Induction of an altered CD40 signaling complex by an antagonistic human monoclonal antibody to CD40.

Blocking the interaction of CD40 with its ligand CD154 is a desirable goal of therapies for preventing and/or ameliorating autoimmune diseases and transplant rejection. CD154-blocking mAbs used in human clinical trials resulted in unanticipated vascular complications, leading to heightened interest in the therapeutic potential of antagonist mAbs specific for human CD40. Abs that do not require physical competition with CD154 to inhibit CD40 signaling have particular therapeutic promise. In this study, we demonstrate that the antagonist anti-human CD40 mAb PG102 fails to trigger CD40-mediated activation, as well as impairs CD154-mediated CD40 activation, via a distinct nonstimulatory CD40 signaling mechanism. PG102 did not induce early CD40-induced signaling events, and it inhibited early kinase and transcription factor activation by CD154 or agonist anti-CD40 mAbs. However, PG102 stimulated normal CD40-mediated TNFR-associated factor (TRAF)2 and TRAF3 degradation. PG102 induced the formation of a CD40 signaling complex that contained decreased amounts of both TRAF2 and TRAF3 and TRAF2-associated signaling proteins. Additionally, PG102-induced CD40 signaling complexes failed to recruit TRAF6 to detergent-insoluble membrane fractions. Fab fragments of PG102, while retaining CD40 binding, did not induce TRAF degradation, nor could they inhibit CD154-stimulated B cell signaling, indicating that CD40 aggregation is required for the signaling inhibition induced by PG102. The antagonistic impact of PG102 on CD40 signaling reveals that the manner of CD40 ligation can determine sharply different outcomes for CD40 signaling and suggests that such information can be used to therapeutically manipulate these outcomes.

Circulating vitamin D binding protein levels are not associated with relapses or with vitamin D status in multiple sclerosis.

BACKGROUND: A low vitamin D status has been associated with multiple sclerosis (MS). Most circulating vitamin D metabolites are bound to vitamin D binding protein (DBP). OBJECTIVES: The purpose of this study was to explore whether there is an association between MS and DBP. METHODS: We compared DBP concentrations in blood samples of controls (n = 30) and subjects with relapsing-remitting MS (RRMS) during remission (n = 29) and relapse (n = 15). Furthermore, we explored correlations of DBP with 25- hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D levels (1,25(OH)2D), and the effect of high-dose vitamin D3 supplementation on DBP levels in RRMS patients (n = 15). RESULTS: DBP-concentration did not differ between the sub-groups measured, and there was no correlation between DBP and vitamin D metabolite concentration within the physiological range. Upon supplementation of high doses vitamin D3, DBP concentration remained unaltered. After supplementation, serum 1,25(OH)2D(R = 0.517, p = 0.049), but not 25(OH)D, correlated positively with DBP. CONCLUSIONS: We found no association between DBP, MS, and vitamin D status within the physiological range. After high-dose vitamin D supplementation, DBP concentrations may be relevant for vitamin D metabolism.

Characterization of skin-resident T cells using a crawl-out method and flow cytometry.

A large fraction of the skin-homing T-cell population resides in the skin even under resting, non-inflammatory conditions. Here, we used a crawl-out culture method to retrieve T cells from human skin and characterized them using flow cytometric analysis. On average, 48000 viable, non-proliferating cells were retrieved per biopsy. We found that human skin contains a larger fraction of IL-17-, IL-4-, IL-10- and IL-22-positive T cells as compared with paired blood samples. Our research indicates that it is feasible to use the crawl-out method in combination with flow cytometry to characterize T-cell subpopulations in patient-derived skin biopsies. This method enables further study of the skin immune system and could function as a valuable tool for evaluation of the effects of immunotherapy in skin diseases.

Repurposing Tamoxifen for Tumor Microenvironment Priming and Enhanced Tumor-Targeted Drug Delivery.

