Anja Windhagen

Modulation of cytokine patterns of human autoreactive T cell clones by a single amino acid substitution of their peptide ligand

We demonstrate that cognate peptide ligands altered at T cell receptor (TCR) contact residues and bound to class II major histocompatability complex can change the cytokine pattern of mature T cell clones. Myelin basic protein peptide 85-99-reactive Th0 T cell clones were stimulated with altered peptide ligands, which acted both as TCR antagonist and induced new mRNA synthesis and protein secretion of TGF-beta 1, while no longer inducing mRNA synthesis or protein secretion of IL-2, IL-4, IL-10, and IFN gamma. The modified peptides failed to induce a detectable calcium flux, p56lck activation, or thymidine incorporation, yet triggered nearly equal amounts of IL-4 secretion in the presence of ionomycin. Antigen-induced modulation of T cell cytokine secretion may be important in regulating the immune response.

Constitutive expression of costimulatory molecules by human microglia and its relevance to CNS autoimmunity

Human microglia constitute the primary residential antigen presenting cells (APCs) in the central nervous system (CNS) and have the capacity of activating myelin reactive T-cells. T-cell activation requires two signals: first is the interaction of the T-cell receptor with the MHC-antigen complex and, secondly, contact of the CD28/CTLA4 T-cell surface molecules with the B7 family of costimulatory molecules on the APCs. We have previously shown high expression of B7.1 in early multiple sclerosis (MS) plaques, suggesting that acute T-cell-mediated CNS inflammation may require local B7.1 upregulation. We have now examined the expression of B7.1 and B7.2 costimulatory molecules on resting ex-vivo human microglia isolated directly from biopsy specimens. We found constitutive expression of B7.2 but not B7.1 on resting microglia, suggesting that B7.2 expression may lead to downregulation of pro-inflammatory Th1 T-cell responses in the normal brain.

Cytokine secretion of myelin basic protein reactive T cells in patients with multiple sclerosis

The objective of this study was to determine whether autoreactive T cells in patients with multiple sclerosis (MS) are polarized and committed in their differentiation to a stable cytokine phenotype or whether the cytokine secretion can be altered. We examined the cytokines secreted by myelin basic protein (MBP) as compared to tetanus toxoid-reactive (TT) T cells in 12 patients with relapsing remitting MS (RR-MS), 9 patients with chronic progressive MS (CP-MS), and 14 normal individuals. A total of 5094 short term T cell lines to MBP and TT were generated in the presence of growth conditions promoting Th1 (IL-12/alpha-IL-4 mAb) or Th2 (IL-4/alpha-IL-12 mAb) cytokine secretion. Antigen-specific cytokine secretion from normals and MS patients could be shifted to a Th1 or Th2 type phenotype depending upon culture conditions, indicating that the phenotype of MBP reactive T cells can be altered even in longstanding chronic progressive MS. There were no significant differences in the cytokine patterns secreted by MBP reactive T cells in patients with MS as compared to normal individuals. However, CP-MS patients tended to have fewer MBP reactive T cells secreting IL-4 when cultured with IL-12/anti-IL-4 mAb and more IFN-gamma secreting MBP reactive T cells when cultured with IL-4/anti-IL-12 mAb as compared to both normal controls and RR-MS, suggesting that cells from these patients might be more polarized or that fewer undifferentiated MBP-reactive cells are present in these individuals. The most striking observation was that in contrast to the RR-MS patients and normal controls, almost none of the MBP reactive T cells secreting cytokines in CP-MS incorporated 3[H]thymidine. This may be due to chronic in vivo stimulation in the presence of IL-12, or because these T cells may have entered a terminally differentiated state. Nonetheless, the ability to alter the cytokine secretion of autoreactive T cell lines even in longstanding autoimmune disease indicates that cytokine therapy might have therapeutic benefits by switching the function of myelin reactive T cells such that they are non-pathogenic.

