Mirjam Korporal

Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis

Immunoregulatory T cells of CD4+CD25+ phenotype suppress T cell function and protect rodents from organ‐specific autoimmune disease. The human counterpart of this subset of T cells expresses high levels of CD25 and its role in human autoimmune disorders is currently under intense investigation. In multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), the activation of circulating self‐reactive T cells with specificity for myelin components is considered to be an important disease initiating event. Here, we investigated whether MS is associated with an altered ability of CD4+CD25high regulatory T cells (Treg) to confer suppression of myelin‐specific immune responses. Whereas Treg frequencies were equally distributed in blood and cerebrospinal fluid of MS patients and did not differ compared to healthy controls, the suppressive potency of patient‐derived CD4+CD25high T lymphocytes was impaired. Their inhibitory effect on antigen‐specific T cell proliferation induced by human recombinant myelin oligodendrocyte protein as well as on immune responses elicited by polyclonal and allogeneic stimuli was significantly reduced compared to healthy individuals. The effect was persistent and not due to responder cell resistance or altered survival of Treg, suggesting that a defective immunoregulation of peripheral T cells mediated by CD4+CD25high T lymphocytes promotes CNS autoimmunity in MS.

Prevalence of Newly Generated Naive Regulatory T Cells (Treg) Is Critical for Treg Suppressive Function and Determines Treg Dysfunction in Multiple Sclerosis

The suppressive function of regulatory T cells (Treg) is impaired in multiple sclerosis (MS) patients. The mechanism underlying the Treg functional defect is unknown. Treg mature in the thymus and the majority of cells circulating in the periphery rapidly adopt a memory phenotype. Because our own previous findings suggest that the thymic output of T cells is impaired in MS, we hypothesized that an altered Treg generation may contribute to the suppressive deficiency. We therefore determined the role of Treg that enter the circulation as recent thymic emigrants (RTE) and, unlike their CD45RO+ memory counterparts, express CD31 as typical surface marker. We show that the numbers of CD31+-coexpressing CD4+CD25+CD45RA+CD45RO−FOXP3+ Treg (RTE-Treg) within peripheral blood decline with age and are significantly reduced in MS patients. The reduced de novo generation of RTE-Treg is compensated by higher proportions of memory Treg, resulting in a stable cell count of the total Treg population. Depletion of CD31+ cells from Treg diminishes the suppressive capacity of donor but not patient Treg and neutralizes the difference in inhibitory potencies between the two groups. Overall, there was a clear correlation between Treg-mediated suppression and the prevalence of RTE-Treg, indicating that CD31-expressing naive Treg contribute to the functional properties of the entire Treg population. Furthermore, patient-derived Treg, but not healthy Treg, exhibit a contracted TCR Vβ repertoire. These observations suggest that a shift in the homeostatic composition of Treg subsets related to a reduced thymic-dependent de novo generation of RTE-Treg with a compensatory expansion of memory Treg may contribute to the Treg defect associated with MS.

Thymic export function and T cell homeostasis in patients with relapsing remitting multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory and possibly autoimmune mediated demyelinating disease of the CNS. Autoimmunity within the CNS may be triggered by dysfunction of peripheral immune tolerance mechanisms via changes in the homeostatic composition of peripheral T cells. We have assessed the release of naive T lymphocytes from the thymus in patients with relapsing remitting MS (RRMS) to identify alterations in the equilibrium of the peripheral T cell compartment. Thymic T cell production was estimated by measuring TCR excision circles (TRECs) as a traceable molecular marker in recent thymic emigrants. A total of 46 treatment-naive patients with active RRMS and 49 gender- and age-matched healthy persons were included in the study. The levels of TREC-expressing CD4+ and CD8+ T lymphocytes were significantly decreased in MS patients, and TREC quantities overall matched those of 30 years older healthy individuals. The average concentrations of TRECs/106 CD4+ and CD8+ T lymphocytes derived from MS patients and healthy donors were 26 × 103/106 and 28 × 103/106 vs 217 × 103/106 and 169 × 103/106, respectively. To account for any influence of T cell proliferation on TREC levels, we assayed T lymphocytes from additional patients with MS and normal individuals for telomere length (n = 20) and telomerase activity (8 MS patients, 16 controls), respectively. There were no significant differences between CD4+ and CD8+ T cells from MS patients and controls. Altogether, our findings suggest that an impaired thymic export function and, as a consequence, altered ability to maintain T cell homeostasis and immune tolerance may play an important pathogenic role in RRMS.