The dense stromal matrix in fibrotic tumors hinders tumor-targeted drug delivery. Tamoxifen (TMX), an estrogen receptor modulator that is clinically used for the treatment of breast cancer, has been shown to reprogram the tumor microenvironment (TME) and to alleviate desmoplasia. We here investigated if TMX, administered in free and nano-formulated form, can be repurposed as a TME remodeling agent to improve tumor accumulation of nano-formulations in pancreatic ductal adenocarcinoma and triple-negative breast cancer mouse models, evaluated using clinical-stage Cy7-labeled core-crosslinked polymeric micelles (CCPM). Under control conditions, we found higher levels of Cy7-CCPM in PANC-1 tumors (16.7 % ID g-1 at 48 h post i.v. injection) than in 4T1 tumors (11.0 % ID g-1). In both models, free and nano-formulated TMX failed to improve CCPM delivery. These findings were congruent with the results from histopathological immunofluorescence analysis of tumor tissue, which indicated that TMX treatment did not significantly change vascularization, perfusion, macrophage infiltration, collagen density, and collagen fiber thickness. Altogether, our results demonstrate that in PANC-1 and 4T1 mouse models, TMX treatment does not contribute to beneficial TME priming and enhanced tumor-targeted drug delivery.

Profiling target engagement and cellular uptake of cRGD-decorated clinical-stage core-crosslinked polymeric micelles.

Polymeric micelles are increasingly explored for tumor-targeted drug delivery. CriPec® technology enables the generation of core-crosslinked polymeric micelles (CCPMs) based on thermosensitive (mPEG-b-pHPMAmLacn) block copolymers, with high drug loading capacity, tailorable size, and controlled drug release kinetics. In this study, we decorated clinical-stage CCPM with the αvß3 integrin-targeted cyclic arginine-glycine-aspartic acid (cRGD) peptide, which is one of the most well-known active targeting ligands evaluated preclinically and clinically. Using a panel of cell lines with different expression levels of the αvß3 integrin receptor and exploring both static and dynamic incubation conditions, we studied the benefit of decorating CCPM with different densities of cRGD. We show that incubation time and temperature, as well as the expression levels of αvß3 integrin by target cells, positively influence cRGD-CCPM uptake, as demonstated by immunofluorescence staining and fluorescence microscopy. We demonstrate that even very low decoration densities (i.e., 1 mol % cRGD) result in increased engagement and uptake by target cells as compared to peptide-free control CCPM, and that high cRGD decoration densities do not result in a proportional increase in internalization. In this context, it should be kept in mind that a more extensive presence of targeting ligands on the surface of nanomedicines may affect their pharmacokinetic and biodistribution profile. Thus, we suggest a relatively low cRGD decoration density as most suitable for in vivo application.

Design, development and clinical translation of CriPec®-based core-crosslinked polymeric micelles.

Nanomedicines are used to improve the efficacy and safety of pharmacotherapeutic interventions. Unraveling the biological behavior of nanomedicines, including their biodistribution and target site accumulation, is essential to establish design criteria that contribute to superior performance. CriPec® technology is based on amphiphilic methoxy-poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide lactate] (mPEG-b-pHPMAmLacn) block copolymers, which are designed to upon self-assembly covalently entrap active pharmaceutical ingredients (API) in core-crosslinked polymeric micelles (CCPM). Key features of CCPM are a prolonged circulation time, high concentrations at pathological sites, and low levels of accumulation in the majority of healthy tissues. Proprietary hydrolysable linkers allow for tunable and sustained release of entrapped API, including hydrophobic and hydrophilic small molecules, as well as peptides and oligonucleotides. Preclinical imaging experiments provided valuable information on their tumor and tissue accumulation and distribution, as well as on uptake by cancer, healthy and immune cells. The frontrunner formulation CPC634, which refers to 65 nm-sized CCPM entrapping the chemotherapeutic drug docetaxel, showed excellent pharmacokinetic properties, safety, tumor accumulation and antitumor efficacy in multiple animal models. In the clinic, CPC634 also demonstrated favorable pharmacokinetics, good tolerability, signs of efficacy, and enhanced localization in tumor tissue as compared to conventional docetaxel. PET imaging of radiolabeled CPC634 showed quantifiable accumulation in âˆ¼50 % of tumors and metastases in advanced-stage cancer patients, and demonstrated potential for use in a theranostic setting even when applied at a companion diagnostic dose. Altogether, the preclinical and clinical results obtained to date demonstrate that mPEG-b-pHPMAmLacn CCPM based on CriPec® technology are a potent, tunable, broadly applicable and well-tolerable platform for targeted drug delivery and improved anticancer therapy.

