Comprehensive antibody and cytokine profiling in hospitalized COVID-19 patients in relation to clinical outcomes in a large Belgian cohort.

The immune response in patients with Coronavirus Disease 2019 (COVID-19) is highly variable and is linked to disease severity and mortality. However, antibody and cytokine responses in the early disease stage and their association with disease course and outcome are still not completely understood. In this large, multi-centre cohort study, blood samples of 434 Belgian COVID-19 hospitalized patients with different disease severities (ranging from asymptomatic/mild to critically ill) from the first wave of the COVID-19 pandemic were obtained. Baseline antibody and cytokine responses were characterized and associations with several clinical outcome parameters were determined. Anti-spike immunoglobulin (Ig)G and IgM levels were elevated in patients with a more severe disease course. This increased baseline antibody response however was associated with decreased odds for hospital mortality. Levels of the pro-inflammatory cytokines IL-6, IP-10 and IL-8, the anti-inflammatory cytokine IL-10 and the antiviral cytokines IFN-α, IFN-ß and IFN-λ1 were increased with disease severity. Remarkably, we found significantly lower levels of IFN-λ2,3 in critically ill patients compared to patients of the moderate and severe disease category. Finally, levels of IL-8, IL-6, IP-10, IL-10, IFN-α, IFN-ß, IFN-γ and IFN-λ1 at baseline were positively associated with mortality, whereas higher IFN-λ2,3 levels were negatively associated with mortality.

IgD-CD27- double negative (DN) B cells: Origins and functions in health and disease.

Human B cells can be divided into four main subsets based on differential expression of immunoglobulin (Ig)D and CD27. IgD-CD27- double negative (DN) B cells make up a heterogeneous group of B cells that have first been described in relation to aging and systemic lupus erythematosus but have been mostly disregarded in B cell research. Over the last few years, DN B cells have gained a lot of interest because of their involvement in autoimmune and infectious diseases. DN B cells can be divided into different subsets that originate via different developmental processes and have different functional properties. Further research into the origin and function of different DN subsets is needed to better understand the role of these B cells in normal immune responses and how they could be targeted in specific pathologies. In this review, we give an overview of both phenotypic and functional properties of DN B cells and provide insight into the currently proposed origins of DN B cells. Moreover, their involvement in normal aging and different pathologies is discussed.

Oligodendroglia-derived extracellular vesicles activate autophagy via LC3B/BAG3 to protect against oxidative stress with an enhanced effect for HSPB8 enriched vesicles.

BACKGROUND: The contribution of native or modified oligodendroglia-derived extracellular vesicles (OL-EVs) in controlling chronic inflammation is poorly understood. In activated microglia, OL-EVs contribute to the removal of cytotoxic proteins following a proteotoxic stress. Intracellular small heat shock protein B8 (HSPB8) sustain this function by facilitating autophagy and protecting cells against oxidative stress mediated cell death. Therefore, secretion of HSPB8 in OL-EVs could be beneficial for neurons during chronic inflammation. However, how secreted HSPB8 contribute to cellular proteostasis remains to be elucidated. METHODS: We produced oligodendroglia-derived EVs, either native (OL-EVs) or HSPB8 modified (OL-HSPB8-EVs), to investigate their effects in controlling chronic inflammation and cellular homeostasis. We analyzed the impact of both EV subsets on either a resting or activated microglial cell line and on primary mixed neural cell culture cells. Cells were activated by stimulating with either tumor necrosis factor-alpha and interleukin 1-beta or with phorbol-12-myristate-13-acetate. RESULTS: We show that OL-EVs and modified OL-HSPB8-EVs are internalized by C20 microglia and by primary mixed neural cells. The cellular uptake of OL-HSPB8-EVs increases the endogenous HSPB8 mRNA expression. Consistently, our results revealed that both EV subsets maintained cellular homeostasis during chronic inflammation with an increase in the formation of autophagic vesicles. Both EV subsets conveyed LC3B-II and BAG3 autophagy markers with an enhanced effect observed for OL-HSPB8-EVs. Moreover, stimulation with either native or modified OL-HSPB8-EVs showed a significant reduction in ubiquitinated protein, reactive oxygen species and mitochondrial depolarization, with OL-HSPB8-EVs exhibiting a more protective effect. Both EV subsets did not induce cell death in the C20 microglia cell line or the primary mixed neural cultures. CONCLUSION: We demonstrate that the functions of oligodendroglia secreted EVs enriched with HSPB8 have a supportive role, comparable to the native OL-EVs. Further development of engineered oligodendroglia derived EVs could be a novel therapeutic strategy in countering chronic inflammation. Video Abstract.

