Circulating B Cells Display Differential Immune Regulatory Molecule Expression in Granulomatosis with Polyangiitis.

Granulomatosis with polyangiitis (GPA) is a B cell-mediated, relapsing, autoimmune disease. There is a need for novel therapeutic approaches and relapse markers to achieve durable remission. B cells express immune regulatory molecules that modulate their activation and maintain tolerance. While recent studies show dysregulation of these molecules in other autoimmune diseases, data on their expression in GPA are limited. This study aimed to map the expression of surface immune regulatory molecules on circulating B cell subsets in GPA and correlate their expression with clinical parameters. Immune regulatory molecule expression on circulating B cell subsets was comprehensively examined in active GPA (n=16), GPA in remission (n=16), and healthy controls (HCs, n=16) cross-sectionally using a 35-color B cell-specific spectral flow cytometry panel. Our supervised and unsupervised in-depth analysis revealed differential expression of inhibitory and stimulatory immune molecules on distinct B cell populations in GPA, with the most notable differences observed in active GPA. These differences include the upregulation of FcγRIIB on non-mature B cells, downregulation of CD21 and upregulation of CD86 on antigen-experienced B cells, and elevated CD22 expression on various populations. Additionally, we found a strong association between FcγRIIB, BTLA, and CD21 expression on specific B cell populations and disease activity in GPA. Together, these findings provide novel insights into the immune regulatory molecule expression profile of B cells in GPA, and could potentially form the foundation for new therapeutic approaches and disease monitoring markers.

Impaired IL-6-induced JAK-STAT signaling in CD4+ T cells associates with longer treatment duration in giant cell arteritis.

INTRODUCTION: The IL-12-IFNγ-Th1 and the IL-6-IL-23-Th17 axes are considered the dominant pathogenic pathways in Giant Cell Arteritis (GCA). Both pathways signal via activation of the downstream JAK/STAT proteins. We hypothesized that phosphorylated STAT (pSTAT) signatures in circulating immune cells may aid to stratify GCA-patients for personalized treatment. METHODS: To investigate pSTAT expression, PBMCs from treatment-naive GCA-patients (n = 18), infection controls (INF, n = 11) and age-matched healthy controls (HC, n = 15) were stimulated in vitro with IL-6, IL-2, IL-10, IFN-γ, M-CSF or GM-CSF, and stained with CD3, CD4, CD19, CD45RO, pSTAT1, pSTAT3, pSTAT5 antibodies, and analyzed by flow cytometry. Serum IL-6, sIL-6-receptor and gp130 were measured by Luminex. The change in percentages of pSTAT3+CD4+T-cells was evaluated at diagnosis and at 3 months and 1-year of follow-up. Kaplan-Meier analyses was used to asses prognostic accuracy. RESULTS: Analysis of IL-6 stimulated immune cell subsets revealed a significant decrease in percentages of pSTAT3+CD4+T-cells of GCA-patients and INF-controls compared to HCs. Following patient stratification according to high (median>1.5 pg/mL) and low (median<1.5 pg/mL) IL-6 levels, we observed a reduction in the pSTAT3 response in GCA-patients with high serum IL-6. Percentages of pSTAT3+CD4+T-cells in patients with high serum IL-6 levels at diagnosis normalized after glucocorticoid (GC) treatment. Importantly, we found that patients with low percentages of pSTAT3+CD4+T-cells at baseline require longer GC-treatment. CONCLUSION: Overall, in GCA, the percentages of in vitro IL-6-induced pSTAT3+CD4+T-cells likely reflect prior in vivo exposure to high IL-6 and may serve as a prognostic marker for GC-treatment duration and may assist improving personalized treatment options in the future.

Targeting NF-κB signaling in B cells as a potential new treatment modality for ANCA-associated vasculitis.

