Kornelis S. M. van der Geest

Disturbed B cell homeostasis in newly diagnosed giant cell arteritis and polymyalgia rheumatica

Several lines of evidence indicate that B cells may be involved in the immunopathology of giant cell arteritis (GCA) and polymyalgia rheumatica (PMR). This study was undertaken to examine the distribution of defined B cell subsets, including effector B (Beff) cells and regulatory B (Breg) cells, in patients with GCA and patients with PMR before and after corticosteroid treatment.

Aging disturbs the balance between effector and regulatory CD4+ T cells

Healthy aging requires an optimal balance between pro-inflammatory and anti-inflammatory immune responses. Although CD4+ T cells play an essential role in many immune responses, few studies have directly assessed the effect of aging on the balance between effector T (Teff) cells and regulatory T (Treg) cells. Here, we determined if and how aging affects the ratio between Treg and Teff cells. Percentages of both naive Treg (nTreg; CD45RA+CD25(int)FOXP3(low)) and memory Treg (memTreg; CD45RA-CD25(high)FOXP3(high)) cells were determined by flow cytometry in peripheral blood samples of healthy individuals of various ages (20-84 years). Circulating Th1, Th2 and Th17 effector cells were identified by intracellular staining for IFN-γ, IL-4 and IL-17, respectively, upon in vitro stimulation with PMA and calcium ionophore. Whereas proportions of nTreg cells declined with age, memTreg cells increased. Both Th1 and Th2 cells were largely maintained in the circulation of aged humans, whereas Th17 cells were decreased. Similar to memTreg cells, the 3 Teff subsets resided primarily in the memory CD4+ T cell compartment. Overall, Treg/Teff ratios were increased in the memory CD4+ T cell compartment of aged individuals when compared to that of young individuals. Finally, the relative increase of memTreg cells in elderly individuals was associated with poor responses to influenza vaccination. Taken together, our findings imply that aging disturbs the balance between Treg cells and Teff cells.

Serum markers associated with disease activity in giant cell arteritis and polymyalgia rheumatica

OBJECTIVE To compare multiple serum markers for their ability to detect active disease in patients with GCA and in those with PMR. METHODS Twenty-six markers related to immune cells that may be involved in GCA and PMR were determined by ELISA and multiplex assay in the serum of 24 newly diagnosed, untreated GCA/PMR patients, 14 corticosteroid (CS)-treated GCA/PMR patients in remission and 13 healthy controls. Receiver operating characteristic analysis with area under the curve and Spearmans correlation coefficients were performed. RESULTS Serum B-cell activating factor (BAFF), CXCL9 and IL-6 were increased in newly diagnosed GCA and PMR patients. Serum CCL2, CCL11, IL-10 and sIL-2R were modulated in GCA patients only and CXCL10 in PMR patients only. BAFF, CXCL9 and IL-6 accurately distinguished newly diagnosed GCA and PMR patients from healthy controls, as shown by area under the curve > 0.80. Upon CS-induced remission, serum BAFF and IL-6 decreased significantly in both GCA and PMR patients, whereas CXCL9 remained high. Serum BAFF and IL-6 correlated strongly with ESR and CRP in GCA and PMR patients. CONCLUSION Among the serum markers tested, BAFF and IL-6 showed the strongest association with disease activity in both GCA and PMR patients. The diagnostic value of these markers should be evaluated in larger, longitudinal studies with GCA and PMR patients, and in patients with infections or other inflammatory conditions.

Circulating CD4+CD161+ T Lymphocytes Are Increased in Seropositive Arthralgia Patients but Decreased in Patients with Newly Diagnosed Rheumatoid Arthritis