Microglial derived extracellular vesicles activate autophagy and mediate multi-target signaling to maintain cellular homeostasis.

Microglia, the immunocompetent cells of the central nervous system (CNS), play an important role in maintaining cellular homeostasis in the CNS. These cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. Accumulating evidence indicates that specifically the cellular exchange of small extracellular vesicles (EVs), participates in physiology and disease through intercellular communication. However, the contribution of microglial-derived extracellular vesicles (M-EVs) to the maintenance of microglia homeostasis and how M-EVs could influence the phenotype and gene function of other microglia subtypes is unclear. In addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to M-EVs is limited. Here, we analysed the effects of M-EVs produced in vitro by either tumour necrosis factor alpha (TNFα) activated or non-activated microglia BV2 cells. We showed that M-EVs are internalized by both mouse and human C20 microglia cells and that the uptake of M-EVs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. Consistently, stimulation of microglia with M-EVs increased the protein expression of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B isoform II (LC3B-II), and promoted autophagic flux in live cells. To elucidate the biological activities occurring at the transcriptional level in C20 microglia stimulated with M-EVs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted RNA sequencing. Inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia stimulated with M-EVs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. In this study, we provide the perspective that a beneficial activity of in vitro cell culture produced EVs could be the modulation of autophagy during cellular stress. Therefore, we use a monoculture system to study microglia-microglia crosstalk which is important in the prevention and propagation of inflammation in the brain. We demonstrate that in vitro produced microglial EVs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homeostasis.

Phenotypic and Ig Repertoire Analyses Indicate a Common Origin of IgD-CD27- Double Negative B Cells in Healthy Individuals and Multiple Sclerosis Patients.

IgD-CD27- double negative (DN) B cells with proinflammatory characteristics are abnormally elevated in a proportion of multiple sclerosis (MS) patients. In this study, the origin and selection characteristics of DN B cells were studied in MS patients and healthy controls (HC). Expression of developmental markers on peripheral blood DN, IgD-CD27+ class-switched memory (CSM) and IgD+CD27- naive B cells of HC (n = 48) and MS patients (n = 96) was determined by flow cytometry. High-throughput adaptive immune receptor repertoire sequencing was performed on peripheral blood DN and CSM B cells of HC and MS patients (n = 3 each). DN B cells from HC and MS patients showed similar phenotypic and Ig repertoire characteristics. Phenotypic analysis indicated a mature state of DN B cells by low CD5, CD10, and CD38 expression. However, the frequency of CD95+ and IgA+ cells was lower in DN versus CSM B cells. DN B cells are Ag experienced, as shown by somatic hypermutation of their Ig genes in adaptive immune receptor repertoire sequencing, although they showed a lower mutation load than CSM B cells. Shared clones were found between DN and CSM B cells, although >95% of the clones were unique to each population, and differences in V(D)J usage and CDR3 physicochemical properties were found. Thus, DN B cells arise in HC and MS patients via a common developmental pathway that is probably linked to immune aging. However, DN and CSM B cells develop through unique differentiation pathways, with most DN B cells representing an earlier maturation state.

Letter to the Editor concerning the article "Application of red light phototherapy in the treatment of radioactive dermatitis in patients with head and neck cancer".