B lineage cells are critically involved in ANCA-associated vasculitis (AAV), evidenced by alterations in circulating B cell subsets and beneficial clinical effects of rituximab (anti-CD20) therapy. This treatment renders a long-term, peripheral B cell depletion, but allows for the survival of long-lived plasma cells. Therefore, there is an unmet need for more reversible and full B lineage cell targeting approaches. To find potential novel therapeutic targets, RNA sequencing of CD27+ memory B cells of patients with active AAV was performed, revealing an upregulated NF-κB-associated gene signature. NF-κB signaling pathways act downstream of various B cell surface receptors, including the BCR, CD40, BAFFR and TLRs, and are essential for B cell responses. Here we demonstrate that novel pharmacological inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signaling) and inhibitor-of-κB-kinase-ß (IKKß, canonical NF-κB signaling) can effectively inhibit NF-κB signaling in B cells, whereas T cell responses were largely unaffected. Moreover, both inhibitors significantly reduced B cell proliferation, differentiation and production of antibodies, including proteinase-3 (PR3) autoantibodies, in B lineage cells of AAV patients. These findings indicate that targeting NF-κB, particularly NIK, may be an effective, novel B lineage cell targeted therapy for AAV and other autoimmune diseases with prominent B cell involvement.

Kv1.3 blockade by ShK186 modulates CD4+ effector memory T-cell activity of patients with granulomatosis with polyangiitis.

OBJECTIVES: Granulomatosis with polyangiitis (GPA) is a chronic relapsing systemic autoimmune vasculitis. Current treatment of GPA is unsatisfactory, as it relies on strong immunosuppressive regimens, with either CYC or rituximab, which reduce the immunogenicity of several vaccines and are risk factors for a severe form of COVID-19. This emphasizes the need to identify new drug targets and to develop treatment strategies with less harmful side effects. Since CD4+ effector memory T cells (TEM) play a key role in the pathogenesis of GPA, we aimed in this study to modulate CD4+TEM cell activity via Kv1.3 blockade using the specific peptide inhibiter, ShK-186. METHODS: Peripheral blood samples from 27 patients with GPA in remission and 16 age- and sex-matched healthy controls (HCs) were pre-incubated in vitro in the presence or absence of ShK-186, followed by stimulation with phorbol myristate acetate, calcium ionophore and brefeldin-A. The effect of ShK-186 on the cytokine production (IFNγ, TNFα, IL-4, IL-17, IL-21) within total and subsets of CD4+ T helper (CD4+TH) cells were assessed using flow cytometry. RESULTS: ShK-186 reduced the expression level of IFNγ, TNFα, IL-4, IL-17 and IL-21 in CD4+TH cells from patients with GPA in vitro. Further analysis performed on sorted CD4+T cell subsets, revealed that ShK-186 predominantly inhibited the cytokine production of CD4+TEM cells. ShK-186 treatment reduced the production of the pro-inflammatory cytokines to the level seen in CD4+ TH cells from HCs. CONCLUSIONS: Modulation of cellular effector function by ShK-186 may constitute a novel treatment strategy for GPA with high specificity and less harmful side effects.

Low density granulocytes and neutrophil extracellular trap formation are increased in incomplete systemic lupus erythematosus.

OBJECTIVE: To investigate the proportion of low-density granulocytes (LDGs), circulating plasma neutrophil extracellular traps (NETs), and serum-induced NET formation in patients with incomplete systemic lupus erythematosus (iSLE) and systemic lupus erythematosus (SLE). METHODS: LDGs were measured cross-sectionally in 18 iSLE patients, 11 SLE patients and 14 healthy controls (HCs), whereas circulating NETs and serum-induced NET formation were assessed in 35 iSLE patients, 41 SLE patients and 16 HCs. LDGs (CD14lowCD15+) were measured in PBMCs using flow cytometry and circulating plasma NETs were measured using anti-myeloperoxidase-DNA, anti-citrullinated histone H3 and anti-elastase-DNA complex ELISAs. Serum-induced NET formation was assessed by incubating healthy neutrophils with serum from iSLE patients, SLE patients or HCs and visualizing NETs with fluorescence microscopy. RESULTS: Proportions of LDGs and circulating plasma NETs were similarly elevated in iSLE and SLE patients compared with those in HCs. Furthermore, patients under hydroxychloroquine (HCQ) treatment had lower proportions of LDGs than those without. Serum from iSLE and SLE patients similarly induced NET formation in healthy neutrophils. In iSLE patients, myeloperoxidase-DNA complexes were correlated with proportions of age-associated B-cells, memory B-cells and negatively with naïve B-cells, while we did not find associations between measures of NETs or serum-induced NET formation and interferon score or clinical parameters. CONCLUSION: These results show that neutrophil dysfunction, including higher proportions of LDGs, and increased NET formation, already occur in iSLE, similar to SLE, despite differences in disease manifestations. Thereby, neutrophil dysfunction may contribute to sustained exposure to autoantigens and autoreactivity in early stages of SLE.