Effect of vitamin D(3) supplementation on peripheral B cell differentiation and isotype switching in patients with multiple sclerosis.

BACKGROUND: Vitamin D has been proposed as a promoter of immune homeostasis in multiple sclerosis (MS). During the past decade, the focus of the effects of vitamin D has been on dendritic cells and on T cells. Since there is an increasing interest in the role of B cells in the pathophysiology of MS, we studied the role of vitamin D on B cells in vivo in patients with MS. OBJECTIVE: We explored the effects of 12 weeks high-dose vitamin D(3) supplementation on peripheral B cell differentiation, immunoglobulin production and levels of B cell activating factor (BAFF) in 15 patients with MS. METHODS: Circulating B cell subsets were characterized by flow cytometry. Plasma immunoglobulin levels were assessed by nephelometry. Plasma BAFF levels were assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Although a significant increase serum 25-hydroxyvitamin D was induced, we found no significant shift in B cell differentiation, isotype switching, or plasma BAFF levels. CONCLUSION: In patients with MS, supplementation of high doses vitamin D(3) does not have substantial effects on phenotypic markers of B cell differentiation in circulating B cells. Future studies may unravel more subtle changes in the B cell compartment, either in the circulation or in the central nervous system.

Acoustic Cluster Therapy (ACT®) enhances accumulation of polymeric micelles in the murine brain.

The restrictive nature of the blood-brain barrier (BBB) prevents efficient treatment of many brain diseases. Focused ultrasound in combination with microbubbles has shown to safely and transiently increase BBB permeability. Here, the potential of Acoustic Cluster Therapy (ACT®), a microbubble platform specifically engineered for theranostic purposes, to increase the permeability of the BBB and improve accumulation of IRDye® 800CW-PEG and core-crosslinked polymeric micelles (CCPM) in the murine brain, was studied. Contrast enhanced magnetic resonance imaging (MRI) showed increased BBB permeability in all animals after ACT®. Near infrared fluorescence (NIRF) images of excised brains 1 h post ACT® revealed an increased accumulation of the IRDye® 800CW-PEG (5.2-fold) and CCPM (3.7-fold) in ACT®-treated brains compared to control brains, which was retained up to 24 h post ACT®. Confocal laser scanning microscopy (CLSM) showed improved extravasation and penetration of CCPM into the brain parenchyma after ACT®. Histological examination of brain sections showed no treatment related tissue damage. This study demonstrated that ACT® increases the permeability of the BBB and enhances accumulation of macromolecules and clinically relevant nanoparticles to the brain, taking a principal step in enabling improved treatment of various brain diseases.

Optical imaging of the whole-body to cellular biodistribution of clinical-stage PEG-b-pHPMA-based core-crosslinked polymeric micelles.

Core-crosslinked polymeric micelles (CCPM) based on PEG-b-pHPMA-lactate are clinically evaluated for the treatment of cancer. We macroscopically and microscopically investigated the biodistribution and target site accumulation of CCPM. To this end, fluorophore-labeled CCPM were intravenously injected in mice bearing 4T1 triple-negative breast cancer (TNBC) tumors, and their localization at the whole-body, tissue and cellular level was analyzed using multimodal and multiscale optical imaging. At the organism level, we performed non-invasive 3D micro-computed tomography-fluorescence tomography (µCT-FLT) and 2D fluorescence reflectance imaging (FRI). At the tissue and cellular level, we performed extensive immunohistochemistry, focusing primarily on cancer, endothelial and phagocytic immune cells. The CCPM achieved highly efficient tumor targeting in the 4T1 TNBC mouse model (18.6 %ID/g), with values twice as high as those in liver and spleen (9.1 and 8.9 %ID/g, respectively). Microscopic analysis of tissue slices revealed that at 48 h post injection, 67% of intratumoral CCPM were localized extracellularly. Phenotypic analyses on the remaining 33% of intracellularly accumulated CCPM showed that predominantly F4/80+ phagocytes had taken up the nanocarrier formulation. Similar uptake patterns were observed for liver and spleen. The propensity of CCPM to primarily accumulate in the extracellular space in tumors suggests that the anticancer efficacy of the formulation mainly results from sustained release of the chemotherapeutic payload in the tumor microenvironment. In addition, their high uptake by phagocytic immune cells encourages potential use for immunomodulatory anticancer therapy. Altogether, the beneficial biodistribution, efficient tumor targeting and prominent engagement of PEG-b-pHPMA-lactate-based CCPM with key cell populations underline the clinical versatility of this clinical-stage nanocarrier formulation.