The aim of this Letter to the Editor was to report some methodological shortcomings in the recently published article "Application of red light phototherapy in the treatment of radioactive dermatitis in patients with head and neck cancer" by Zhang et al. There are some issues regarding the incomplete photobiomodulation (PBM) parameters, the chosen outcome measures, and some missing reference articles. In conclusion, the results of this study should be interpreted with caution and further research is necessary.

Biophysical skin measurements to evaluate the effectiveness of photobiomodulation therapy in the prevention of acute radiation dermatitis in breast cancer patients.

PURPOSE: The purpose of this study was to evaluate objectively the effectiveness of photobiomodulation therapy (PBMT) for the prevention of acute radiation dermatitis (ARD) by using biophysical skin measurements. METHODS: A randomized, placebo-controlled trial with 120 breast cancer patients who underwent an identical radiotherapy (RT) regimen post-lumpectomy was performed (TRANSDERMIS trial). Patients were randomized to receive PBM (808 nm CW/905 nm pulsed, 168 mW/cm2, spot size 19.6 cm2, fluence 4 J/cm2) or placebo treatments from the first day of RT (2×/week). Biophysical skin measurements were collected to assess the skin pigmentation and barrier function. Measurements were collected at the first day of RT, a RT dose of 40 Gray (Gy), and the end of RT (66 Gy). RESULTS: The incidence of moist desquamation was significantly higher in the control than in the PBMT group at the end of RT (30 vs. 7%, respectively, odds ratio = 6, p = 0.004). The biophysical skin measures showed that the mean percentage change from the baseline transepidermal water loss (TEWL), erythema, and melanin values was significantly higher in the control than in the PBMT group at the end of RT (ps < 0.05). Logistic regression analysis revealed that the risk on moist desquamation was significantly increased for patients with a large (> 800 cc) breast volume (odds ratio = 4, p = 0.017). CONCLUSIONS: This is the first randomized controlled trial demonstrating by objective measurements that PBMT is effective in reducing the incidence of moist desquamation in breast cancer patients undergoing RT. Additionally, a large breast volume is an important risk factor for the development of moist desquamation.

Prevention of acute radiodermatitis by photobiomodulation: A randomized, placebo-controlled trial in breast cancer patients (TRANSDERMIS trial).

OBJECTIVE: Acute radiodermatitis (RD) is a distressing and painful skin reaction that occurs in 95% of the patients undergoing radiotherapy (RT). The aim of this study was to evaluate the effectiveness of photobiomodulation therapy (PBMT) in the prevention of acute RD in breast cancer (BC) patients undergoing RT. METHODS: This study was a randomized, placebo-controlled trial including 120 BC patients that underwent an identical RT regimen post-lumpectomy. Patients were randomly assigned to the laser therapy (LT) or placebo group, with 60 patients in each group. Laser or placebo treatments were applied 2 days a week, immediately after the RT session, starting at the first day of RT. PBMT was delivered using a class IV MLS® M6 laser that combines two synchronized laser diodes in the infrared range (808-905 nm) with a fixed energy density (4 J/cm2 ). Skin reactions were scored based on the criteria of the Radiation Therapy Oncology Group (RTOG) and the Radiation-Induced Skin Reaction Assessment Scale (RISRAS). The patients completed the Skindex-16 questionnaire to evaluate their quality of life. All the measurements were collected at the first day, at a RT dose of 40 Gray (Gy), and at the end of RT (total dose 66 Gy). RESULTS: At a RT dose of 40 Gy, there was no significant difference between the groups in the distribution of RTOG grades. However, at the end of RT the severity of the skin reactions significantly differed between the two groups (P = 0.004), with a larger percentage of patients experiencing RTOG grade 2 or higher (e.g., moist desquamation) in the placebo group (30% vs. 6.7%, for the placebo and laser group, resp.). The objective RISRAS score confirmed these results. In addition, the Skindex-16 and RISRAS subjective score demonstrated that the patients' quality of life was significantly better in the LT than in the control group. CONCLUSIONS: The results of this trial show that PBMT is an effective tool to prevent the development of grade 2 acute RD or higher in BC patients. In addition, it also reduces the patients' symptoms related to RD. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.