Improved understanding of the immune events discriminating between seropositive arthralgia and clinical synovitis is of key importance in rheumatology research. Ample evidence suggests a role for Th17 cells in rheumatoid arthritis. We hypothesized that CD4+CD161+ cells representing Th17 lineage cells may be modulated prior to or after development of clinical synovitis. Therefore, in a cross-sectional study, we investigated the occurrence of CD4+CD161+ T-cells in seropositive arthralgia patients who are at risk for developing rheumatoid arthritis and in newly diagnosed rheumatoid arthritis patients. In a prospective study, we evaluated the effect of methotrexate treatment on circulating CD4+CD161+ T-cells. Next, we assessed if these cells can be detected at the level of the RA joints. Precursor Th17 lineage cells bearing CD161 were found to be increased in seropositive arthralgia patients. In contrast, circulating CD4+CD161+T-cells were decreased in newly diagnosed rheumatoid arthritis patients. The decrease in CD4+CD161+ T-cells correlated inversely with C-reactive protein and with the 66 swollen joint count. Methotrexate treatment led to normalization of CD4+CD161+ T-cells and reduced disease activity. CD4+CD161+ T cells were readily detected in synovial tissues from both early and late-stage rheumatoid arthritis. In addition, synovial fluid from late-stage disease was found to be enriched for CD4+CD161+ T-cells. Notably, synovial fluid accumulated CD4+CD161+T-cells showed skewing towards the Th1 phenotype as evidenced by increased interferon-γ expression. The changes in peripheral numbers of CD4+CD161+ T-cells in seropositive arthralgia and early rheumatoid arthritis and the enrichment of these cells at the level of the joint predict a role for CD4+CD161+ T-cells in the early immune events leading to clinical synovitis. Our findings may add to the development of RA prediction models and provide opportunities for early intervention.

Different Scoring Methods of FDG PET/CT in Giant Cell Arteritis : Need for Standardization

Abstract Giant cell arteritis (GCA) is the most frequent form of vasculitis in persons older than 50 years. Cranial and systemic large vessels can be involved. [18F] fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) is increasingly used to diagnose inflammation of the large arteries in GCA. Unfortunately, no consensus exists on the preferred scoring method. In the present study, we aim to define the optimal FDG PET/CT scoring method for GCA diagnosis using temporal artery biopsy and clinical diagnosis as the reference method. FDG PET/CT scans of GCA patients (12 glucocorticoid-naive, 6 on glucocorticoid treatment) and 3 control groups (inflammatory, atherosclerotic, and normal controls) were evaluated. We compared 2 qualitative visual methods (i.e. (1a) first impression and (1b) vascular uptake versus liver uptake) and 4 semiquantitative methods ((2a) SUVmax aorta, (2b) SUVmax aorta-to-liver ratio, (2c) SUVmax aorta-to-superior-caval-vein ratio, and (2d) SUVmax aorta-to-inferior-caval-vein ratio). FDG uptake pattern (diffuse or focal) and presence of arterial calcifications were also scored. Diagnostic accuracy of the visual method vascular versus liver uptake (1b) was highest when the cut-off point “vascular uptake higher than liver uptake” (sensitivity 83%, specificity 91%) was used. Sensitivity increased to 92% when patients on glucocorticoids were excluded from the analysis. Regarding the semiquantitative methods, the aorta-to-liver ratio (2b) with a cutoff of 1.03 had the highest diagnostic accuracy, with a sensitivity and specificity of 69% and 92%, respectively. Sensitivity increased to 90% when patients on glucocorticoids were excluded. The number of vascular segments with diffuse FDG uptake pattern was significantly higher in GCA patients without glucocorticoid use compared with all control patient groups. CRP was not significantly different between positive and negative FDG PET scans in the GCA group. Visual vascular uptake higher than liver uptake resulted in the highest diagnostic accuracy for the detection of GCA, especially in combination with a diffuse FDG uptake pattern. Of the semiquantitative methods, the aorta-to-liver SUVmax ratio (cutoff point = 1.03) had the highest diagnostic accuracy. The diagnostic accuracy increased when patients using glucocorticoids were excluded from the analyses.

Low-affinity TCR engagement drives IL-2-dependent post-thymic maintenance of naive CD4+T cells in aged humans

Insight into the maintenance of naive T cells is essential to understand defective immune responses in the context of aging and other immune compromised states. In humans, naive CD4+ T cells, in contrast to CD8+ T cells, are remarkably well retained with aging. Here, we show that low‐affinity TCR engagement is the main driving force behind the emergence and accumulation of naive‐like CD4+ T cells with enhanced sensitivity to IL‐2 in aged humans. In vitro, we show that these CD45RA+CD25dimCD4+ T cells can develop from conventional naive CD25−CD4+ T cells upon CD3 cross‐linking alone, in the absence of costimulation, rather than via stimulation by the homeostatic cytokines IL‐2, IL‐7, or IL‐15. In vivo, TCR engagement likely occurs in secondary lymphoid organs as these cells were detected in lymph nodes and spleen where they showed signs of recent activation. CD45RA+CD25dimCD4+ T cells expressed a broad TCRVβ repertoire and could readily differentiate into functional T helper cells. Strikingly, no expansion of CD45RA+CD25dimCD8+ T cells was detected with aging, thereby implying that maintenance of naive CD4+ T cells is uniquely regulated. Our data provide novel insight into the homeostasis of naive T cells and may guide the development of therapies to preserve or restore immunity in the elderly.