The Impact of Different Anesthetics on the Distribution and Cytotoxic Function of NK Cell Subpopulations: An In Vitro Study.

Only some subpopulations of natural killer (NK) cells have cytotoxic functionality, and the effects of anesthetics on these subpopulations are unknown. This study aimed to evaluate the in vitro effects of various anesthetics, both alone and in combination, on the distribution and cytotoxic function of NK cells and their subpopulations. Peripheral blood mononuclear cells (PBMCs) from eight healthy volunteers were treated for 4 h in vitro with dexmedetomidine, remifentanil, lidocaine, propofol, sevoflurane, and combinations in clinically relevant concentrations or left untreated. Flow cytometry was used to quantify the percentage of sampled NK cells and evaluate their distribution (CD56brightCD16neg, CD56brightCD16dim, CD56dimCD16neg, CD56dimCD16bright, and CD56negCD16bright) and cytotoxicity (Granzyme B (GrzB) and perforin) of NK cell subpopulations. Although the percentage of total NK cells did not change following exposure to anesthesia, the most important cytotoxic subpopulation (CD56dimCD16bright NK cells) decreased after exposure to both propofol (-3.58%, p = 0.045) and sevoflurane (-16.10%, p = 0.008) alone, and most combinations, especially in combination with lidocaine (propofol with lidocaine (-9.66%, p = 0.002) and sevoflurane with lidocaine (-21.90%, p < 0.001)). Dexmedetomidine and remifentanil had no effect on CD56dimCD16bright NK cells. Furthermore, no anesthetic regimen or combination altered the expression of GrzB and perforin in NK cells or NK cell subpopulations. In short, propofol and sevoflurane suppressed the highly cytotoxic phenotype (CD56dimCD16bright) of NK cells, with those exposed to sevoflurane combinations showing greater reductions. Immunosuppression was intensified with the inclusion of lidocaine in the anesthetic regimen.

Cytokine producing B-cells and their capability to polarize macrophages in giant cell arteritis.

OBJECTIVE: The lack of disease-specific autoantibodies in giant cell arteritis (GCA) suggests an alternative role for B-cells readily detected in the inflamed arteries. Here we study the cytokine profile of tissue infiltrated and peripheral blood B-cells of patients with GCA. Moreover, we investigate the macrophage skewing capability of B-cell-derived cytokines. METHODS: The presence of various cytokines in B-cell areas in temporal artery (n = 11) and aorta (n = 10) was identified by immunohistochemistry. PBMCs of patients with GCA (n = 11) and polymyalgia rheumatica (n = 10), and 14 age- and sex-matched healthy controls (HC) were stimulated, followed by flow cytometry for cytokine expression in B-cells. The skewing potential of B-cell-derived cytokines (n = 6 for GCA and HC) on macrophages was studied in vitro. RESULTS: The presence of IL-6, GM-CSF, TNFα, IFNγ, LTß and IL-10 was documented in B-cells and B-cell rich areas of GCA arteries. In vitro, B-cell-derived cytokines (from both GCA and HC) skewed macrophages towards a pro-inflammatory phenotype with enhanced expression of IL-6, IL-1ß, TNFα, IL-23, YKL-40 and MMP-9. In vitro stimulated peripheral blood B-cells from treatment-naïve GCA patients showed an enhanced frequency of IL-6+ and TNFα+IL-6+ B-cells compared to HCs. This difference was no longer detected in treatment-induced remission. Erythrocyte sedimentation rate positively correlated with IL-6+TNFα+ B-cells. CONCLUSION: B-cells are capable of producing cytokines and steering macrophages towards a pro-inflammatory phenotype. Although the capacity of B-cells in skewing macrophages is not GCA specific, these data support a cytokine-mediated role for B-cells in GCA and provide grounds for B-cell targeted therapy in GCA.

Phenotypic, transcriptomic and functional profiling reveal reduced activation thresholds of CD8+ T cells in giant cell arteritis.