Age-Associated B Cells with Proinflammatory Characteristics Are Expanded in a Proportion of Multiple Sclerosis Patients.

Immune aging occurs in the elderly and in autoimmune diseases. Recently, IgD-CD27- (double negative, DN) and CD21-CD11c+ (CD21low) B cells were described as age-associated B cells with proinflammatory characteristics. This study investigated the prevalence and functional characteristics of DN and CD21low B cells in multiple sclerosis (MS) patients. Using flow cytometry, we demonstrated a higher proportion of MS patients younger than 60 y with peripheral expansions of DN (8/41) and CD21low (9/41) B cells compared with age-matched healthy donors (1/33 and 2/33, respectively), which indicates an increase in age-associated B cells in MS patients. The majority of DN B cells had an IgG+ memory phenotype, whereas CD21low B cells consisted of a mixed population of CD27- naive, CD27+ memory, IgG+, and IgM+ cells. DN B cells showed similar (MS patients) or increased (healthy donors) MHC-II expression as class-switched memory B cells and intermediate costimulatory molecule expression between naive and class-switched memory B cells, indicating their potential to induce (proinflammatory) T cell responses. Further, DN B cells produced proinflammatory and cytotoxic cytokines following ex vivo stimulation. Increased frequencies of DN and CD21low B cells were found in the cerebrospinal fluid of MS patients compared with paired peripheral blood. In conclusion, a proportion of MS patients showed increased peripheral expansions of age-associated B cells. DN and CD21low B cell frequencies were further increased in MS cerebrospinal fluid. These cells could contribute to inflammation by induction of T cell responses and the production of proinflammatory cytokines.

The isotype repertoire of antibodies against novel UH-RA peptides in rheumatoid arthritis.

BACKGROUND: Recently, autoantibodies against novel UH-RA peptides (UH-RA.1 and UH-RA.21) were identified as candidate biomarkers for patients with rheumatoid arthritis (RA) who are seronegative for the current diagnostic markers rheumatoid factor and anticitrullinated protein antibodies. Previously, screening for anti-UH-RA autoantibodies was based on measuring the immunoglobulin (Ig) G response. We aimed to investigate whether measurement of other isotypes could improve the performance of diagnostic testing. In addition, assigning the isotype profile might provide valuable information on effector functions of the antibodies. METHODS: The isotype profile of antibodies against UH-RA.1 and UH-RA.21 was studied. The IgG, IgM, and IgA classes, together with the 4 different IgG subclasses, were determined in 285 patients with RA, 88 rheumatic control subjects, and 90 healthy control subjects. RESULTS: Anti-UH-RA.1 antibodies were primarily of the IgM isotype and twice as prevalent as IgG (IgG3-dominated) and IgA. RA sensitivity when testing for anti-UH-RA.1 IgM was shown to be higher than when testing for the IgG isotype: 18 % versus 9 % sensitivity when RA specificity was set to 90 %. Within antibodies against UH-RA.21, IgG and IgA were more common than IgM. Different anti-UH-RA.21 IgG subclasses were found, with the highest prevalence found for IgG2. Combined testing for IgG and IgA slightly increased RA sensitivity of UH-RA.21-specific antibody testing to 27 % compared with solely testing for IgG (23 %). Notably, a higher number of anti-UH-RA.21 antibody isotypes was related to increased levels of erythrocyte sedimentation rate. Finally, for both antibody responses, the full antibody isotype use was demonstrated in early and seronegative disease. CONCLUSIONS: The isotype distribution of anti-UH-RA.1 and anti-UH-RA.21 antibodies was successfully outlined, and, for antibodies against UH-RA.1, we found that isotype-specific testing might have implications for diagnostic testing. The exact mechanisms by which the different antibody isotypes act still have to be unraveled.

Autoantibodies to two novel peptides in seronegative and early rheumatoid arthritis.

