that crossreact with myelin proteins. GA-specific, Th2-modulatory cells may play an important role in mediating the effect of the drug in vivo.

Inhibition of LTi Cell Development by CD25 Blockade Is Associated with Decreased Intrathecal Inflammation in Multiple Sclerosis

Daclizumab inhibition of inflammation in human autoimmunity is associated with blockade of lymphoid tissue inducer cell development. The Innate Talent of Daclizumab Watching an Olympic medalist excel at his or her chosen event is a thing of beauty. Yet when it comes to analyzing these superior performances, opinions differ on the relative contributions of innate talent versus targeted training. In the debilitating autoimmune disease multiple sclerosis (MS), T lymphocytes—adaptive immune cells—have long been thought to be one of the main culprits. However, a new study by Perry et al. suggests that the humanized monoclonal antibody daclizumab decreases MS-associated inflammation partly through its inhibitory effects on innate rather than adaptive lymphoid cells. Daclizumab has a direct but limited ability to block the function of CD25, a receptor that is up-regulated on activated T cells, including those involved in autoimmune disease. In the new work, the authors turned their attention to innate lymphoid cells (ILCs), components of the other branch of the immune system, and found higher numbers of circulating ILCs in untreated MS patients compared with daclizumab-treated ones. Indeed, daclizumab therapy modified this cell population, skewing it toward a more immunoregulatory cell type. Thus, daclizumab may indirectly modulate activated T cells by altering the regulatory effects of ILCs on the adaptive immune system. Therapies that specifically target ILCs represent a new avenue of therapeutic research for MS and perhaps other autoimmune diseases as well. If this research crosses the translational finish line, MS patients will take home the gold. Genetic polymorphisms in the interleukin-2 receptor α (IL-2Rα) chain (CD25) locus are associated with several human autoimmune diseases, including multiple sclerosis (MS). Blockade of CD25 by the humanized monoclonal antibody daclizumab decreases MS-associated inflammation but has surprisingly limited direct inhibitory effects on activated T cells. The present study describes unexpected effects of daclizumab therapy on innate lymphoid cells (ILCs). The number of circulating retinoic acid receptor–related orphan receptor γt–positive ILCs, which include lymphoid tissue inducer (LTi) cells, was found to be elevated in untreated MS patients compared to healthy subjects. Daclizumab therapy not only decreased numbers of ILCs but also modified their phenotype away from LTi cells and toward a natural killer (NK) cell lineage. Mechanistic studies indicated that daclizumab inhibited differentiation of LTi cells from CD34+ hematopoietic progenitor cells or c-kit+ ILCs indirectly, steering their differentiation toward immunoregulatory CD56bright NK cells through enhanced intermediate-affinity IL-2 signaling. Because adult LTi cells may retain lymphoid tissue–inducing capacity or stimulate adaptive immune responses, we indirectly measured intrathecal inflammation in daclizumab-treated MS patients by quantifying the cerebrospinal fluid chemokine (C-X-C motif) ligand 13 and immunoglobulin G index. Both of these inflammatory biomarkers were inhibited by daclizumab treatment. Our study indicates that ILCs are involved in the regulation of adaptive immune responses, and their role in human autoimmunity should be investigated further, including their potential as therapeutic targets.

Evolution of the blood–brain barrier in newly forming multiple sclerosis lesions

Multiple sclerosis (MS) lesions develop around small, inflamed veins. New lesions enhance with gadolinium on magnetic resonance imaging (MRI), reflecting disruption of the blood–brain barrier (BBB). Single time point results from pathology and standard MRI cannot capture the spatiotemporal expansion of lesions. We investigated the development and expansion of new MS lesions, focusing on the dynamics of BBB permeability.

Myelin-associated oligodendrocytic basic protein: identification of an encephalitogenic epitope and association with multiple sclerosis.

Myelin-associated oligodendrocytic basic protein (MOBP) is an abundant myelin constituent expressed exclusively by oligodendrocytes, the myelin-forming cells of the CNS. We report that MOBP causes experimental allergic encephalomyelitis and is associated with multiple sclerosis. First, we note that purified recombinant MOBP inoculated into SJL/J mice produces CNS disease. Tests of overlapping peptides spanning the murine MOBP molecule map the encephalitogenic site to amino acids 37–60. MOBP-induced experimental allergic encephalomyelitis shows a severe clinical course and is characterized by a prominent CD4+ T lymphocyte infiltration and a lesser presence of CD8+ T cells and microglia/macrophages around vessels and in the white matter of the CNS. Second, PBL obtained from patients with relapsing/remitting multiple sclerosis mount a proliferative response to human MOBP, especially at amino acids 21–39. This response equals or exceeds the response to myelin basic protein and an influenza virus hemagglutinin peptide, both serving as internal controls. Thus, a novel myelin Ag, MOBP aa 37–60, plays a role in rodent autoimmune CNS disease, and its human MOBP counterpart is associated with the human demyelinating disease multiple sclerosis.