Validated programmed cell death ligand 1 immunohistochemistry assays (E1L3N and SP142) reveal similar immune cell staining patterns in melanoma when using the same sensitive detection system.

AIMS: Tumour cell and/or immune cell programmed cell death ligand 1 (PD-L1) expression is considered as a potential biomarker for anti-PD1 and anti-PD-L1 immunotherapy. Currently, different PD-L1 assays are used. This study aims to compare the staining patterns of two PD-L1 antibody clones in melanoma metastases and correlate them with PD-L1 mRNA expression. METHODS AND RESULTS: The immunohistochemistry assays were optimized and validated independently on a Ventana Benchmark Ultra (Ventana Medical Systems Inc., Tucson, AZ, USA) (E1L3N) and XT (SP142), using the same detection system. In total, 46 melanoma metastases were stained with both validated immunohistochemistry assays. Stained slides were digitized for qualitative and semi-quantitative evaluation; done by pathologist and semi-automated software analysis. A subset of 21 melanoma metastases was selected for quantification of the PD-L1 mRNA expression. Accuracy and precision criteria were met for both assays. PD-L1 protein and mRNA expression showed remarkably good Spearman's coefficients of 0.90 (E1L3N) and 0.87 (SP142). Despite the remarkable correlation between both PD-L1 assays in expression patterns and quantification values (ρ > 0.90), E1L3N showed significantly more tumour cell staining than SP142. CONCLUSIONS: E1L3N and SP142 IHC assays were optimized and validated successfully and independently for sensitive and accurate PD-L1 detection. Concordance was best for immune cell scoring, while E1L3N tended to detect more tumour cells. Determination of the clinically relevant cut-off values for immune cell versus tumour cell detection requires further research.

Apoptotic cell death and efferocytosis in atherosclerosis.

Apoptotic cell death is an important feature of atherosclerotic plaques, and it seems to exert both beneficial and detrimental effects depending on the cell type and plaque stage. Because late apoptotic cells can launch proatherogenic inflammatory responses, adequate engulfment of apoptotic cells (efferocytosis) by macrophages is important to withstand atherosclerosis progression. Several efferocytosis systems, composed of different phagocytic receptors, apoptotic ligands, and bridging molecules, can be distinguished. Because phagocytes in atherosclerotic plaques are very much solicited, a fully operative efferocytosis system seems to be an absolute requisite. Indeed, recent studies demonstrate that deletion of just 1 of the efferocytosis pathways aggravates atherosclerosis. This review discusses the role of apoptosis in atherosclerosis and general mechanisms of efferocytosis, to end with indirect and direct indications of the significance of effective efferocytosis in atherosclerosis.

Decreased numbers of peripheral blood dendritic cells in patients with coronary artery disease are associated with diminished plasma Flt3 ligand levels and impaired plasmacytoid dendritic cell function.

We investigated whether activation of circulating DCs (dendritic cells) or levels of Flt3L (FMS-like tyrosine kinase 3 ligand) and GM-CSF (granulocyte/macrophage colony-stimulating factor), haematopoietic growth factors important for DC differentiation, could account for reduced blood DC numbers in CAD (coronary artery disease) patients. Concentrations of Flt3L and GM-CSF were measured in plasma from CAD patients (n = 15) and controls (n = 12). Frequency and phenotype of mDCs (myeloid dendritic cells) and pDCs (plasmacytoid dendritic cells) were analysed by multicolour flow cytometry in fresh blood, and after overnight incubation with TLR (Toll-like receptor)-4 or -7 ligands LPS (lipopolysaccharide) or IQ (imiquimod). DC function was measured by IL (interleukin)-12 and IFN (interferon)-α secretion. Circulating numbers of CD11c+ mDCs and CD123+ pDCs and frequencies of CD86+ and CCR-7+ (CC chemokine receptor type 7) mDCs, but not pDCs, were declined in CAD. In addition, plasma Flt3L, but not GM-CSF, was lower in patients and positively correlated with blood DC counts. In response to LPS, mDCs up-regulated CD83 and CD86, but CCR-7 expression and IL-12 secretion remained unchanged, similarly in patients and controls. Conversely, pDCs from patients had lower CD83 and CCR-7 expression after overnight incubation and had a weaker IQ-induced up-regulation of CD83 and IFN-α secretion. In conclusion, our results suggest that reduced blood DC counts in CAD are, at least partly, due to impaired DC differentiation from bone marrow progenitors. Decreased levels of mDCs are presumably also explained by activation and subsequent migration to atherosclerotic plaques or lymph nodes. Although mDCs are functioning normally, pDCs from patients appeared to be both numerically and functionally impaired.