OBJECTIVES: Despite recent progress in biomarker discovery for RA diagnostics, still over one-third of RA patients-and even more in early disease-present without RF or ACPA. The aim of this study was to confirm the presence of previously identified autoantibodies to novel Hasselt University (UH) peptides in early and seronegative RA. METHODS: Screening for antibodies against novel UH peptides UH-RA.1, UH-RA.9, UH-RA.14 and UH-RA.21, was performed in two large independent cohorts. Peptide ELISAs were developed to screen for the presence of antibodies to UH-RA peptides. First, 292 RA patients (including 39 early patients), 90 rheumatic and 97 healthy controls from UH were studied. Antibody reactivity to two peptides (UH-RA.1 and UH-RA.21) was also evaluated in 600 RA patients, 309 patients with undifferentiated arthritis and 157 rheumatic controls from the Leiden Early Arthritis Clinic cohort. RESULTS: In both cohorts, 38% of RA patients were seronegative for RF and ACPA. Testing for autoantibodies to UH-RA.1 and UH-RA.21 reduced the serological gap from 38% to 29% in the UH cohort (P = 0.03) and from 38% to 32% in the Leiden Early Arthritis Clinic cohort (P = 0.01). Furthermore, 19-33% of early RA patients carried antibodies to these peptides. Specificities in rheumatic controls ranged from 82 to 96%. Whereas antibodies against UH-RA.1 were related to remission, anti-UH-RA.21 antibodies were associated with inflammation, joint erosion and higher tender and swollen joint counts. CONCLUSION: This study validates the presence of antibody reactivity to novel UH-RA peptides in seronegative and early RA. This might reinforce current diagnostics and improve early diagnosis and intervention in RA.

The prevalence of vertebral fractures in spondyloarthritis: relation to disease characteristics, bone mineral density, syndesmophytes and history of back pain and trauma.

INTRODUCTION: An increased risk of vertebral fracture (VF) is one of the extra-articular manifestations of spondyloarthropathy (SpA). The prevalence of moderate to severe VFs visualized by radiography (Rx) in patients with SpA in daily practice is unknown until imaging of the full spine is available, as most VFs do not present with clinical signs and symptoms of an acute fracture. METHODS: We evaluated the prevalence of VFs (>25% loss in height) on available Rx and dual-energy X-ray absorptiometry (DXA) images in 390 consecutive patients with SpA in daily practice. We assessed their association with disease characteristics, bone mineral density, the modified Stoke Ankylosing Spondylitis Spinal Score, and history of trauma. RESULTS: Forty-six patients (11.8%) had Rx VF (56.4% men, 93.5% in the thoracic spine), and 44.5% had multiple VFs. Compared with patients without VF, patients with VF were older (52.2 vs. 47.3 years, p < 0.01; range 25-84 years), had lower femoral neck T-scores (-1.1 vs. -0.7; p < 0.05), and had a marginally higher modified Stoke Ankylosing Spondylitis Spinal Score (11.7 vs. 7.0; p = 0.06). Among patients with VFs, 15.2% had a history of trauma with acute back pain (p < 0.001 vs. no VF). The reliability of DXA for diagnosing radiographic VFs was high (κ 0.90). CONCLUSIONS: Moderate to severe VFs are found in more than 10% of patients with SpA before the age of 40 years in 5% of women and 9% in men. Most VFs are located in the thoracic region, are related to low femoral neck bone mineral density and to stiffening of the spine, and are only rarely related to trauma history. DXA is a useful alternative for diagnosing VFs.

Real-time analysis of dual-display phage immobilization and autoantibody screening using quartz crystal microbalance with dissipation monitoring.