SF Treg cells transcribing high levels of Bcl-2 and microRNA-21 demonstrate limited apoptosis in RA

OBJECTIVE The aim of this study was to investigate the turnover of Treg cells in the SF of RA patients. METHODS Treg cells were enumerated in peripheral blood and SF of RA patients and analysed by flow cytometry for expression of the proliferation marker Ki-67 and binding of the apoptosis marker annexin V. Sorted Treg cells of RA patients were analysed for expression of anti-apoptotic regulators Bcl-2 and microRNA-21 (miR-21) by RT-PCR. RESULTS Treg cells displaying a memory phenotype were abundant in the SF of RA patients. SF Treg cells more frequently expressed the proliferation marker Ki-67 than conventional T cells. Only few SF Treg cells were apoptotic, as indicated by limited annexin V staining of these cells. SF Treg cells displayed high transcription levels of Bcl-2 and miR-21 in comparison with SF conventional T cells and peripheral blood Treg cells. CONCLUSION Treg cells with a memory phenotype accumulate in the SF of RA patients. These Treg cells have a high proliferative activity and demonstrate little apoptosis. The latter finding could be explained by high transcription of Bcl-2 and miR-21 in SF Treg cells.

Quantifying Distribution of Flow Cytometric TCR-Vβ Usage with Economic Statistics

Measuring changes of the T cell receptor (TCR) repertoire is important to many fields of medicine. Flow cytometry is a popular technique to study the TCR repertoire, as it quickly provides insight into the TCR-Vβ usage among well-defined populations of T cells. However, the interpretation of the flow cytometric data remains difficult, and subtle TCR repertoire changes may go undetected. Here, we introduce a novel means for analyzing the flow cytometric data on TCR-Vβ usage. By applying economic statistics, we calculated the Gini-TCR skewing index from the flow cytometric TCR-Vβ analysis. The Gini-TCR skewing index, which is a direct measure of TCR-Vβ distribution among T cells, allowed us to track subtle changes of the TCR repertoire among distinct populations of T cells. Application of the Gini-TCR skewing index to the flow cytometric TCR-Vβ analysis will greatly help to gain better understanding of the TCR repertoire in health and disease.

Age-Associated Differences in MiRNA Signatures Are Restricted to CD45RO Negative T Cells and Are Associated with Changes in the Cellular Composition, Activation and Cellular Ageing.

MicroRNAs (miRNAs) have emerged as important players in the regulation of T-cell functionality. However, comprehensive insight into the extent of age-related miRNA changes in T cells is lacking. We established miRNA expression patterns of CD45RO- naïve and CD45RO+ memory T-cell subsets isolated from peripheral blood cells from young and elderly individuals. Unsupervised clustering of the miRNA expression data revealed an age-related clustering in the CD45RO- T cells, while CD45RO+ T cells clustered based on expression of CD4 and CD8. Seventeen miRNAs showed an at least 2-fold up- or downregulation in CD45RO- T cells obtained from young as compared to old donors. Validation on the same and independent samples revealed a statistically significant age-related upregulation of miR-21, miR-223 and miR-15a. In a T-cell subset analysis focusing on known age-related phenotypic changes, we showed significantly higher miR-21 and miR-223 levels in CD8+CD45RO-CCR7- TEMRA compared to CD45RO-CCR7+ TNAIVE-cells. Moreover, miR-21 but not miR-223 levels were significantly increased in CD45RO-CD31- post-thymic TNAIVE cells as compared to thymic CD45RO-CD31+ TNAIVE cells. Upon activation of CD45RO- TNAIVE cells we observed a significant induction of miR-21 especially in CD4+ T cells, while miR-223 levels significantly decreased only in CD4+ T cells. Besides composition and activation-induced changes, we showed a borderline significant increase in miR-21 levels upon an increasing number of population doublings in CD4+ T-cell clones. Together, our results show that ageing related changes in miRNA expression are dominant in the CD45RO- T-cell compartment. The differential expression patterns can be explained by age related changes in T-cell composition, i.e. accumulation of CD8+ TEMRA and CD4+ post-thymic expanded CD31- T cells and by cellular ageing, as demonstrated in a longitudinal clonal culture model.