Glatiramer acetate (GA) induces IL-13/IL-5 secretion in naive T cells

In order to define possible mechanisms of immunomodulation by glatiramer acetate (GA), we investigated the primary in vitro cytokine response of peripheral blood mononuclear cells (PBMCs) and T-cell subpopulations. In PBMCs from healthy subjects and untreated patients with multiple sclerosis (MS) GA-induced T-cell proliferation and mRNA expression/cytokine, secretion of IL-13 and IL-5 but not of IL-10, TGF-beta or IL-12, IL-4 was detected at the mRNA level only. IFN-gamma was induced in a few subjects at very low concentrations. The response to GA was driven by the CD4(+)/CD45RA(+) T-cell subpopulation and was mediated by T-cell receptor (TCR) engagement as determined by anti-TCR blocking antibodies. The findings are compatible with the hypothesis that GA functions as partial or weak TCR-agonist activating naive T cells to produce the Th2 cytokines IL-13 and IL-5.

Effects of interferon-β on co-signaling molecules: upregulation of CD40, CD86 and PD-L2 on monocytes in relation to clinical response to interferon-β treatment in patients with multiple sclerosis

Interferon-beta (IFN-β) reduces disease activity in a subgroup of patients with relapsing remitting multiple sclerosis (MS). The mechanism of action as well as the pathophysiological basis of responsiveness to IFN-β is not well understood. Since T-cell activation plays an important part in the pathophysiology of MS, we here investigated the effect of IFN-β on the expression of co-signaling pathways (CD28—CD80/CD86, CD154—CD40, ICOS—ICOSL, PD-1—PD-L1/2) in MS patients and correlated the results with the clinical response to IFN-β in individual patients. Expression of co-signaling molecules was measured by flow cytometry in vitro on peripheral blood mononuclear cells after incubation with IFN-β, and in vivo in whole blood samples of 32 untreated and 24 IFN-β treated MS patients, including 13 patients longitudinal. IFN-β treatment induced upregulation of CD40, CD80, CD86, PD-L1 and PD-L2 on monocytes as well as PD-L1 on CD4+-T-cells in vitro and in vivo. IFN-β treated MS patients were grouped into responders and non-responders on the basis of Kurtzkés EDSS (expanded disability status scale) progression and relapse rate. Upregulation of CD40, CD86 and PD-L2 on monocytes was associated with treatment response to IFN-β (P < 0.001, P = 0.028 and P = 0.028, respectively). Our results show that IFN-β upregulates co-stimulatory as well as co-inhibitory molecules in vitro and in vivo implicating that modulation of the balance between positive and negative co-stimulatory signals might be an important part of the mechanism of action of IFN-β in MS. Upregulation of the expression of CD40, CD86 and PD-L2 may be useful as a predictive marker for clinical response to IFN-β treatment at early timepoints during IFN-β therapy. Multiple Sclerosis 2008; 14: 166—176. http://msj.sagepub.com

Interferon-β increases the stimulatory capacity of monocyte-derived dendritic cells to induce IL-13, IL-5 and IL-10 in autologous T-cells

Dendritic cells (DCs) are key regulators of immune responses and have been associated with autoimmunity in animal models and human disease. The effects of interferon beta (IFN-beta), an immunomodulatory cytokine used in multiple sclerosis (MS) therapy, on DCs are not well understood. Monocyte-derived DCs at different stages of maturation were stimulated with IFN-beta and DC-phenotype and stimulatory function were measured. IFN-beta inhibited the development of DCs at early stages but enhanced DC maturation. Moreover, IFN-beta enhanced the capacity of DCs to stimulate autologous T-cells to secrete IL-13, IL-10 and IL-5. Thus, IFN-beta has both immunostimulatory and immunosuppressive effects on DCs depending on the stage of maturation.

Human polymorphonuclear neutrophils express a B7-1-like molecule.

Polymorphonuclear neutrophils (PMN) are part of the innate immune system and are first‐line effector cells in acute inflammatory responses. On activation PMNs secrete cytokines and oxygen metabolites that might be involved in the regulation of the acquired immune response. We show here that peripheral blood PMNs constitutively express a B7‐1‐like molecule as detected by immunostaining with several B7‐1 antibodies. Reverse transcriptase‐polymerase chain reaction using three sets of primers spanning different regions of B7‐1 indicate dissimilarities at the mRNA level. B7‐1 mRNA is expressed in bone marrow cells and lipopolysaccharide (LPS)‐stimulated but not in unstimulated PMNs. The B7‐1‐like molecule is localized to the cytoplasmic granules and translocated to the cell surface after stimulation with LPS or interleukin‐12 in some donors. Binding of CTLA4‐Ig suggests that the B7‐1‐like molecule can interact with functional B7 ligand and might be important in the immunobiology of PMNs. J. Leukoc. Biol. 66: 945–952; 1999.