Over the last three decades, phage display technology has been used for the display of target-specific biomarkers, peptides, antibodies, etc. Phage display-based assays are mostly limited to the phage ELISA, which is notorious for its high background signal and laborious methodology. These problems have been recently overcome by designing a dual-display phage with two different end functionalities, namely, streptavidin (STV)-binding protein at one end and a rheumatoid arthritis-specific autoantigenic target at the other end. Using this dual-display phage, a much higher sensitivity in screening specificities of autoantibodies in complex serum sample has been detected compared to single-display phage system on phage ELISA. Herein, we aimed to develop a novel, rapid, and sensitive dual-display phage to detect autoantibodies presence in serum samples using quartz crystal microbalance with dissipation monitoring as a sensing platform. The vertical functionalization of the phage over the STV-modified surfaces resulted in clear frequency and dissipation shifts revealing a well-defined viscoelastic signature. Screening for autoantibodies using antihuman IgG-modified surfaces and the dual-display phage with STV magnetic bead complexes allowed to isolate the target entities from complex mixtures and to achieve a large response as compared to negative control samples. This novel dual-display strategy can be a potential alternative to the time consuming phage ELISA protocols for the qualitative analysis of serum autoantibodies and can be taken as a departure point to ultimately achieve a point of care diagnostic system.

Circulating Follicular Regulatory T Cells Are Defective in Multiple Sclerosis.

Follicular regulatory T cells (TFR) have been extensively characterized in mice and participate in germinal center responses by regulating the maturation of B cells and production of (auto)antibodies. We report that circulating TFR are phenotypically distinct from tonsil-derived TFR in humans. They have a lower expression of follicular markers, and display a memory phenotype and lack of high expression of B cell lymphoma 6 and ICOS. However, the suppressive function, expression of regulatory markers, and FOXP3 methylation status of blood TFR is comparable with tonsil-derived TFR. Moreover, we show that circulating TFR frequencies increase after influenza vaccination and correlate with anti-flu Ab responses, indicating a fully functional population. Multiple sclerosis (MS) was used as a model for autoimmune disease to investigate alterations in circulating TFR. MS patients had a significantly lower frequency of circulating TFR compared with healthy control subjects. Furthermore, the circulating TFR compartment of MS patients displayed an increased proportion of Th17-like TFR. Finally, TFR of MS patients had a strongly reduced suppressive function compared with healthy control subjects. We conclude that circulating TFR are a circulating memory population derived from lymphoid resident TFR, making them a valid alternative to investigate alterations in germinal center responses in the context of autoimmune diseases, and TFR impairment is prominent in MS.

Heat-transfer-method-based cell culture quality assay through cell detection by surface imprinted polymers.

Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time.

IL-15 amplifies the pathogenic properties of CD4+CD28- T cells in multiple sclerosis.

CD4(+)CD28(-) T cells arise through repeated antigenic stimulation and are present in diseased tissues of patients with various autoimmune disorders, including multiple sclerosis (MS). These cells are believed to have cytotoxic properties that contribute to the pathogenic damaging of the target organ. Endogenous cues that are increased in the diseased tissue may amplify the activity of CD4(+)CD28(-) T cells. In this study, we focused on IL-15, a cytotoxicity-promoting cytokine that is increased in the serum and cerebrospinal fluid of MS patients. Using immunohistochemistry, we demonstrate that IL-15 is mainly produced by astrocytes and infiltrating macrophages in inflammatory lesions of MS patients. Moreover, in vitro transmigration studies reveal that IL-15 selectively attracts CD4(+)CD28(-) T cells of MS patients, but not of healthy individuals. IL-15 further induces the expression of chemokine receptors and adhesion molecules on CD4(+)CD28(-) T cells, as investigated using flow cytometry, resulting in enhanced migration over a monolayer of human brain endothelial cells. Finally, flow cytometric analyses revealed that IL-15 increases the proliferation and production of GM-CSF, expression of cytotoxic molecules (NKG2D, perforin, and granzyme B), and degranulation capacity of CD4(+)CD28(-) T cells. Taken together, these findings indicate that increased peripheral and local levels of IL-15 amplify the pathogenic potential of CD4(+)CD28(-) T cells, thus contributing to tissue damage in MS brain lesions.