OBJECTIVES: Evidence from temporal artery tissue and blood suggests involvement of CD8+ T cells in the pathogenesis of GCA, but their exact role is poorly understood. Therefore, we performed a comprehensive analysis of circulating and lesional CD8+ T cells in GCA patients. METHODS: Circulating CD8+ T cells were analysed for differentiation status (CD45RO, CCR7), markers of activation (CD69 and CD25) and proliferation (Ki-67) in 14 newly diagnosed GCA patients and 18 healthy controls by flow cytometry. Proliferative capacity of CD8+ T cells upon anti-CD3 and anti-CD3/28 in vitro stimulation was assessed. Single-cell RNA sequencing of peripheral blood mononuclear cells of patients and controls (n = 3 each) was performed for mechanistic insight. Immunohistochemistry was used to detect CD3, CD8, Ki-67, TNF-α and IFN-γ in GCA-affected tissues. RESULTS: GCA patients had decreased numbers of circulating effector memory CD8+ T cells but the percentage of Ki-67-expressing effector memory CD8+ T cells was increased. Circulating CD8+ T cells from GCA patients demonstrated reduced T cell receptor activation thresholds and displayed a gene expression profile that is concurrent with increased proliferation. CD8+ T cells were detected in GCA temporal arteries and aorta. These vascular CD8+ T cells expressed IFN-γ but not Ki-67. CONCLUSION: In GCA, circulating effector memory CD8+ T cells demonstrate a proliferation-prone phenotype. The presence of CD8+ T cells in inflamed arteries seems to reflect recruitment of circulating cells rather than local expansion. CD8+ T cells in inflamed tissues produce IFN-γ, which is an important mediator of local inflammatory responses in GCA.

Immune Modulatory Effects of Nonsteroidal Anti-inflammatory Drugs in the Perioperative Period and Their Consequence on Postoperative Outcome.

Nonsteroidal anti-inflammatory drugs are among the most commonly administered drugs in the perioperative period due to their prominent role in pain management. However, they potentially have perioperative consequences due to immune-modulating effects through the inhibition of prostanoid synthesis, thereby affecting the levels of various cytokines. These effects may have a direct impact on the postoperative outcome of patients since the immune system aims to restore homeostasis and plays an indispensable role in regeneration and repair. By affecting the immune response, consequences can be expected on various organ systems. This narrative review aims to highlight these potential immune system-related consequences, which include systemic inflammatory response syndrome, acute respiratory distress syndrome, immediate and persistent postoperative pain, effects on oncological and neurologic outcome, and wound, anastomotic, and bone healing.

Distribution of monocytes subpopulations in the peripheral blood from patients with Behçet's disease - Impact of disease status and colchicine use.

The innate immune response has a predominant role in Behçet's disease (BD) pathogenesis, but few studies have assessed monocytes in BD. This study aims to evaluate the profile of monocytes subsets in the peripheral blood of BD patients and healthy controls (HC). Monocytes subsets were identified as classical (CD14+CD16-), intermediate (CD14+CD16dim), and non-classical (CD14dimCD16high) subsets. Patients with BD presented a lower number of total monocytes (p = 0.020) and a lower number (p < 0.0001) of circulating classical monocytes than HC. In contrast, the number of intermediate monocytes was higher in BD patients than HC (p < 0.0001). In BD patients, no associations were observed with the severity of clinical manifestations or therapy. Colchicine was associated with a higher number of non-classical monocytes (p = 0.035). In conclusion, BD patients present an altered distribution of monocytes subsets with a reduction of classical and an increase of intermediate subsets.

Association of the CXCL9-CXCR3 and CXCL13-CXCR5 axes with B-cell trafficking in giant cell arteritis and polymyalgia rheumatica.

OBJECTIVE: B-cells are present in the inflamed arteries of giant cell arteritis (GCA) patients and a disturbed B-cell homeostasis is reported in peripheral blood of both GCA and the overlapping disease polymyalgia rheumatica (PMR). In this study, we aimed to investigate chemokine-chemokine receptor axes governing the migration of B-cells in GCA and PMR. METHODS: We performed Luminex screening assay for serum levels of B-cell related chemokines in treatment-naïve GCA (n = 41), PMR (n = 31) and age- and sex matched healthy controls (HC, n = 34). Expression of chemokine receptors on circulating B-cell subsets were investigated by flow cytometry. Immunohistochemistry was performed on GCA temporal artery (n = 14) and aorta (n = 10) and on atherosclerosis aorta (n = 10) tissue. RESULTS: The chemokines CXCL9 and CXCL13 were significantly increased in the circulation of treatment-naïve GCA and PMR patients. CXCL13 increased even further after three months of glucocorticoid treatment. At baseline CXCL13 correlated with disease activity markers. Peripheral CXCR3+ and CXCR5+ switched memory B-cells were significantly reduced in both patient groups and correlated inversely with their complementary chemokines CXCL9 and CXCL13. At the arterial lesions in GCA, CXCR3+ and CXCR5+ B-cells were observed in areas with high CXCL9 and CXCL13 expression. CONCLUSION: Changes in systemic and local chemokine and chemokine receptor pathways related to B-cell migration were observed in GCA and PMR mainly in the CXCL9-CXCR3 and CXCL13-CXCR5 axes. These changes can contribute to homing and organization of B-cells in the vessel wall and provide further evidence for an active involvement of B-cells in GCA and PMR.