Dendritic cells in human atherosclerosis: from circulation to atherosclerotic plaques.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease with atherosclerotic plaques containing inflammatory infiltrates predominantly consisting of monocytes/macrophages and activated T cells. More recent is the implication of dendritic cells (DCs) in the disease. Since DCs were demonstrated in human arteries in 1995, numerous studies in humans suggest a role for these professional antigen-presenting cells in atherosclerosis. AIM: This paper focuses on the observations made in blood and arteries of patients with atherosclerosis. In principal, flow cytometric analyses show that circulating myeloid (m) and plasmacytoid (p) DCs are diminished in coronary artery disease, while immunohistochemical studies describe increased intimal DC counts with evolving plaque stages. Moreover, mDCs and pDCs appear to behave differently in atherosclerosis. Yet, the origin of plaque DCs and their relationship with blood DCs are unknown. Therefore, several explanations for the observed changes are postulated. In addition, the technical challenges and discrepancies in the research field are discussed. FUTURE: Future studies in humans, in combination with experimental animal studies will unravel mechanisms leading to altered blood and plaque DCs in atherosclerosis. As DCs are crucial for inducing but also dampening immune responses, understanding their life cycle, trafficking and function in atherosclerosis will determine potential use of DCs in antiatherogenic therapies.

Human C-reactive protein activates monocyte-derived dendritic cells and induces dendritic cell-mediated T-cell activation.

OBJECTIVE: Recent studies proposed a pathogenic role for C-reactive protein (CRP), an independent predictor of cardiovascular disease (CVD), in atherosclerosis. Therefore, we tested whether CRP may modulate dendritic cell (DC) function, because these professional antigen-presenting cells have been implicated in atherogenesis. METHODS AND RESULTS: Human monocyte-derived immature DCs were cultured with human CRP (0 to 60 microg/mL) for 24 hours. Thereafter, activation markers were measured by flow-cytometry and DCs were cocultured with CFSE-labeled lymphocytes to measure T-cell proliferation and interferon (IFN)-gamma secretion after 8 days. Exposure to 60 microg/mL CRP (n=5) induced an activated cell morphology and significant (CD40 increase MFI 5.23+/-0.28, P<0.01 paired t test; CD80 6.18+/-0.51, P<0.01) to modest (CD83 1.38+/-0.17, P<0.05, CCR7 1.60+/-0.29, P=0.05) upregulation of DC activation markers. The expression of CD86 and HLA-DR was high, but not affected. T-lymphocytes incubated with CRP-pulsed DCs displayed increased IFN-gamma secretion and proliferation (P<0.001). DC activation was concentration-dependent and detected from 2 mug/mL CRP; the maximum effect was equivalent to that seen with 0.1 microg/mL lipopolysaccharide (LPS). Polymyxin B abolished the LPS response, without influencing CRP effects. Finally, immunohistochemistry could demonstrate DC/CRP colocalization in human atherosclerotic lesions. CONCLUSIONS: These findings suggest that CRP in plaques or found circulating in CVD patients can influence DC function during atherogenesis.

Optimal Evaluation of Programmed Death Ligand-1 on Tumor Cells Versus Immune Cells Requires Different Detection Methods.

CONTEXT: - The benefit of programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) as a method to select patients who may benefit from programmed death receptor-1 (PD-1)/PD-L1 immunotherapies remains uncertain in many tumor indications. OBJECTIVES: - To compare the commercially available, approved PD-L1 IHC assays (22C3, 28-8, SP142, SP263), specifically identifying the changes in staining output created by altering the detection method. DESIGN: - This pilot study investigates the respective PD-L1 kit assay staining patterns and related scoring of tumor cells and immune cells on lung carcinoma and melanoma. Furthermore, the influence of the detection method (platform and related reagents) on PD-L1 antibody performance is studied. RESULTS: - The SP142 kit reveals more immune cell staining but less tumor cell staining than the other PD-L1 kits. Alternatively, the 22C3 and 28-8 kits show good tumor cell sensitivity, but less pronounced immune cell staining, even in tonsil. Tumor cell staining by the SP263 kit is comparable to that of 22C3 and 28-8 kits, while immune cell staining is better. Strikingly, the selection of the detection method has a major impact on the sensitivity of the assay for PD-L1 detection per cell type. Switching the detection method of the kits could largely circumvent the observed staining differences. CONCLUSIONS: - The diverse sensitivities caused by the choice of the detection method should be taken into consideration when selecting PD-L1 kits or developing PD-L1 IHC laboratory-developed tests. When using alternative kits or laboratory-developed tests, it is strongly recommended to reestablish their clinical utility per therapeutic agent or compare them with the original kit.