Leukemia inhibitory factor tips the immune balance towards regulatory T cells in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), for which current treatments are unable to prevent disease progression. Based on its neuroprotective and neuroregenerating properties, leukemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) cytokine family, is proposed as a novel candidate for MS therapy. However, its effect on the autoimmune response remains unclear. In this study, we determined how LIF modulates T cell responses that play a crucial role in the pathogenesis of MS. We demonstrate that expression of the LIF receptor was strongly increased on immune cells of MS patients. LIF treatment potently boosted the number of regulatory T cells (Tregs) in CD4(+) T cells isolated from healthy controls and MS patients with low serum levels of IL-6. Moreover, IL-6 signaling was reduced in the donors that responded to LIF treatment in vitro. Our data together with previous findings revealing that IL-6 inhibits Treg development, suggest an opposing function of LIF and IL-6. In a preclinical animal model of MS we shifted the LIF/IL-6 balance in favor of LIF by CNS-targeted overexpression. This increased the number of Tregs in the CNS during active autoimmune responses and reduced disease symptoms. In conclusion, our data show that LIF downregulates the autoimmune response by enhancing Treg numbers, providing further impetus for the use of LIF as a novel treatment for MS and other autoimmune diseases.

Compositional changes of B and T cell subtypes during fingolimod treatment in multiple sclerosis patients: a 12-month follow-up study.

BACKGROUND AND OBJECTIVE: The long term effects of fingolimod, an oral treatment for relapsing-remitting (RR) multiple sclerosis (MS), on blood circulating B and T cell subtypes in MS patients are not completely understood. This study describes for the first time the longitudinal effects of fingolimod treatment on B and T cell subtypes. Furthermore, expression of surface molecules involved in antigen presentation and costimulation during fingolimod treatment are assessed in MS patients in a 12 month follow-up study. METHODS: Using flow cytometry, B and T cell subtypes, and their expression of antigen presentation, costimulation and migration markers were measured during a 12 month follow-up in the peripheral blood of MS patients. Data of fingolimod-treated MS patients (n = 49) were compared to those from treatment-naive (n = 47) and interferon-treated (n = 27) MS patients. RESULTS: In the B cell population, we observed a decrease in the proportion of non class-switched and class-switched memory B cells (p<0.001), both implicated in MS pathogenesis, while the proportion of naive B cells was increased during fingolimod treatment in the peripheral blood (PB) of MS patients (p<0.05). The remaining T cell population, in contrast, showed elevated proportions of memory conventional and regulatory T cells (p<0.01) and declined proportions of naive conventional and regulatory cells (p<0.05). These naive T cell subtypes are main drivers of MS pathogenesis. B cell expression of CD80 and CD86 and programmed death (PD) -1 expression on circulating follicular helper T cells was increased during fingolimod follow-up (p<0.05) pointing to a potentially compensatory mechanism of the remaining circulating lymphocyte subtypes that could provide additional help during normal immune responses. CONCLUSIONS: MS patients treated with fingolimod showed a change in PB lymphocyte subtype proportions and expression of functional molecules on T and B cells, suggesting an association with the therapeutic efficacy of fingolimod.

Construction of helper plasmid-mediated dual-display phage for autoantibody screening in serum.

M13 filamentous bacteriophage has been used in displaying disease-specific antibodies, biomarkers, and peptides. One of the major drawbacks of using phage in diagnostic assays is the aspecific adsorption of proteins leading to a high background signal and decreasing sensitivity. To deal with this, we developed a genetically pure, exchangeable dual-display phage system in which biomarkers and streptavidin-binding protein (SBP) are displayed at opposite ends of the phage. This approach allows for sample purification, using streptavidin-coated magnetic beads resulting in a higher sensitivity of signal detection assays. Our dual-display cassette system approach also allows for easy exchange of both the anchor protein (SBP) and the displayed biomarker. The presented principle is applied for the detection of antibody reactivity against UH-RA.21 which is a good candidate biomarker for rheumatoid arthritis (RA). The applicability of dual-display phage preparation using a helper plasmid system is demonstrated, and its increased sensitivity in phage ELISA assays using patient serum samples is shown.

Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor.

Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR-MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR-MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR-MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein-Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR-MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR-MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR-MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected.