Analysis of combinatorial chemokine receptor expression dynamics using multi-receptor reporter mice

Inflammatory chemokines and their receptors are central to the development of inflammatory/immune pathologies. The apparent complexity of this system, coupled with lack of appropriate in vivo models, has limited our understanding of how chemokines orchestrate inflammatory responses and has hampered attempts at targeting this system in inflammatory disease. Novel approaches are therefore needed to provide crucial biological, and therapeutic, insights into the chemokine-chemokine receptor family. Here, we report the generation of transgenic multi-chemokine receptor reporter mice in which spectrally-distinct fluorescent reporters mark expression of CCRs 1, 2, 3 and 5, key receptors for myeloid cell recruitment in inflammation. Analysis of these animals has allowed us to define, for the first time, individual and combinatorial receptor expression patterns on myeloid cells in resting and inflamed conditions. Our results demonstrate that chemokine receptor expression is highly specific, and more selective than previously anticipated.

Altered circulating CCR6+and CXCR3+ T cell subsets are associated with poor renal prognosis in MPO-ANCA-associated vasculitis

Background Effector memory T cells are pivotal effectors of adaptive immunity with enhanced migration characteristics and are involved in the pathogenesis of ANCA-associated vasculitis (AAV). The diversity of effector memory T cells in chemokine receptor expression has been well studied in proteinase 3 (PR3)-AAV. However, few studies have been conducted in myeloperoxidase (MPO)-AAV. Here, we characterized chemokine receptor expression on effector memory T cells from patients with active MPO-AAV. Methods Clinical data from newly diagnosed MPO-AAV patients and healthy subjects were collected and analyzed. Human peripheral blood mononuclear cells (PBMCs) isolated from patients with active MPO-AAV were analyzed by flow cytometry. The production of effector memory T cell-related chemokines in serum was assessed by ELISA. Results We observed decreased percentages of CD4+ and CD8+ T cells in the peripheral blood, accompanied by a significant decrease in CCR6-expressing T cells but an increase in CXCR3+ T cells, in active MPO-AAV. Furthermore, the decrease in CCR6 and increase in CXCR3 expression were mainly limited to effector memory T cells. Consistent with this finding, the serum level of CCL20 was increased. In addition, a decreasing trend in the TEM17 cell frequency, with concomitant increases in the frequencies of CD4+ TEM1 and CD4+ TEM17.1 cells, was observed when T cell functional subsets were defined by chemokine receptor expression. Moreover, the proportions of peripheral CD8+ T cells and CD4+ TEM subsets were correlated with renal prognosis and inflammatory markers. Conclusions Our data indicate that dysregulated chemokine receptor expression on CD4+ and CD8+ effector memory T cells and aberrant distribution of functional CD4+ T cell subsets in patients with active MPO-AAV have critical roles related to kidney survival.

Chemokine receptor expression on T-cells in the blood of patients with normal body weight or obesity before abdominoplasty

Целью работы было определение уровня экспрессии хемокиновых рецепторов на лимфоцитах периферической крови пациентов при проведении операции абдоминопластики, а также сравнение этих показателей у пациентов с ожирением и с нормальной массой тела. Методы. В работе проанализированы данные 92 женщин в возрасте от 18 до 56 лет, которым в клинике пластической и эстетической хирургии «Фрау Клиник 1» проведена операция абдоминопластики. Кровь брали натощак перед операцией, мононуклеары выделяли на градиенте плотности, клетки окрашивали моноклоанльными антителами, конъюгированными с флуоресцентными красителями. Интенсивность флуоресценции измеряли на проточном цитометре FACSCalibur по программе SimulSet. Статистический анализ проводили по программе «Биостатистика». Результаты. Было показано, что у больных с ожирением в периферической крови достоверно повышен уровень CD4+ Т-клеток, Т-хелперов первого порядка (Th1), регуляторных Т-клеток (Treg), наивных Т-клеток и Т-клеток памяти. Экспрессия хемокиновых рецепторов CXCR4, CCR6, CCR7, CCR9 достоверно повышена на субпопуляции CD4+ Т-лимфоцитов при ожирении. Также было установлено, что ожирение ассоциировано с повышением хемокиновых рецепторов CCR5, CX3CR1, CXCR3 на CD8+ цитотоксических лимфоцитах, что отражает их способность отвечать на регуляторные хемокины и аккумулироваться в жировой ткани. Заключение. Полученные результаты свидетельствуют об изменении в системе регуляции хемокинов и их рецепторов при ожирении, что является патогенетической основой для развития локального воспаления в жировой ткани и может оказывать негативное влияние на развитие осложнений после операции абдоминопластики. The aim of this study was to determine the level of chemokine receptor expression on peripheral blood lymphocytes of patients undergoing abdominoplasty and to compare these data in obese patients and with data in patients with normal body weight. Methods. We analyzed the data of 92 women aged 18 to 56 years who underwent abdominoplasty surgery at the Plastic and Aesthetic Surgery “Frau Klinik 1”. Blood was collected during fasting before surgery. Mononuclear cells were isolated on a density gradient and stained with monoclonal antibodies conjugated with fluorescent dyes. Fluorescence intensity was measured on a FACSCalibur flow cytometer using the SimulSet program. Data were analyzed statistically with Biostatistics software. Results. In obese patients, the peripheral blood levels of CD4+ T-cells, T-helper cells 1 (Th1), regulatory T-cells (Treg), naive T-cells, and memory T-cells were significantly increased (p < 0.05). Also in obese patients, expression of chemokine receptors CXCR4, CCR6, CCR7, and CCR9 was elevated on the CD4+ T-lymphocyte subpopulation. It was found that obesity is associated with an increased expression of the chemokine receptors CCR5, CX3CR1, and CXCR3 on CD8+ cytotoxic lymphocytes, which reflects their ability to respond to regulatory chemokines and to be accumulated in the adipose tissue. Conclusion. The results indicate a change in regulation of chemokines and their receptors in obesity. This is the pathogenetic basis for developing local inflammation in adipose tissue, and this regulatory change may facilitate the development of complications following abdominoplasty surgery.

SLAMF7 and IL-6R define distinct cytotoxic versus helper memory CD8+ T cells

The prevailing ‘division of labor’ concept in cellular immunity is that CD8+ T cells primarily utilize cytotoxic functions to kill target cells, while CD4+ T cells exert helper/inducer functions. Multiple subsets of CD4+ memory T cells have been characterized by distinct chemokine receptor expression. Here, we demonstrate that analogous CD8+ memory T-cell subsets exist, characterized by identical chemokine receptor expression signatures and controlled by similar generic programs. Among them, Tc2, Tc17 and Tc22 cells, in contrast to Tc1 and Tc17 + 1 cells, express IL-6R but not SLAMF7, completely lack cytotoxicity and instead display helper functions including CD40L expression. CD8+ helper T cells exhibit a unique TCR repertoire, express genes related to skin resident memory T cells (TRM) and are altered in the inflammatory skin disease psoriasis. Our findings reveal that the conventional view of CD4+ and CD8+ T cell capabilities and functions in human health and disease needs to be revised.

Innate-like Chemokine Receptor Profile and Migratory Behaviour By Terminally Differentiated and Educated NK Cells

Natural Killer (NK) cells play an essential role in cancer surveillance and have a unique capability of spontaneous cytotoxicity against cancer cells. The human NK cell repertoire is functionally diversified through a tightly regulated differentiation process characterized by an early transition from CD56bright to CD56dim NK cells, followed by coordinated changes in expression of inhibitory receptors, including NKG2A and killer cell immunoglobulin-like receptors (KIR). The acquisition of self HLA class I binding KIRs during NK cell differentiation tunes the cytotoxic potential of NK cells in a process termed education, characterized by increased loading of granzyme B in dense core granules. Although NK cell differentiation and education are critical determinants of the functional potential of the cell, little is known about how these events shape the migratory behavior of NK cells. To mediate appropriate and directed immune response against cancer, NK cells must be capable of migration to the tumor site. This process is mediated by chemokines, which guide cell migration by binding to their specific receptors. For example, in multiple myeloma, CXCR3 and CCR5 ligands (MIG, IP-10, and MIP-1a) are significantly upregulated in the bone marrow compared to healthy controls, affecting the composition of immune cells in the tumor microenvironment. In order to delineate the homing patterns of distinct NK cell subsets, we used high-dimensional flow cytometry combined with functional assays to map the NK cell chemokine receptor expression and migratory behavior. We screened resting and cytokine/feeder cell stimulated peripheral blood NK cells for the expression of a panel of 20 chemokine receptors (A). Based on CD56, CD57, NKG2A, and KIR expression, NK cells were divided into 6 phenotypically and functionally distinct subsets that were ordered according to their differentiation status (B). We found that the expression of CX3CR1, CXCR1, CXCR2, and CMKLR1 gradually increased during differentiation, whereas the expression of CXCR3, CCR7, and CCR5 was lower in more differentiated NK cells. CXCR4, CCR4, and CCR2 expression was relatively uniform across all subsets. Interestingly, CCR1 and CXCR6 were expressed mainly on less differentiated NKG2A+ CD56dim NK cells (B). Next, we stratified the chemokine receptor expression on mature KIR+ NK cells based on the expression of self (educated) or non-self KIR (uneducated). Educated NK cells expressed CXCR1, CX3CR1, CCR5, and CMKLR1 at higher levels than the uneducated NK cells. Conversely, CXCR3 was expressed at lower levels on educated NK cells (C). No difference was observed for CXCR2 expression. To determine whether the observed differences in chemokine receptor expression translate into altered chemokine responsiveness between the subsets, we combined the transwell system with multicolor flow cytometry. We found that the chemokine-induced migration capability of NK cells correlated closely with the expression level of corresponding chemokine receptor, leading to subset specific responses to various chemokine gradients (D). The present results show that peripheral blood NK cell chemokine receptor profile changes in a coordinated fashion during NK cell differentiation and is further influenced by the expression of self-specific KIR. Interestingly, receptors which expression declines during NK cell differentiation (CCR5, CCR7, and CXCR3) are commonly associated with adaptive T cell responses to viruses, whereas receptors that are upregulated along the differentiation axis (CXCR1, CXCR2, CX3CR1, CMKLR1) are typical for neutrophils and macrophages as a part of the innate immune response. Thus, our results suggest that NK cell differentiation and education processes together shape the NK cell migratory capabilities to promote homing of the most functional NK cell subsets to the site of inflammation and serve as the first line of defense in the immune response to pathogens and tumors. Figure Disclosures Malmberg: Fate Therapeutics: Consultancy, Patents & Royalties; Vycellix: Membership on an entity's Board of Directors or advisory committees.