The impact of exercise on the variation of serum free light chains

Measurement of serum free light chains (sFLC) has become an established method for screening, prognostic evaluation, and monitoring of multiple myeloma and related monoclonal gammopathies [1]. In recent years it was shown that non-clonal elevations of sFLC, measured as the κand λFLC sum (ΣFLC), can act as a global marker of immune stimulation, and may be linked to severity of immune disease [2]. In a cohort of 15,859 individuals all aged above 50 years, Dispenzieri et al. found that ΣFLC is a significant predictor of worse overall survival in persons without plasma cell disorders [3]. sFLC as such is regarded a biomarker of frailty in the elderly. Due to the well-defined clinical role of sFLC, the longterm biological variation of monoclonal FLC in patients with monoclonal gammopathies [4] and polyclonal FLC in healthy controls were recently assessed [5]. Immune status is not solely influenced by pathogen exposure or inherent immune disorders, other factors such as exercise also play a role. Acute exercise, e.g., induces immune stimulation, which is reflected by transiently increased concentrations of sensitive inflammation markers [6]. Whether or not exercise can affect FLC concentrations has to our knowledge not been assessed before. In the present study, we hypothesized that exerciseinduced immune stimulation may affect sFLC concentrations. To test this hypothesis we monitored sFLC concentrations in 37 elderly volunteers who participated in the Nijmegen Four Days Marches and walked 30 km at 4 consecutive days at a self-selected pace. Thirty-seven elderly men and women (age 76–86 years) of the 2013 Nijmegen Four Days Marches (an annual walking event in The Netherlands) volunteered to participate in our study. The experimental set-up to determine the demographic characteristics of the volunteers, their health status and exercise characteristics are described previously [7]. Baseline blood measurements were performed 12–36 h preceding the start. Immediately after finishing on the fourth day, all baseline measurements were repeated. FLC measurements were measured in batches of frozen and anonymized sera. Sera aliquots were stored at –20 °C directly after collection and thawed directly before analysis. Serum FLC analysis was performed on the SPAplus instrument using Freelite reagents (The Binding Site Ltd, Birmingham, UK). ΣFLC was calculated as the sum of the individual measurements of both κFLC and λFLC. Values are presented as means ± standard deviation. *Corresponding author: Joannes F.M. Jacobs, PhD, MD, Radboud University Medical Center, Department of Laboratory Medicine, Laboratory Medical Immunology (route 469), Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands, Phone: +31 24 3617414, Fax: +31 24 3619415, E-mail: H.Jacobs@Radboudumc.nl; and Department of Laboratory Medicine, Laboratory Medical Immunology, Radboud University Medical Center, Nijmegen, The Netherlands; and Department of Tumor Immunology, Radboud University Medical Center, Nijmegen, The Netherlands Thijs M.H. Eijsvogels: Department of Physiology, Radboud University Medical Center, Nijmegen, The Netherlands; and Henry Low Heart Center, Department of Cardiology, Hartford Hospital, Hartford, CT, USA Kornelis S.M. van der Geest and Annemieke M.H. Boots: Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, Groningen, The Netherlands; and Groningen Research initiative on healthy Ageing and Immune Longevity (GRAIL), Groningen, The Netherlands Hans J.P.M. Koenen and Irma Joosten: Department of Laboratory Medicine, Laboratory Medical Immunology, Radboud University Medical Center, Nijmegen, The Netherlands Colin A. Hutchison: Renal Institute of Birmingham, University Hospital Birmingham, Birmingham, UK; and Hawke’s Bay District Health Board, Hawke’s Bay, New Zealand Maria T.E. Hopman: Department of Physiology, Radboud University Medical Center, Nijmegen, The Netherlands