High angiopoietin-2 levels associate with arterial inflammation and long-term glucocorticoid requirement in polymyalgia rheumatica.

OBJECTIVES: PMR frequently co-occurs with GCA. So far, a simple biomarker for detecting concomitant arterial inflammation in PMR patients is lacking. Furthermore, biomarkers predicting disease course in PMR are awaited. We here investigated the diagnostic and prognostic value of acute-phase markers (ESR, CRP, IL-6, serum amyloid A) and angiogenesis markers (VEGF, soluble Tie2, angiopoietin-1, angiopoietin-2) in isolated PMR and PMR/GCA overlap patients. METHODS: We prospectively included 39 treatment-naïve PMR patients, of whom 10 patients also showed evidence of large vessel GCA by PET-CT. Age-matched healthy controls (n = 32) and infection controls (n = 13) were included for comparison. Serum marker levels were measured by an ELISA or Luminex assay. Receiver operating characteristic and Kaplan-Meier analyses were used to asses diagnostic and prognostic accuracy, respectively. RESULTS: All acute-phase and angiogenesis markers, except angiopoietin-1, were higher in isolated PMR patients than in healthy controls. Angiopoietin-2, ESR and soluble Tie-2 were significantly higher in patients with PMR/GCA overlap than in isolated PMR patients. Angiopoeietin-2, but not soluble Tie2, outperformed ESR and CRP in discriminating patients with and without overlapping GCA (area under the curve: 0.90; sensitivity: 100%; specificity: 76%). Moreover, high angiopoietin-2 levels were associated with long-term glucocorticoid requirement. CONCLUSION: Assessment of angiopoietin-2 at baseline may assist diagnosis of concomitant vasculitis in PMR. Moreover, high levels of angiopoietin-2 were associated with an unfavourable disease course in isolated PMR patients. These findings imply that angiopoietin-2 is an interesting diagnostic and prognostic biomarker in PMR.

Proportions of B-cell subsets are altered in incomplete systemic lupus erythematosus and correlate with interferon score and IgG levels.

OBJECTIVES: Incomplete SLE (iSLE) patients display symptoms typical for SLE but have insufficient criteria to fulfil the diagnosis. Biomarkers are needed to identify iSLE patients that will progress to SLE. IFN type I activation, B-cell-activating factor (BAFF) and B-cell subset distortions play an important role in the pathogenesis of SLE. The aim of this cross-sectional study was to investigate whether B-cell subsets are altered in iSLE patients, and whether these alterations correlate with IFN scores and BAFF levels. METHODS: iSLE patients (n = 34), SLE patients (n = 41) with quiescent disease (SLEDAI ≤4) and healthy controls (n = 22) were included. Proportions of B-cell subsets were measured with flow cytometry, IFN scores with RT-PCR and BAFF levels with ELISA. RESULTS: Proportions of age-associated B-cells were elevated in iSLE patients compared with healthy controls and correlated with IgG levels. In iSLE patients, IFN scores and BAFF levels were significantly increased compared with healthy controls. Also, IFN scores correlated with proportions of switched memory B-cells, plasma cells and IgG levels, and correlated negatively with complement levels in iSLE patients. CONCLUSION: In this cross-sectional study, distortions in B-cell subsets were observed in iSLE patients and were correlated with IFN scores and IgG levels. Since these factors play an important role in the pathogenesis of SLE, iSLE patients with these distortions, high IFN scores, and high levels of IgG and BAFF may be at risk for progression to SLE.

Angiogenic T cells are decreased in people with type 2 diabetes mellitus and recruited by the dipeptidyl peptidase-4 inhibitor Linagliptin: A subanalysis from a randomized, placebo-controlled trial (RELEASE study).