New concepts on the pathogenesis of autoimmune diseases: a role for immune homeostasis, immunoregulation, and immunosenescence.

Complex autoimmune diseases are chronic conditions initiated by a loss of immunological tolerance to self-antigens. Despite many years of intense investigation, the etiopathogenic mechanisms initiating these chronic inflammatory conditions remain undefined. The relatively high prevalence and the clustering of multiple autoimmune diseases within individuals or within families suggest that rather common immune mechanisms may facilitate the development of autoimmune diseases. However, specific genetic or environmental factors seem to be necessary for triggering disease. Clearly, immune mechanisms, which are intended to protect from disease, fail or become dysregulatcd. In this review, we discuss the mechanisms of immune homeostasis, immune regulation, and immunosenescence. We focus on the merit, consequences, and/or shortcomings of these mechanisms and speculate on their possible involvement in autoimmune-disease development.

Compromised CD4+ CD25(high) regulatory T-cell function in patients with relapsing-remitting multiple sclerosis is correlated with a reduced frequency of FOXP3-positive cells and reduced FOXP3 expression at the single-cell level.

CD4+ CD25(high) regulatory T cells (Tregs) of patients with relapsing-remitting (RR) multiple sclerosis (MS), in contrast to those of patients with secondary progressive (SP) MS, show a reduced suppressive function. In this study, we analysed forkhead box P3 (FOXP3) at the single-cell level in MS patients and controls (healthy individuals and patients with other neurological diseases) by means of intracellular flow cytometry. Our data revealed a reduced number of peripheral blood CD4+ CD25(high) FOXP3+ T cells and lower FOXP3 protein expression per cell in RR-MS patients than in SP-MS patients and control individuals, which was correlated with the suppressive capacity of Tregs in these patients. Interestingly, interferon (IFN)-beta-treated RR-MS patients showed restored numbers of FOXP3+ Tregs. Furthermore, a higher percentage of CD4+ CD25(high) FOXP3+ Tregs in RR-MS patients, as compared with controls and SP-MS patients, expressed CD103 and CD49d, adhesion molecules involved in T-cell recruitment towards inflamed tissues. This was consistent with a significantly increased number of CD27+ CD25(high) CD4+ T cells in the cerebrospinal fluid (CSF), as compared with peripheral blood, in RR-MS patients. Taken together, these data show aberrant FOXP3 expression at the single-cell level correlated with Treg dysfunction in RR-MS patients. Our results also suggest that Tregs accumulate in the CSF of RR-MS patients, in an attempt to down-regulate local inflammation in the central nervous system.

A CFSE based assay for measuring CD4+CD25+ regulatory T cell mediated suppression of auto-antigen specific and polyclonal T cell responses.

CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as suppressors of immune mediated reactions. The analysis of Treg function in patients with autoimmune, allergic or oncogenic diseases has emerged over the past years. In the present study we describe a CFSE based protocol to measure Treg mediated suppression of CD4(+) T cells. Measuring Treg suppressive capacity towards proliferation of anti-CD3 Ab stimulated CD4(+)CD25(-) T cells in coculture experiments by means of a CFSE based and a classical [(3)H]thymidine incorporation assay gave similar results, provided that CD4(+)CD25(+) T cells were anergic. However, when CD4(+)CD25(+) T cells proliferated upon mitogenic stimulation, data obtained by the CFSE assay allowed the detection of a significant Treg suppression whereas this was clearly underestimated using the [(3)H]thymidine assay. In addition, an indirect CFSE based method was developed to analyze antigen specific responses of total CD4(+) T cells and Treg depleted CD4(+) T cells (i.e. CD4(+)CD25(-) T cells). Our results indicate that, in healthy individuals, CD4(+) T cell responses against the multiple sclerosis (MS) auto-antigens, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), were increased in Treg depleted CD4(+) T cells as compared to total CD4(+) T cells. Our initial data suggest that Tregs in MS patients show an impaired suppression of myelin reactive T cells when compared to healthy controls. Moreover, this experimental setup permits the measurement of cytokine production of the antigen proliferated CFSE(low) T cells by additional flow cytometric analyses. In conclusion, the described CFSE based Treg suppression assay is a valuable tool to study suppressor T cells in (auto)immune disorders.

Immune regulatory effects of high dose vitamin D3 supplementation in a randomized controlled trial in relapsing remitting multiple sclerosis patients receiving IFNß; the SOLARIUM study.

Multiple sclerosis (MS) is characterized by a disturbed immune homeostasis and low serum vitamin D levels are associated with an increased disease activity. While vitamin D has been hypothesized to promote the maintenance of immune homeostasis, vitamin D supplementation could be of benefit to patients with MS. The SOLAR study investigated the effects of high dose vitamin D3 supplementation on clinical outcomes in a randomized controlled trial. Here we present the immune regulatory effects, investigated in the SOLARIUM sub-study. Thirty Dutch relapsing remitting (RR) MS patients treated with IFNß-1a received high dose vitamin D3 supplementation and 23 patients received placebo during a period of 48weeks. Lymphocytes were phenotypically characterized by flow cytometry and in vitro cytokine secretion was assessed in the presence or absence of 1,25(OH)2D3 using Luminex technology. Changes in immune regulatory parameters were determined within subjects as well as between treatment groups. The proportion of cells in the immune regulatory cell compartment (nTreg, iTreg and Breg) was not altered upon high dose vitamin D3 supplementation. Proportions of T helper subsets were not affected by vitamin D3, except for the proportion of IL4+ Th cells, which decreased in the placebo but not in the vitamin D3 group. T cell cytokine secretion increased, most pronounced for IL5 and latency activated protein of TGFß, in the placebo group but not in the vitamin D3 group. Lymphocytes remained equally reactive to in vitro 1,25(OH)2D3. In conclusion, high dose vitamin D3 supplementation did not result in a relative increase in lymphocytes with a regulatory phenotype. However, this study supports the hypothesis that vitamin D contributes to the maintenance of immune homeostasis by preventing further disturbance of the T cell compartment early in the disease course of MS.

Impaired activation-induced telomerase activity in PBMC of early but not chronic rheumatoid arthritis patients.

Although telomerase activity is important in normal immune function, it is unclear whether telomerase or telomerase (dys)regulation plays a role in the pathogenic immune response in autoimmune diseases like rheumatoid arthritis (RA). In this study, we evaluated the dynamics of the activation-induced human telomerase reverse transcriptase (hTERT) response in RA patients and non-RA controls. The expression of the catalytic subunit of telomerase, hTERT, was measured in peripheral blood mononuclear cells (PBMC) of RA patients and controls after in vitro stimulation with anti-CD3 monoclonal antibody (mAb) using real-time PCR. Anti-CD3 mAb stimulation induced activation and proliferation of the T cells in all populations studied. In early RA patients with a disease duration of less than 1 year, the activation-induced hTERT mRNA levels were found to be reduced as compared to healthy controls (HC). Chronic RA patients, with a disease duration of more than 1 year, did not show these impaired hTERT mRNA levels after stimulation with anti-CD3 mAb. Decreased hTERT mRNA levels were also found in multiple sclerosis patients and patients suffering from flu-like symptoms, indicating that these deviations are not disease-specific. The impaired activation-induced hTERT response in PBMC may be a general response of the immune cells in cases of acute or chronic immune activation, presumably to control unwanted clonal expansions and to maintain the diversity of the TCR repertoire. Our results also indicate that clonal T cell expansions, described in RA, are probably not mediated by an elevated potency to express hTERT.