Stereology: a simplified and more time-efficient method than planimetry for the quantitative analysis of vascular structures in different models of intimal thickening.

OBJECTIVE: Semiautomatic computer-assisted planimetry is currently the standard morphometric technique in models of intimal thickening. We evaluated stereological point counting as an alternative method for the measurement of cross-sectional vascular areas (lumen, intima, and media) by comparing precision, efficiency, and variance components. METHODS: Sections from murine atherosclerotic aorta (n = 21), stented rabbit iliac arteries (n = 30), and porcine coronary arteries (n = 30) were analyzed at two institutes using both techniques. To determine reproducibility, porcine arteries were measured twice. RESULTS: Area measurements showed almost identical means and standard deviations for planimetry and stereology [e.g., intima (mm2): 0.10+/-0.11 vs. 0.12+/-0.12 (mouse), 0.60+/-0.16 vs. 0.60+/-0.15 (rabbit), and 1.92+/-1.52 vs. 1.97+/-1.61 (pig)]. Deming regression and Bland-Altman plots demonstrated a good agreement between both techniques that was not influenced by artery size. Both methods exhibited excellent repeatability, although planimetry (-0.18+/-0.27) was more precise than stereology (-0.17+/-0.47; variance, P < .01, Levene test). In addition, intraoperator variance (error inherent to the technique) was greater for stereology (1.6-15.8% vs. 4.8-33.5%), whereas interoperator variance (error between institutes) was very small for both methods (0.1-0.9% vs. 0.1-1.7%). Indeed, biologic variability was, by far, the most important variance component in all measurements (84-98% vs. 65-95%). Finally, stereology required 20% (mouse; P < .05) to 40% (pig; P < .001) less time to complete analysis. CONCLUSION: The quantification of vascular structures by planimetry and stereology yielded comparable results in all models of intimal thickening, but stereology proved to be less time-consuming. Therefore, study design may dictate the most appropriate choice of technique.

Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease.

BACKGROUND: Dendritic cells are potent antigen-presenting and immune modulating cells that have been implicated in the development of atherosclerosis. In human blood, two distinct lineages are distinguished: plasmacytoid dendritic cells and myeloid dendritic cells. Although dendritic cells have been described in atherosclerotic plaques, no information exists concerning circulating blood dendritic cells in atherosclerosis. This study aims to evaluate the number of circulating dendritic cells in patients with coronary artery disease. The relation with the extent of coronary artery disease, the clinical syndrome and with a marker of inflammation will be documented. METHODS: Patients with angiographically proven coronary artery disease (n=18) and age and sex-matched controls (n=18) were included. Myeloid dendritic cells and plasmacytoid dendritic cells were detected with the specific blood dendritic cell antigens, blood dendritic cell antigen-1 and blood dendritic cell antigen-2, respectively. RESULTS: Absolute and relative numbers of circulating plasmacytoid dendritic cells were significantly lower in patients with coronary artery disease (5722+/-601/ml and 0.08+/-0.01%) than in controls (12,640+/-1289/ml and 0.21+/-0.02%). Plasmacytoid dendritic cells were more decreased in patients with troponin-positive unstable coronary syndromes than in patients with low troponin values, and tended to be lower in more extensive coronary artery disease. Absolute myeloid dendritic cells numbers tended to be reduced in patients, whereas relative numbers were significantly decreased: 11,857+/-1895/ml versus 15,226+/-928/ml and 0.17+/-0.03% versus 0.26+/-0.01% in controls. CONCLUSIONS: The present study shows a significant decrease of circulating blood dendritic cell antigen-2 positive plasmacytoid dendritic cells in patients with coronary artery disease. The decrease tended to be more pronounced in unstable coronary syndromes and extensive coronary artery disease, suggesting a possible role of dendritic cells in plaque progression and rupture.

Potential use of dendritic cells for anti-atherosclerotic therapy.