Angiogenic T (Tang) cells are mediators of vascular repair, and are characterized by surface expression of CXCR4. This receptor for stromal cell-derived factor-1α (SDF-1α) is cleaved by dipeptidyl peptidase-4 (DPP-4). Tang cell levels were investigated in people with type 2 diabetes mellitus (T2DM) compared with matched healthy controls and after treatment with the DPP-4 inhibitor Linagliptin. People with T2DM were randomized to 5 mg/day Linagliptin (n = 20) or placebo (n = 21) for 26 weeks. Tang cell frequency was identified in peripheral blood mononuclear cells (CD3+ CD31+ CXCR4+ ) and levels of endothelial progenitor cells (EPCs) (CD34+ CD133+ KDR+ ) were also assessed in whole blood. Circulating Tang cell levels were significantly lower in people with T2DM compared with the healthy control group. SDF-1α levels increased significantly in Linagliptin-treated people with T2DM compared to placebo, and a trend was observed in change of Tang cell levels, while EPC count did not change. In conclusion, circulating Tang cell levels were considerably lower in people with T2DM, while a trend was observed in recruitment of Tang cells after 26 weeks of treatment with Linagliptin. These data suggest that DPP-4 inhibitors may potentially exert beneficial effects on bone marrow-driven vascular repair.

Effects of propofol and dexmedetomidine with and without remifentanil on serum cytokine concentrations in healthy volunteers: a post hoc analysis.

BACKGROUND: Anaesthetic agents are likely to alter circulating cytokine concentrations. Because preceding studies have not been able to exclude the contribution of surgical trauma, perioperative stress, or both to circulating cytokine concentrations, the effects of anaesthesia remain unclear. The aim of this study was to quantify serum cytokines in healthy volunteers administered i.v. anaesthetic agents in the absence of surgical trauma and perioperative stress. METHODS: Serum samples obtained during previous standardised studies from healthy volunteers were compared before and 6-8 h after induction of anaesthesia with propofol (n=31), propofol/remifentanil (n=30), dexmedetomidine (n=17) or dexmedetomidine/remifentanil (n=15). Anaesthetic regimens were standardised and volunteers did not undergo any surgical intervention. Serum concentrations of interleukin (IL)2, IL4, IL6, IL10, IL17, IL18, IL21, IL22, IL23, C-X-C motif ligand 8, interferon gamma, E-selectin, L-selectin, major histocompatibility complex class I chain-polypeptide-related sequence (MIC)A, MICB, Granzyme A, and Granzyme B were quantified using a multiplexed antibody-based assay (Luminex). RESULTS: Samples were obtained from volunteers of either sex aged 18-70 yr. After anaesthesia with propofol alone, concentrations of IL4 (P=0.012), IL6 (P=0.027), IL21 (P=0.035), IL22 (P=0.002), C-X-C motif ligand 8 (P=0.004), MICB (P=0.046), and Granzyme A (P=0.045) increased. After anaesthesia with propofol and remifentanil, IL17 (P=0.013), interferon gamma (P=0.003), and MICA (P=0.001) decreased, but IL6 (P=0.006) and L-selectin (P=0.001) increased. After dexmedetomidine alone, IL18 (P=0.002), L-selectin (P=0.017), E-selectin (P=0.002), and Granzyme B (P=0.023) decreased. After dexmedetomidine with remifentanil no changes were observed. CONCLUSIONS: In healthy volunteers not undergoing surgery, different i.v. anaesthesia regimens were associated with differential effects on circulating cytokines.

Effect of age and sex on immune checkpoint expression and kinetics in human T cells.