Premature immunosenescence in rheumatoid arthritis and multiple sclerosis patients.

Patients with T-cell-mediated autoimmune diseases show immune system abnormalities that resemble the typical characteristics of autoimmune dysfunction described in the elderly. In addition, the incidence of autoimmune disease increases with advancing age. To evaluate whether patients with rheumatoid arthritis (RA) and multiple sclerosis (MS) have premature immuno-senescence, we measured two indicators of aging: the number of T-cell-receptor excision circles (TRECs) and the percentage of CD4+CD28(null) T cells. We studied them in the peripheral blood mononuclear cells (PBMCs) of 60 RA patients, 32 MS patients, and 40 healthy controls (HCs). We found that TREC numbers were lower in RA and MS patients than in age-matched HCs, indicating premature thymic involution. Moreover, a subset of these patients contained age-inappropriate high frequencies of CD4+CD28(null) T cells. This study provides evidence of premature immune system senescence in both RA and MS patients. Premature aging could be a risk factor for developing autoimmune disorders in genetically predisposed individuals in a susceptible environment.

Th17 expansion in granulomatosis with polyangiitis (Wegener's): the role of disease activity, immune regulation and therapy.

INTRODUCTION: In autoimmune diseases, IL-17 producing T-cells (Th17), a pro-inflammatory subset of T-cells, are pathophysiologically involved. There is little knowledge on the role of Th17 cells in granulomatosis with polyangiitis (GPA). In the present study, we investigated Th17 cells, Tregs and subsets of circulating Th17 cells in GPA and related results to disease activity. METHODS: 42 GPA patients in remission, 18 with active disease and 14 healthy controls (HC) were enrolled. Th17 cells, their subsets and regulatory T-cells were determined by intracellular fluorescence activated cell sorter (FACS). Data are given as mean percentage ±SD of total T-helper-cells. RESULTS: Th17 cells are expanded in active and quiescent GPA as compared to HC (1.7±1.4% vs. 0.7 ±0.3%, P = 0.006 and 1.9 ±1.5% vs. 0.7 ±0.3%, P<0.0001). Th17 expansion is stable over time and does not decline when remission is achieved. However, a negative association of Th17 cells and steroid dosage is observed (r=-0.46, P = 0.002). The Th17 expansion was not balanced by Tregs as indicated by skewed Th17/Treg ratios in active and quiescent GPA. Th17 subsets co-producing IFNγ or IL-10 are significantly increased in GPA. GPA patients in remission not receiving maintenance therapy have significantly more IL-10/IL-17A double positive T-cells than HC (0.0501 ±0.031% vs. 0.0282 ±0.016%, P = 0.007). CONCLUSIONS: We provide evidence for a persistent, unbalanced expansion of Th17 cells and Th17 subsets which seems to be independent of disease activity. Maintenance therapy reduces -but does not normalize- Th17 expansion.

Expression of NLRP3 inflammasome and T cell population markers in adipose tissue are associated with insulin resistance and impaired glucose metabolism in humans.