Glatiramer acetate improves regulatory T-cell function by expansion of naive CD4(+)CD25(+)FOXP3(+)CD31(+) T-cells in patients with multiple sclerosis.

Naturally occurring regulatory T-cells (Treg) exhibit impaired function in patients with relapsing-remitting multiple sclerosis (RRMS) resulting from an age-inappropriate disproportion between prevalences of newly generated CD31-coexpressing naive Treg and long-lived memory Treg in the periphery. Recent evidence suggests that the immunomodulatory action of glatiramer acetate (GA) includes effects on Treg function and frequencies. We prospectively assessed suppressive activities and frequencies of Treg and Treg subsets in 15 patients with RRMS undergoing long-term therapy with GA. Treatment for up to six months reconstituted naive Treg and increased total Treg numbers with concomitant reversion of the Treg defect.

Interferon Beta–Induced Restoration of Regulatory T-Cell Function in Multiple Sclerosis Is Prompted by an Increase in Newly Generated Naive Regulatory T Cells

BACKGROUND Naturally occurring regulatory T (T(reg)) cells are functionally impaired in patients with relapsing-remitting multiple sclerosis. We recently showed that prevalences of newly generated CD31-coexpressing naive T(reg) cells (recent thymic emigrant-T(reg) cells) are critical for suppressive function of circulating T(reg) cells, and a shift in the homeostatic composition of T(reg)-cell subsets related to a reduced de novo generation of recent thymic emigrant-T(reg) cells may contribute to the multiple sclerosis (MS)-related T(reg)-cell dysfunction. Interferon beta, an immunomodulatory agent with established efficacy in MS, lowers relapse rates and slows disease progression. Emerging evidence suggests that T(reg)-cell suppressive capacity may increase in patients with MS undergoing treatment with interferon beta, although the mechanisms mediating this effect are uncertain. OBJECTIVE To evaluate the effect of interferon beta treatment on the suppressive activity and the homeostasis of circulating T(reg) cells in patients with MS. PARTICIPANTS Twenty patients with relapsing-remitting MS and 18 healthy control subjects. INTERVENTIONS Administration of interferon beta. MAIN OUTCOME MEASURES Effect of interferon beta on T(reg)-cell homeostasis and suppressive capacity. RESULTS Suppressive capacities of T(reg) cells were consistently upregulated at 3 and 6 months after treatment with interferon beta. The restoration of T(reg)-cell function was paralleled by increased naive recent thymic emigrant-T(reg) cells and a coincidental reduction in memory T(reg) cells. CONCLUSION The increase in T(reg)-cell inhibitory capacity mediated by interferon beta treatment can be explained by its effect on the homeostatic balance within the T(reg) cell compartment.