BACKGROUND: Immune checkpoints are crucial molecules in maintaining a proper immune balance. Even though age and sex are known to have effects on the immune system, the interplay between age, sex and immune checkpoint expression by T cells is not known. The aim of this study was to determine whether age and sex affect immune checkpoint expression by T cells and if age and sex affect the kinetics of immune checkpoint expression following ex vivo stimulation. In this study, whole blood samples of 20 healthy young adults (YA, 9 males and 11 females) and 20 healthy older adults (OA, 9 males and 11 females) were stained for lymphocyte lineage markers and immune checkpoints and frequencies of CD28+, PD-1+, VISTA+ and CD40L+ T cells were determined. Immune checkpoint expression kinetics were studied following ex vivo anti-CD3/anti-CD28 stimulation of T cells from young and older healthy adults. RESULTS: We report an age-associated increase of CD40L + CD4+ and CD40L + CD8+ T-cell frequencies, whereas CD40+ B-cell frequencies were decreased in older adults, suggesting modulation of the CD40L-CD40 interaction with age. Immune checkpoint expression kinetics revealed differences in magnitude between CD4+ and CD8+ T cells independent of age and sex. Further analysis of CD4+ T-cell subsets revealed an age-associated decrease of especially PD-1 + CD4+ memory T cells which tracked with the female sex. CONCLUSION: Collectively, our results demonstrate that both age and sex modulate expression of immune checkpoints by human T cells. These findings may have implications for optimising vaccination and immune checkpoint immunotherapy and move the field towards precision medicine in the management of older patient groups.

Circulating CD24hiCD38hi regulatory B cells correlate inversely with the ThEM17 cell frequency in granulomatosis with polyangiitis patients.

OBJECTIVES: To investigate whether there is a direct relation between expanded proportions of Th17 effector memory (ThEM17) cells and regulatory B cells (Bregs) in peripheral blood of granulomatosis with polyangiitis (GPA) patients. METHODS: Frequencies of Bregs and ThEM17 cells, as well as ThEM1 cells, were determined by flow cytometry in blood samples from 42 GPA patients in remission and 18 matched healthy controls (HCs). The Breg frequency was defined as CD24hiCD38hiCD19+ cells. ThEM17 cells were defined as CCR6+CXCR3-CCR4+ cells and ThEM1 cells as CCR6-CXCR3+CCR4- cells within the CD3+CD4+CD45RO+CCR7- population. In addition, CD3+CD4+ Th cells from 9 GPA patients were co-cultured in vitro with either total B cells or a Breg-depleted B cell fraction. Cultured cells were stimulated with Staphylococcus Enterotoxin B (SEB) and CpG-oligodeoxynucleotides (CpG-ODN). Th17- (IL-17+) and Th1 cell (IFNγ+) frequencies were determined at baseline and day 5 upon restimulation with phorbol myristate acetate (PMA) and Ca-I. RESULTS: A decreased Breg frequency was found in treated GPA patients, whereas an increased ThEM17 cell frequency was observed in treated and untreated GPA patients compared with HCs. Additionally, a decreased ThEM1 cell frequency was seen in untreated GPA patients compared with HCs. In untreated GPA patients circulating Breg frequencies correlated negatively with ThEM17 cells (r = -0.533; P = 0.007) and positively with ThEM1 cells (r = -0.473; P = 0.015). The co-culture experiments revealed a significant increase in the frequency of IL-17+ Th cells in Breg-depleted samples (median: 3%; range: 1-7.5%) compared with Breg-undepleted samples (P = 0.002; undepleted samples median: 2.1%; range: 0.9-6.4%), whereas no difference in the frequency of IFNγ+ Th cells in Breg-depleted cultures was observed (undepleted median: 11.8%; range: 2.8-21% vs Breg-depleted median: 12.2%; range: 2.6-17.6%). CONCLUSION: Bregs modulate ThEM17 responses in GPA patients. Future studies should elaborate on clinical and therapeutical implications of the Breg-Th17 interaction in GPA patients.

Markers of angiogenesis and macrophage products for predicting disease course and monitoring vascular inflammation in giant cell arteritis.

