Vissia Viglietta

Loss of Functional Suppression by CD4+CD25+ Regulatory T Cells in Patients with Multiple Sclerosis

CD4+CD25+ regulatory T cells contribute to the maintenance of peripheral tolerance by active suppression because their deletion causes spontaneous autoimmune diseases in mice. Human CD4+ regulatory T cells expressing high levels of CD25 are suppressive in vitro and mimic the activity of murine CD4+CD25+ regulatory T cells. Multiple sclerosis (MS) is an inflammatory disease thought to be mediated by T cells recognizing myelin protein peptides. We hypothesized that altered functions of CD4+CD25hi regulatory T cells play a role in the breakdown of immunologic self-tolerance in patients with MS. Here, we report a significant decrease in the effector function of CD4+CD25hi regulatory T cells from peripheral blood of patients with MS as compared with healthy donors. Differences were also apparent in single cell cloning experiments in which the cloning frequency of CD4+CD25hi T cells was significantly reduced in patients as compared with normal controls. These data are the first to demonstrate alterations of CD4+CD25hi regulatory T cell function in patients with MS.

Inhibition of Human CD4+CD25+high Regulatory T Cell Function

CD4+CD25+high T cells are potent regulators of autoreactive T cells. However, it is unclear how regulatory CD4+CD25+high cells discriminate between desirable inflammatory immune responses to microbial Ags and potentially pathologic responses by autoreactive T cells. In this study, an in vitro model was created that allowed differential activation of regulatory CD4+CD25+high and responder CD4+ T cells. If CD4+CD25+high regulatory cells were strongly activated, they maintained suppressive effector function for only 15 h, while stimulation with weaker TCR stimuli produced regulatory cells that were suppressive until 60 h after activation. In contrast, strongly activated CD4+ responder T cells were resistant to regulation at all time points, while weakly stimulated CD4+ cells were sensitive to suppression until 38 or 60 h after activation depending upon the strength of the stimulus. The extent of suppression mediated by CD4+CD25+high cells also depended on the strength of stimulation in an Ag-specific system. Thus, the stronger the TCR signal, the more rapidly and more completely the responder cells become refractory to suppression.

Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein

The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte glycoprotein (MOG) by selective unfolding of the antigen domain. The tetramer radioimmunoassay (RIA) was more sensitive for MOG autoantibody detection than other methodologies, including monomer-based RIA, ELISA or fluorescent-activated cell sorting (FACS). Autoantibodies from individuals with acute disseminated encephalomyelitis (ADEM) selectively bound the folded MOG tetramer, whereas sera from mice with experimental autoimmune encephalomyelitis induced with MOG peptide immunoprecipitated only the unfolded tetramer. MOG-specific autoantibodies were identified in a subset of ADEM but only rarely in adult-onset MS cases, indicating that MOG is a more prominent target antigen in ADEM than MS.

Antigen microarrays identify unique serum autoantibody signatures in clinical and pathologic subtypes of multiple sclerosis

Multiple sclerosis (MS) is a chronic relapsing disease of the central nervous system (CNS) in which immune processes are believed to play a major role. To date, there is no reliable method by which to characterize the immune processes and their changes associated with different forms of MS and disease progression. We performed antigen microarray analysis to characterize patterns of antibody reactivity in MS serum against a panel of CNS protein and lipid autoantigens and heat shock proteins. Informatic analysis consisted of a training set that was validated on a blinded test set. The results were further validated on an independent cohort of relapsing–remitting (RRMS) samples. We found unique autoantibody patterns that distinguished RRMS, secondary progressive (SPMS), and primary progressive (PPMS) MS from both healthy controls and other neurologic or autoimmune driven diseases including Alzheimers disease, adrenoleukodystropy, and lupus erythematosus. RRMS was characterized by autoantibodies to heat shock proteins that were not observed in PPMS or SPMS. In addition, RRMS, SPMS, and PPMS were characterized by unique patterns of reactivity to CNS antigens. Furthermore, we examined sera from patients with different immunopathologic patterns of MS as determined by brain biopsy, and we identified unique antibody patterns to lipids and CNS-derived peptides that were linked to each type of pathology. The demonstration of unique serum immune signatures linked to different stages and pathologic processes in MS provides an avenue to monitor MS and to characterize immunopathogenic mechanisms and therapeutic targets in the disease.

GAD65-reactive T cells are activated in patients with autoimmune type 1a diabetes

Insulin-dependent type 1 diabetes is an autoimmune disease mediated by T lymphocytes recognizing pancreatic islet cell antigens. Glutamic acid decarboxylase 65 (GAD65) appears to be an important autoantigen in the disease. However, T cells from both patients with type 1 diabetes and healthy subjects vigorously proliferate in response to GAD65 stimulation ex vivo, leading us to postulate that the critical event in the onset of human diabetes is the activation of autoreactive T cells. Thus, we investigated whether GAD65-reactive T cells in patients with diabetes functioned as previously activated memory T cells, no longer requiring a second, costimulatory signal for clonal expansion. We found that in patients with new-onset type 1 diabetes, GAD65-reactive T cells were strikingly less dependent on CD28 and B7-1 costimulation to enter into cell cycle and proliferate than were equivalent cells derived from healthy controls. We hypothesize that these autoreactive T cells have been activated in vivo and have differentiated into memory cells, suggesting a pathogenic role in type 1 diabetes. In addition, we observed different effects with selective blockade of either B7-1 or B7-2 molecules; B7-1 appears to deliver a negative signal by engaging CTLA-4, while B7-2 engagement of CD28 upregulates T cell proliferation and cytokine secretion.

Age-Dependent B Cell Autoimmunity to a Myelin Surface Antigen in Pediatric Multiple Sclerosis

Multiple sclerosis (MS) typically manifests in early to mid adulthood, but there is increasing recognition of pediatric-onset MS, aided by improvements in imaging techniques. The immunological mechanisms of disease are largely unexplored in pediatric-onset MS, in part because studies have historically focused on adult-onset disease. We investigated autoantibodies to myelin surface Ags in a large cohort of pediatric MS cases by flow cytometric labeling of transfectants that expressed different myelin proteins. Although Abs to native myelin oligodendrocyte glycoprotein (MOG) were uncommon among adult-onset patients, a subset of pediatric patients had serum Abs that brightly labeled the MOG transfectant. Abs to two other myelin surface Ags were largely absent. Affinity purification of MOG Abs as well as competition of binding with soluble MOG documented their binding specificity. Such affinity purified Abs labeled myelin and glial cells in human CNS white matter as well as myelinated axons in gray matter. The prevalence of such autoantibodies was highest among patients with a very early onset of MS: 38.7% of patients less than 10 years of age at disease onset had MOG Abs, compared with 14.7% of patients in the 10- to 18-year age group. B cell autoimmunity to this myelin surface Ag is therefore most common in patients with a very early onset of MS.

Natalizumab treatment is associated with peripheral sequestration of proinflammatory T cells

Background: Natalizumab is an antibody directed against integrin α4 that reduces disease activity in patients with multiple sclerosis (MS) by blocking migration of T and B cells into the CNS. The goal of this study was to characterize the effects of natalizumab treatment on cytokine production and expression of activation markers, costimulatory molecules, and trafficking determinants on CD4+ and CD8+ T cells. Methods: In a longitudinal study, we investigated the expression of surface makers and cytokine expression on peripheral blood lymphocytes from 28 patients with MS who started natalizumab treatment and were followed for 1 year. A mixed effects model was used to compare pretreatment to on-treatment measurements. Results: The frequency of CD4+ T cells producing interferon-γ, tumor necrosis factor, and interleukin (IL)-17 upon anti-CD3 stimulation increased 6 months after initiation of natalizumab treatment and remained elevated throughout the follow-up. The frequency of CD4+ T cells expressing CD25, HLA-DR, and CCR6 ex vivo was increased at one or more time points during treatment. Among CD8+ T cells, the frequency of cells producing IL-2 and IL-17 after stimulation was increased during natalizumab treatment, as was the frequency of CD8+ T cells expressing CD58 and CCR5 ex vivo. The increase in the frequency of activated cells could not be replicated by in vitro exposure to natalizumab. Conclusion: Natalizumab treatment increases the percentage of activated leukocytes producing proinflammatory cytokines in blood, presumably due to sequestration of activated cells in the peripheral circulation.

CTLA4Ig treatment in patients with multiple sclerosis: an open-label, phase 1 clinical trial.

Background: The modulation of costimulatory pathways represents an original therapeutic approach to regulate T cell–mediated autoimmune diseases by preventing or reducing autoantigen-driven T-cell activation in humans. Autoreactive CD4+ T cells play a critical role in initiating the immune response leading to the chronic inflammation and demyelination characteristic of multiple sclerosis (MS). Methods: We used IV infusions of CTLA4Ig to block the CD28/B7 T-cell costimulatory pathway in a phase 1 dose-escalation study in MS. Sixteen patients with relapsing–remitting MS received a single CTLA4Ig infusion and were monitored for up to 3 months after treatment. In an extension study, four additional subjects received four doses of CTLA4Ig. Results: CTLA4Ig was well tolerated in patients with MS, and most adverse events were rated as mild. Immunologic assessment of the patients showed a reduction in myelin basic protein (MBP) proliferation within 2 months of infusion and decreased interferon-γ production by MBP-specific lines. Conclusions: Inhibiting costimulatory molecule interactions by using CTLA4Ig seems safe in multiple sclerosis (MS), and the immunologic effects suggest that it may be a promising approach to regulate the inflammatory process associated with MS. GLOSSARY: 1M = 1 month after infusion; 2M = 2 months after infusion; 3M = 3 months after infusion; 8D = 8 days after infusion; AE = adverse event; AI = Ambulation Index; APC = antigen-presenting cell; BL = baseline; BL2 = second baseline; BPF = brain parenchymal fraction; CTLA-4 = cytotoxic T lymphocyte–associated gene 4; EDSS = Expanded Disability Status Scale; FDA = Food and Drug Administration; Gd+ = gadolinium-enhanced; hMBP = human myelin basic protein; IDO = indolamine 2,3-dioxygenase; IFN = interferon; IL = interleukin; MBP = myelin basic protein; MS = multiple sclerosis; MSFC = Multiple Sclerosis Functional Composite; PBMC = peripheral blood mononuclear cell; RA = rheumatoid arthritis; TE = echo time; TR = repetition time; URI = upper respiratory infection; UTI = urinary tract infection; WBC = white blood cell.

Cytometric profiling in multiple sclerosis uncovers patient population structure and a reduction of CD8low cells

As part of a biomarker discovery effort in peripheral blood, we acquired an immunological profile of cell-surface markers from healthy control and untreated subjects with relapsing-remitting MS (RRMS). Fresh blood from each subject was screened ex vivo using a panel of 50 fluorescently labelled monoclonal antibodies distributed amongst 56 pools of four antibodies each. From these 56 pools, we derived an immunological profile consisting of 1018 features for each subject in our analysis using a systematic gating strategy. These profiles were interrogated in an analysis with a screening phase (23 patients) and an extension phase (15 patients) to identify cell populations in peripheral blood whose frequency is altered in untreated RRMS subjects. A population of CD8(low)CD4(-) cells was identified as being reduced in frequency in untreated RRMS subjects (P = 0.0002), and this observation was confirmed in an independent sample of subjects from the Comprehensive Longitudinal Investigation of MS at the Brigham & Womens Hospital (P = 0.002). This reduction in the frequency of CD8(low)CD4(-) cells is also observed in 38 untreated subjects with a clinically isolated demyelination syndrome (CIS) (P = 0.0006). We also show that these differences may be due to a reduction in the CD8(low)CD56(+)CD3(-)CD4(-) subset of CD8(low) cells, which have a natural killer cell profile. Similarities between untreated CIS and RRMS subjects extend to broader immunological profiles: consensus clustering of our data suggests that there are three distinct populations of untreated RRMS subjects and that these distinct phenotypic categories are already present in our sample of untreated CIS subjects. Thus, our large-scale immunophenotyping approach has yielded robust evidence for a reduction of CD8(low)CD4(-) cells in both CIS and RRMS in the absence of treatment as well as suggestive evidence for the existence of immunologically distinct subsets of subjects with a demyelinating disease.

Loss of IL-4 Secretion from Human Type 1a Diabetic Pancreatic Draining Lymph Node NKT Cells

Altered frequency and function of peripheral invariant NKT (iNKT) cells have been implicated in the regulation of murine and human type 1a diabetes. To examine regulatory cells from the site of drainage of autoinflammatory tissue and autoantigenic T cell priming in diabetes, we directly cloned iNKT cells from human pancreatic draining lymph nodes (PLN). From 451 T cell clones from control and diabetic PLN, we derived 55 iNKT cells by two methods and analyzed function by cytokine secretion. iNKT cell clones isolated from control PLN secreted IL-4 and IFN-γ upon TCR stimulation. For type 1a diabetic subjects, PLN iNKT cell clones from three samples secreted IFN-γ and no IL-4. In a rare recent onset diabetic sample with islet-infiltrating CD4+ T cells, the phenotype of PLN iNKT cell clones was mixed. From normal and diabetic PLN, one-third of CD1d tetramer+-sorted T cell clones were reactive with CD1d transfectants or proliferated/secreted cytokine in response to α-galactosylceramide-pulsed PBMCs; tetramer-staining T cell clones from diabetic PLN did not secrete IL-4. This is the first report directly examining iNKT cells from lymph nodes draining the site of autoimmunological attack in humans; iNKT cells were altered in cytokine secretion as previously reported for circulating iNKT cells in human type 1a diabetes.