B cells undergo unique compartmentalized redistribution in multiple sclerosis

Increasing evidence fosters the role of B cells (BC) in multiple sclerosis (MS). The compartmentalized distribution of BC in blood and cerebrospinal fluid (CSF) is incompletely understood. In this study, we analyzed BC-patterns and BC-immunoreactivity at these sites during active and during stable disease and the impact of disease modifying drugs (DMD) on peripheral BC-homeostasis. For this purpose we assessed BC-subsets in blood and CSF from patients with clinically isolated syndrome (CIS), relapsing remitting MS (RRMS), rheumatoid arthritis (RA), and healthy controls (HC) by flow cytometric detection of whole (W-BC), naïve, transitional (TN-BC), class-switched memory (CSM-BC), unswitched memory (USM-BC), double-negative memory (DNM-BC) BC-phenotypes, plasma blasts (PB), and plasma cells (PC). FACS-data were correlated with BC-specific chemotactic activities in CSF, intrathecal CXCL13-levels, and immunoreactivity of peripheral W-BC. Our study revealed that frequencies of systemic CSM-BC/USM-BC became contracted in active CIS/MS while proportions of naive BC, TN-BC and DNM-BC were reciprocally expanded. Moreover, the shifted BC-composition promoted reduced immunoreactivity of W-BC and resolved during remission. Cross-over changes in CSF included privileged accumulation of CSM-BC linked to intrathecal CXCL13-concentrations and expansion of PB/PC. Treatment with interferon-beta and natalizumab evoked distinct though differing redistribution of circulating BC-subsets. We conclude that symptomatic CIS and MS are accompanied by distinctive changes in peripheral and CSF BC-homeostasis. The privileged reciprocal distribution between naïve versus CSM-phenotypes in both compartments together with the marked chemotactic driving force towards BC prompted by CSF supernatants renders it likely that CSF BC are mainly recruited from peripheral blood during active CIS/MS, whereas constantly low percentages of circulating PB/PC and their failure to respond to migratory stimuli favors intrathecal generation of antibody secreting cells. Notably, BC-redistribution closely resembles alterations detectable in systemic autoimmunity associated with active RA and impacts BC-function Together with unique effects of DMDs on BC-homeostasis these findings underline the important role of BC in MS.

The interleukin-7 receptor α chain contributes to altered homeostasis of regulatory T cells in multiple sclerosis.

Treg homeostasis is disturbed in multiple sclerosis (MS). Frequencies of recent thymic emigrant (RTE)‐Treg are reduced and the disparity between RTE‐Treg and long‐lived memory Treg coincides with the MS‐associated Treg defect, as shown previously. Recent studies demonstrate that IL‐7 and thymic stromal lymphopoietin (TSLP) are critical for Treg maturation. Therefore, altered signaling through their receptors (IL‐7R, TSLP receptor (TSLPR)), sharing the IL‐7Rα‐chain (IL‐7Rα), might contribute to impaired Treg development. Using blood samples from 56 patients with MS and 33 healthy controls, we assessed IL‐7Rα‐expression on conventional T cells; frequencies, phenotypes and suppressive activities of Treg, plasma levels of IL‐7 and soluble IL‐7Rα; and screened for MS‐associated IL‐7RA gene polymorphism rs6897932. Moreover, we determined Treg expressing two different TCR Vα‐chains designating thymus‐originated cells. As TSLP/TSLPR signaling in thymic myeloid dendritic cells (MDCs) promotes Treg differentiation, we measured TSLPR expression on peripheral MDCs to indirectly test whether altered TSLPR expression might add to compromised Treg neogenesis. We found reduced IL‐7Rα expression on conventional T cells and upregulated IL‐7 plasma levels together with reduction of RTE‐Treg frequencies and Treg function in MS, without clear genetic influence. Decreased IL‐7Rα expression in MS correlated with declined dual‐receptor‐Treg and reduced MDC TSLPR expression, indicating contracted thymic Treg output. We suggest that altered IL‐7R/TSLPR signaling contributes to impaired Treg neogenesis in MS, which is compensated by expanded memory‐Treg and finally results in dysfunctional Treg.

Exposure of NK cells to intravenous immunoglobulin induces IFNγ release and degranulation but inhibits their cytotoxic activity

The mechanisms underlying the modulation of Natural Killer (NK) cell functions by intravenous immunoglobulin (IVIg) are poorly understood. Using an ex vivo whole blood assay system we demonstrate that IVIg suppresses NK cell cytotoxicity. This was paralleled by IVIg-induced degranulation of CD56(bright), CD16(positive) NK cells, reduced expression of CD16 and elevated IFN gamma release. To assess whether these findings also occur in vivo we analyzed whole blood before and after IVIg therapy of patients. Following IVIg treatment the number of NK cells in peripheral blood dropped significantly. We observed reduced CD16 expression, elevated IFN gamma-amounts in plasma, reduced NK cell cytotoxicity, and granzyme B release into the plasma, confirming our in vitro data. These effects on the functions of NK cells describe a novel immunomodulatory effect of IVIg. The in vitro assays employed here could represent informative test systems to monitor effects of in vivo IVIg treatment at an individual level.