The chronic inflammatory nature of atherosclerosis is nowadays widely accepted. Dendritic cells (DCs) are likely to play a crucial role in directing innate and adaptive immunity against altered (self-)antigens, such as oxidized low density lipoproteins (oxLDL). DCs are found in early lesions and their numbers become even higher when the lesion progresses. DCs are most abundant in areas of neovascularization where they are often found near T cells. All stages from precursors to fully mature DCs are present in human plaques. Treatment of atherosclerosis is currently based on reducing risk factors, e.g. by use of statins and beta-blockers. Some of these pharmacological agents also show anti-inflammatory properties and consequently can affect DC function. Yet, many patients remain at risk for acute coronary events, and new therapies to treat atherosclerosis are needed. One therapeutic strategy is based on isolation of patient's DCs that are then pulsed with appropriate antigen(s) ex vivo, e.g. (immunogenic components of) oxLDL or total extract of atherosclerotic plaque tissue, and returned to the blood stream. Other approaches to ensure immune protection include generation of tolerogenic DCs, or using DCs to deplete detrimental Th1 or Th17 cells. However, the future lies in direct targeting of DCs by manipulating functions of different DC subsets. Therefore, it would be useful to isolate plaque-resident DCs to be able to identify unique antigen(s) on their surface. The challenge is to selectively identify regulatory molecules and novel therapies to inhibit DC migration and function during atherogenesis, without affecting normal DC function under physiological conditions.

MFGE8 inhibits inflammasome-induced IL-1ß production and limits postischemic cerebral injury.

Milk fat globule-EGF 8 (MFGE8) plays important, nonredundant roles in several biological processes, including apoptotic cell clearance, angiogenesis, and adaptive immunity. Several recent studies have reported a potential role for MFGE8 in regulation of the innate immune response; however, the precise mechanisms underlying this role are poorly understood. Here, we show that MFGE8 is an endogenous inhibitor of inflammasome-induced IL-1ß production. MFGE8 inhibited necrotic cell-induced and ATP-dependent IL-1ß production by macrophages through mediation of integrin ß(3) and P2X7 receptor interactions in primed cells. Itgb3 deficiency in macrophages abrogated the inhibitory effect of MFGE8 on ATP-induced IL-1ß production. In a setting of postischemic cerebral injury in mice, MFGE8 deficiency was associated with enhanced IL-1ß production and larger infarct size; the latter was abolished after treatment with IL-1 receptor antagonist. MFGE8 supplementation significantly dampened caspase-1 activation and IL-1ß production and reduced infarct size in wild-type mice, but did not limit cerebral necrosis in Il1b-, Itgb3-, or P2rx7-deficient animals. In conclusion, we demonstrated that MFGE8 regulates innate immunity through inhibition of inflammasome-induced IL-1ß production.

Humoral and cellular immune responses in atherosclerosis: spotlight on B- and T-cells.

Despite more than 1 million basic and clinical investigation reports on the mechanism and clinical outcome of cardiovascular events, the pathogenesis of this multi factorial disease is still incompletely understood, which is illustrated by the fact that it is still the leading cause of death in the western world. Over the decades it has been well approved that in addition to lipid dysfunction and arterial lipid accumulation, inflammation and autoimmune responses are major factors in directing the initiation and progression of atherosclerosis, the underlying cause of cardiovascular diseases. Atherosclerosis involves both humoral and cellular compartments of innate and adaptive immunity making it a very complex disease. This review discusses the innate and adaptive immune responses in atherosclerosis, with a focus on T- and B-cell mediated processes.

Immunohistochemical characterisation of dendritic cells in human atherosclerotic lesions: possible pitfalls.

BACKGROUND: Previously we demonstrated decreased blood myeloid (m) and plasmacytoid (p) dendritic cell (DC) counts in atherosclerotic patients. Therefore, we examined whether DCs, in particular DC precursors, accumulate in human plaques. METHODS: Blood DC antigen (BDCA)-1, CD11c (mDCs), BDCA-2, CD123 (pDCs), langerin, fascin, S-100 (immature/mature DCs), and CD1a and CD83 (mature DCs) were investigated by immunohistochemistry of carotid arteries obtained by endarterectomy (EAS, frozen n = 11, fixed n = 11) or autopsy (fixed, n = 87). RESULTS: Fascin and S-100 required formaldehyde fixation, other markers needed cryo-preservation. BDCA-1, BDCA-2, langerin, and S-100 appeared specific for intimal DCs, unlike CD123 and fascin (staining endothelial cells), CD11c and CD1a (staining monocytes, foam cells) or CD83 (staining lymphocytes). BDCA-1 and BDCA-2 cells were detected in EAS, preferentially near microvessels. S-100 cells increased successively from intimal thickening, via pathological intimal thickening, fibrous cap atheroma and finally complicated plaques. Fascin cells followed the same pattern, but were more abundant. However, in lesions containing microvessels (complicated plaques, plaque shoulders and most EAS) this was partly explained by fascin positive endothelial cells. Even complicated plaques contained relatively few mature CD83 DCs. CONCLUSIONS: Accumulation of BDCA-1 and BDCA-2 around neovessels showed that mDCs and pDCs are recruited to advanced plaques, which is in line with the previously described decline of circulating blood DCs in patients with coronary artery disease. Unexpectedly, several DC markers yielded false positive signals. Hence, some accounts on numbers, trafficking and activation of DCs in atherosclerotic plaques may require re-evaluation.