OBJECTIVE: GCA, a systemic vasculitis, is characterized by an IL-6-dependent acute-phase response. This response is typically suppressed by treatment rendering CRP/ESR unreliable for monitoring vascular inflammation. Also, there are no accurate biomarkers predicting a non-favourable disease course. Here we investigated macrophage products and markers of angiogenesis as biomarkers for prognosis and monitoring of vascular inflammation. METHODS: Forty-one newly diagnosed, glucocorticoid-naive GCA patients were prospectively followed for relapses and glucocorticoid requirement for a median of 30 months (range 0-71). Serum markers at baseline and during follow-up were compared with 33 age-matched healthy controls and 13 infection controls. Concentrations of IL-6, serum amyloid A, soluble CD163, calprotectin, YKL-40, VEGF, angiopoietin-1 and -2 and sTie2 were determined by ELISA/Luminex assay. RESULTS: Serum concentrations of all markers, but not angiopoietin-1, were elevated in GCA patients at baseline when compared with healthy controls. High VEGF (P = 0.0025) and angiopoietin-1 (P = 0.0174) and low YKL-40 (P = 0.0369) levels at baseline were predictive of a short time to glucocorticoid-free remission. Elevated angiopoietin-2 levels were associated with an imminent relapse during treatment (P < 0.05). IL-6 correlated strongly with acute-phase markers and soluble CD163 but not with markers of angiogenesis, YKL-40 or calprotectin. Glucocorticoid treatment down-modulated all markers except for calprotectin and YKL-40. Tissue expression of markers in temporal arteries was confirmed. CONCLUSION: Markers of angiogenesis at baseline and during treatment predict GCA disease course, suggesting utility in patient stratification for glucocorticoid-sparing therapy. Calprotectin and YKL-40 are candidate markers for monitoring vessel wall inflammation.

Evidence for enhanced Bruton's tyrosine kinase activity in transitional and naïve B cells of patients with granulomatosis with polyangiitis.

OBJECTIVES: To determine Bruton's tyrosine kinase (BTK) protein and phosphorylation levels in B cell subsets of granulomatosis with polyangiitis (GPA) patients and to investigate the effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production. METHODS: BTK protein and phosphorylation levels were determined by flow cytometry in peripheral blood B cells of 29 untreated GPA patients [9 active and 20 remission GPA patients (10 ANCA- and 10 ANCA+)], 9 age- and sex-matched healthy controls (HCs) and 9 untreated active RA patients. The effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production was determined in the same donors in peripheral blood mononuclear cell cultures. RESULTS: BTK protein levels were significantly increased in transitional and naïve B cells of active GPA and RA patients compared with remission GPA patients and HCs. Both B cell subsets of active patients were more sensitive to B cell receptor stimulation, as BTK and phospholipase Cγ2 phosphorylation were increased in these patients. In vitro BTK blockade had profound effects on B cell cytokine production, plasma cell formation and (auto)antibody production in both GPA patients and HCs. Interestingly, the effect of BTK blockade was less pronounced in active GPA patients, possibly due to increased activation of B cells. CONCLUSION: We show that BTK protein and phosphorylation levels are most profoundly increased in newly emerging B cells of active GPA patients compared with remission patients. BTK blockade greatly inhibits in vitro B cell effector functions in GPA patients and HCs. These promising data identify BTK as an interesting novel therapeutic target in the treatment of GPA.

Urinary and serum soluble CD25 complements urinary soluble CD163 to detect active renal anti-neutrophil cytoplasmic autoantibody-associated vasculitis: a cohort study.

Background: Early detection of renal involvement in anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is of major clinical importance to allow prompt initiation of treatment and limit renal damage. Urinary soluble cluster of differentiation 163 (usCD163) has recently been identified as a potential biomarker for active renal vasculitis. However, a significant number of patients with active renal vasculitis test negative using usCD163. We therefore studied whether soluble CD25 (sCD25), a T cell activation marker, could improve the detection of renal flares in AAV. Methods: sCD25 and sCD163 levels in serum and urine were measured by enzyme-linked immunosorbent assay in 72 patients with active renal AAV, 20 with active extrarenal disease, 62 patients in remission and 18 healthy controls. Urinary and blood CD4+ T and CD4+ T effector memory (TEM) cell counts were measured in 22 patients with active renal vasculitis. Receiver operating characteristics (ROC) curves were generated and recursive partitioning was used to calculate whether usCD25 and serum soluble CD25 (ssCD25) add utility to usCD163. Results: usCD25, ssCD25 and usCD163 levels were significantly higher during active renal disease and significantly decreased after induction of remission. A combination of usCD25, usCD163 and ssCD25 outperformed all individual markers (sensitivity 84.7%, specificity 95.1%). Patients positive for sCD25 but negative for usCD163 (n = 10) had significantly higher C-reactive protein levels and significantly lower serum creatinine and proteinuria levels compared with the usCD163-positive patients. usCD25 correlated positively with urinary CD4+ T and CD4+ TEM cell numbers, whereas ssCD25 correlated negatively with circulating CD4+ T and CD4+ TEM cells. Conclusion: Measurement of usCD25 and ssCD25 complements usCD163 in the detection of active renal vasculitis.