Spontaneous Intracranial Hypotension: Correlation of Imaging Findings with Clinical Features

Background: Spontaneous intracranial hypotension (SIH) is increasingly recognized as a clinically variable and likely underdiagnosed syndrome caused by non-traumatic CSF leaks. The aim of this study was to correlate the findings of imaging studies – magnetic resonance imaging (MRI), radionuclide cisternography – with clinical features and CSF pressure in SIH in order to improve the diagnostic yield and management in patients with SIH. Methods: Clinical case study of 10 consecutive cases of SIH, MRI, radio-isotope cisternography. Results: 5 out of 10 patientshad unusual clinical symptoms of SIH(2 subdural haematomas, 1 gait ataxia, 1 tinnitus, 1 haemodialysis-associated headache). In 7 patients pachymeningeal gadolinium enhancement was detected in MRI accompanied by a reduced CSF opening pressure. In contrast, the 3 patients with normal MRI also had a normal CSF pressure. Radio-isotope cisternography was abnormal in all patients tested. There was no correlation between the severity of clinical symptoms and MRI or radionuclide cisternography findings. Conclusions: The spectrum of clinical symptoms and imaging findings in SIH is highly variable. There- fore the diagnosis of SIH is often delayed. Radio-isotope cisternography is an important additional diagnostic method to detect CSF leaks or pathological kinetics of radio-isotope movement particularly in cases with normal MRI findings.

Costimulatory molecules B7-1 and B7-2 on CSF cells in multiple sclerosis and optic neuritis.

The aberrant expression of B7 costimulatory molecules is involved in the pathogenesis of autoimmune diseases and overexpression of B7-1 was found in inflammatory multiple sclerosis (MS) lesions. We here report that costimulatory molecules B7-1 and B7-2 are expressed on cerebrospinal fluid (CSF) monocytes and B-lymphocytes from patients with MS, optic neuritis (ON) and other inflammatory central nervous system (CNS) diseases. In patients with ON but not MS, increased expression of B7-2 was detected as compared to non-inflammatory controls. The expression of B7-1 in MS and ON patients correlates with disease duration but not with relapses in patients with MS indicating a role in early disease but not as a reliable marker of disease activity at later stages of MS.

Switch of autoreactive human T cell clones from a TH2 to a TGFβ1 secreting phenotype with low affinity T-cell receptor stimulating peptide/MHC complex

Tncra1~utic intervention in autoimmtme disease.s would ideally employ the andgouic ep~tope which is the target of the aumimmnne wocess to induce deletion, anoraye~ mpgmssim~ q~gif~mlty maUive ~ . Previous w~kf~z~n our lab has shown that mAbs to edhesiou molecule~, though no{ capable of inhibiting the inflammatiou of passive EAE, did in fact wovide a degree of protectiou against active EAE in rats. We ~ ask if the ~ could be used in coujunctiou with specific antigen (without adjuvant) to induce tolex~ce (re~tance) to EAE indectio~. Here we deacttbe the buinction of tolenmce by a sovan day infusion of MBP in-saline in ~mbinatiou with three ip injections of antt-CDlla (LFA-I) mAbs. This bd~ef 10realmant induces resistance to EAE induction which persisU; for at iemtema~zmh. AmI~CD~IC, A M I ~ ~ , ~ _ ~ when given in the same gestman nm did it synergizc witb anti-CD1 la when tbe two were ad~sinistea~ tosetber. H.httological lesions in MBP-CFA dmllenged ~ t tam slm~,~d adair se almence of le~doas or mild ~ , winch when ixeP~t was coufined to the manthg¢~ of the lower spinal cord. Lymplmode cell pmlife~aiou to MBP following challenge of tolerant rats was unaltered indicating that MBP reactive cells had not beco dekted. The tolerizing ~ e n t did not prime rats for antibody woductiou to MBP and total antibody level in tolerant rats following challenge was signiflc~lly lower than controls. There was aLso a shift in the isotype of mtib~ly IgOdlged ~ an 18G2a:IgGl ratio of 2:1 to 1:1 which w.as due m an increase in 18G1 productions. This result suggests a possible alteranon m CD4 + THI,TH2 profile in tolerant rats. Such deviation could account for the resistance to EAE h~inctiou.