Recent studies in rodents indicate that the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome and a proinflammatory shift in the T cell population in adipose tissue (AT) contribute to AT inflammation and insulin resistance. We investigated: (1) the interplay between the NLRP3 inflammasome and T cell populations in abdominal subcutaneous AT in obese and lean humans in relation to AT inflammatory processes, and (2) involvement of the NLRP3 inflammasome and T cell populations in insulin resistance. Abdominal subcutaneous AT biopsies were collected in 10 obese men with impaired glucose tolerance and 9 lean normal glucose tolerant age-matched controls. AT gene expression of NLRP3 inflammasome-related genes and markers of T cell populations, chemoattraction, macrophage infiltration and other aspects of inflammation were examined. Furthermore, we examined systemic adaptive immune activation and insulin sensitivity (hyperinsulinemic-euglycemic clamp). CASPASE-1 mRNA and the proportion of T(h)1 transcripts (TBX21/CD3ɛ) were significantly higher in AT from obese compared with lean subjects. CASPASE-1 expression and a relative increase in T(h)1 transcripts in AT were strongly associated with insulin resistance and impairments in glucose homeostasis. Gene expression of NLRP3, CASPASE-1, CD3ɛ (pan T cells), TBX21 (T(h)1 cells) and RORC (T(h)17 cells) was positively, whereas GATA3 (T(h)2 cells) was inversely correlated with AT inflammation. Our data suggest that NLRP3 inflammasome activation and a T(h)1 shift in the T cell population in AT of obese subjects is related to insulin resistance and impaired glucose metabolism, which may be explained by AT inflammatory processes.

Intracellular IL-10 detection in T cells by flowcytometry: the use of protein transport inhibitors revisited.

In the past two decades, interleukin-10 (IL-10) has gained much attention as an important regulatory cytokine involved in self-tolerance. Functional assessment of IL-10 producing immune cells is traditionally done by stimulation and measurement of cytokine production by flowcytometry. Thereby a protein transport inhibitor like monensin is used to accumulate the cytokine of interest intracellularly. In this study we elaborated on the monensin effect on cytokine detection and focused on IL-10 detection in human T cells. Peripheral blood mononuclear cells (PBMC) of 32 study subjects were isolated and stimulated with PMA/ionomycin, in the absence and presence of monensin, and stained intracellularly for IFN-γ, IL-4, IL-10 and IL-17A. Our results re-established that detection of IFN-γ+ and IL-4+ T cells benefited from the presence of monensin during stimulation. However, stimulation in the presence of monensin yielded lower proportions of IL-10+ T cells (0.45% (0.28-0.80) versus 0.80% (0.50-1.50) of CD4+ T cells, p<0.01), although monensin addition did result in an increased MFI (2431 (1273-4959) versus 1928 (1147-3760), p<0.01). Detectable fractions of IL-17A+ CD4+ T cells were not affected by monensin. A shorter incubation time, but not lower monensin concentrations, was effective in improving the detection of IL-10+ T cells. We found a strong correlation between the fraction of IL-10+ CD4+ T cells in the presence and absence of monensin (R=0.80 p<0.01). Next to this, also the detection of IL-10+ NK-T cells and IL-10+ monocytes, but not IL-10+ B cells, is impaired in the presence of monensin. This study shows that the effect of monensin on cytokine accumulation is time and cytokine dependent. Due to the use of monensin, previous research may have underestimated the number of IL-10+ leukocytes or may even have not been able to detect them at all. It is important to consider this for future research or when interpreting historical IL-10 data.

Neutrophils and T cells: bidirectional effects and functional interferences.

Polymorphonuclear cells (PMN) are widely recognized as sophisticated killers during microbial infections. In recent years, PMN have been shown to interact and functionally interfere with other cells of the immune system. In this study, we investigated PMN-T cell interactions in an in vitro co-culture system. A relative increase in T cells in the co-culture was associated with the upregulation of CD66b expression on PMN. In addition, PMN were found to dose-dependently impair anti-CD3 induced CD4(+) T cell activation, proliferation and viability. In a transwell co-culture system, proliferation of T cells was, however, enhanced which illustrates that suppression was contact-dependent. The addition of an arginase-inhibitor or blocking antibodies against calprotectin, but not myeloperoxidase (MPO), partially restored T cell proliferation. Furthermore, the presence of PMN in the co-culture dose-dependently increased the fraction of IFN-γ and IL-17 producing T cells and decreased the percentage of IL-10 producing CD4(+) T cells. Altogether, these data show that there is cross-talk between PMN and T cells which, in non-inflammatory conditions, results in the effects described above. Further studies should investigate PMN-T cell functional interference in inflammatory situations and clarify the importance of this PMN-T cell cross-talk in the regulation of the immune response.