Similar sensitivity of regulatory T cells towards CD95L-mediated apoptosis in patients with multiple sclerosis and healthy individuals

Impaired suppressive function of CD4(+)CD25(high) regulatory T cells (T(reg)) has been reported as a novel pathogenetic mechanism in Multiple sclerosis (MS). We addressed if high apoptosis sensitivity of MS-T(reg) could explain this functional T(reg) defect. T(reg) from treatment-naïve MS patients showed high sensitivity towards CD95Ligand-mediated apoptosis and exhibited enhanced cell death to IL-2 and TCR-signal deprivation. Since susceptibility of T(reg) to cell death was similar in MS patients and healthy controls, this cannot explain the inhibitory dysfunction of T(reg) associated with MS. Furthermore, as cell death is not enhanced, therapeutic expansion of MS-T(reg)in vitro should be a reasonable and novel therapeutic option.

Mononeuropathy multiplex as a result of treatment with interferon-α and ribavirin in a patient with hepatitis C

Hepatitis C may be complicated by systemic mixed cryoglobulinemic (MC) vasculitis, and in some cases by a polyarteritis nodosa (PAN)-type non-cryoglobulinemic vasculitis [1]. Treatment with interferon-a (IFN-a) and ribavirin mostly is associated with an improvement of vasculitic symptoms. In some cases, exacerbation and rarely new onset of vasculitis of the peripheral nervous system have been described [2–5]. So far, only one patient with hepatitis C, who developed MC-vasculitic bilateral and predominantly sensory axonal neuropathy under treatment with the polyethylene glycol (PEG)-conjugated form of IFN-a (PEG-IFN-a) and ribavirin [2] has been published. On the other hand, a recent study revealed no signs of peripheral neurotoxicity in PEGIFN-a treated hepatitis C patients [6]. Here, we report a 43-year-old male patient, who was diagnosed with hepatitis C type 1 and treated with ribavirin and PEG-IFN-a 5 years later because of an excessive viral load (180 lg s.c., every week). At this time, the patient had no neurological symptoms and liver enzymes were normal. There was also no evidence of a systemic autoimmune disease. Two weeks after initiation of therapy, he experienced paraesthesias in the right hand and both legs rapidly followed by weakness of the right abductor pollicis brevis muscle. Nerve conduction velocity of the median nerve was reduced on the right side and electromyography showed a neurogenic pattern. Hepatitis C viral load was not tested at this time. After 4 weeks of PEGIFN-a and ribavirin treatment hepatitis C virus was undetectable. Two weeks later, the patient suffered from intensive pain of the right arm and sudden weakness of both arms and both legs. On neurological examination, he had severe motor and sensory dysfunction affecting the territories of the right median, left ulnar, left peroneal and right tibial nerves. Laboratory tests revealed monoclonal gammopathy of IgM kappa type with Bence-Jones protein in urine and an increased rheumatoid factor (RF). Cerebrospinal fluid was normal. Mononeuropathy multiplex (MMp) was diagnosed and PEG-IFN-a and ribavirin treatment was stopped immediately. The patient was treated with intravenous immunoglobulin (IVIG) (initially 60 g, followed by 40 g i.v. every 6 weeks) and pregabaline (600 mg daily) against the neuropathic pain. Six months after initiation of PEGIFN-a and ribavirin treatment, the patient was clinically stabilized but still had weakness of the right arm, both hands and a hypoesthesia in the territories of the left ulnar nerve, right median nerve and both legs. Motor and sensory nerve conduction