Expression of dendritic cell markers CD11c/BDCA-1 and CD123/BDCA-2 in coronary artery disease upon activation in whole blood.

OBJECTIVES: Previous in vivo studies on dendritic cell (DC) enumeration in coronary artery disease (CAD) were not always consistent. Therefore, we investigated by flow cytometry whether this was due to CAD-related differences in expression of subset markers for myeloid (m)DCs (blood DC antigen (BDCA)-1, CD11c) and plasmacytoid (p)DCs (BDCA-2, CD123), before and after in vitro stimulation with Toll-like receptor ligands. RESULTS: Our data showed that circulating DCs decline in CAD, irrespective of the DC subset marker that was used for enumeration. Upon in vitro activation, BDCA-2 was downregulated, whereas CD11c and CD123 were upregulated. This implies that the expression ratios CD11c/BDCA-1 and CD123/BDCA-2 can assess DC activation. Comparing these ratios between controls and CAD patients showed no differences in blood DC activation in both groups. CONCLUSIONS: This study suggests that when different DC numbers are found between two study populations, the DC activation status from both groups always needs to be verified, since a decrease in BDCA-2(+) pDCs or an increase in CD11c(+) mDCs or CD123(+) pDCs can be due to the altered expression of these markers during activation. Given that CD11c, BDCA-1, CD123 and BDCA-2 are more abundantly expressed on blood DCs than typical activation markers like CD83, CD86 or CCR-7, the use of the ratios is an easy and reliable way to determine DC activation in whole blood assays.

Changes in blood dendritic cell counts in relation to type of coronary artery disease and brachial endothelial cell function.

BACKGROUND: Recently we reported a decline of circulating myeloid (m) and plasmacytoid (p) dendritic cells (DCs) in patients with coronary artery disease (CAD). This study also determined the total blood DC numbers and focused on effects of extent (one vs. three-vessel disease) and type (stable vs. unstable) of CAD, and on endothelial cell function. METHODS: Patients undergoing diagnostic coronarography were enrolled in four groups: control patients (atypical chest pain, <50% narrowing, n=15), stable one-vessel (n=15), stable three-vessel (n=15), and unstable one-vessel CAD (n=16). Total blood DCs were identified as lineage (lin) and HLADR, and DC subtypes with blood DC antigen (BDCA)-1 for mDCs and BDCA-2 for pDCs. Flow-mediated dilatation (FMD) was measured in the brachial artery. RESULTS: Numbers of total blood DCs, mDCs and pDCs declined in CAD patients compared with control patients, but without differences between the CAD groups. Interleukin-6 and high sensitivity C-reactive protein displayed inverse associations with mDCs. A FMD below the median of the study population, use of beta-blockers or of lipid-lowering drugs was associated with increased mDCs, whereas pDCs were similar. Interestingly, the effects of drugs and FMD were additive with that of CAD. CONCLUSION: This study indicates that lower blood DCs do not result from medication intake or endothelial dysfunction, and are an overall systemic effect of atherosclerosis rather than CAD type (stable or unstable) or number of stenotic coronary arteries. In view of discrete associations with cytokines, FMD, beta-blockers and statins, mDCs and pDCs seem to behave differently and may influence inflammation during atherosclerosis in different ways.

B cell depletion reduces the development of atherosclerosis in mice.

B cell depletion significantly reduces the burden of several immune-mediated diseases. However, B cell activation has been until now associated with a protection against atherosclerosis, suggesting that B cell-depleting therapies would enhance cardiovascular risk. We unexpectedly show that mature B cell depletion using a CD20-specific monoclonal antibody induces a significant reduction of atherosclerosis in various mouse models of the disease. This treatment preserves the production of natural and potentially protective anti-oxidized low-density lipoprotein (oxLDL) IgM autoantibodies over IgG type anti-oxLDL antibodies, and markedly reduces pathogenic T cell activation. B cell depletion diminished T cell-derived IFN-gamma secretion and enhanced production of IL-17; neutralization of the latter abrogated CD20 antibody-mediated atheroprotection. These results challenge the current paradigm that B cell activation plays an overall protective role in atherogenesis and identify new antiatherogenic strategies based on B cell modulation.