Inflammaging phenotype in rhesus macaques is associated with a decline in epithelial barrier-protective functions and increased pro-inflammatory function in CD161-expressing cells.

The development of chronic inflammation, called inflammaging, contributes to the pathogenesis of age-related diseases. Although it is known that both B and T lymphocyte compartments of the adaptive immune system deteriorate with advancing age, the impact of aging on immune functions of Th17-type CD161-expressing innate immune cells and their role in inflammaging remain incompletely understood. Here, utilizing the nonhuman primate model of rhesus macaques, we report that a dysregulated Th17-type effector function of CD161+ immune cells is associated with leaky gut and inflammatory phenotype of aging. Higher plasma levels of inflammatory cytokines IL-6, TNF-α, IL-1ß, GM-CSF, IL-12, and Eotaxin correlated with elevated markers of gut permeability including LPS-binding protein (LBP), intestinal fatty acid binding protein (I-FABP), and sCD14 in aging macaques. Further, older macaques displayed significantly lower frequencies of circulating Th17-type immune cells comprised of CD161+ T cell subsets, NK cells, and innate lymphoid cells. Corresponding with the increased markers of gut permeability, production of the type-17 cytokines IL-17 and IL-22 was impaired in CD161+ T cell subsets and NK cells, along with a skewing towards IFN-γ cytokine production. These findings suggest that reduced frequencies of CD161+ immune cells along with a specific loss in Th17-type effector functions contribute to impaired gut barrier integrity and systemic inflammation in aging macaques. Modulating type-17 immune cell functions via cytokine therapy or dietary interventions towards reducing chronic inflammation in inflammaging individuals may have the potential to prevent or delay age-related chronic diseases and improve immune responses in the elderly population.

Production of IL-17 by MAIT Cells Is Increased in Multiple Sclerosis and Is Associated with IL-7 Receptor Expression.

Multiple sclerosis (MS) is a T cell-driven inflammatory disease of the CNS. Research on T cell subsets involved in MS pathogenesis has mainly focused on classical CD4+ T cells, especially Th17 cells, as they produce the proinflammatory, MS-associated cytokine IL-17. However, the abundant unconventional mucosal-associated invariant T (MAIT) cells are also able to produce IL-17. MAIT cells are characterized by high CD161 expression and a semi-invariant Vα7.2 TCR, with which they recognize bacterial and yeast Ags derived from the riboflavin (vitamin B2) metabolism. In this study, we characterized MAIT cells from the peripheral blood of MS patients in comparison with healthy individuals with respect to their type-17 differentiation. We found a specific increase of IL-17+ MAIT cells as well as an increased expression of retinoic acid-related orphan receptor (ROR)γt and CCR6 in MAIT cells from MS patients, whereas the expression of T cell activation markers HLA-DR and CD38 was not different. IL-17 production by MAIT cells furthermore correlated with the surface expression level of the IL-7 receptor α-chain (CD127), which was significantly increased on MAIT cells from MS patients in comparison with healthy individuals. In summary, our findings indicate an augmented type-17 differentiation of MAIT cells in MS patients associated with their IL-7 receptor surface expression, implicating a proinflammatory role of these unconventional T cells in MS immunopathology.

High-level expression and purification of soluble form of human natural killer cell receptor NKR-P1 in HEK293S GnTI- cells.

Human natural killer receptor protein 1 (NKR-P1, CD161, gene klrb1) is a C-type lectin-like receptor of natural killer (NK) cells responsible for recognition of its cognate protein ligand lectin-like transcript 1 (LLT1). NKR-P1 is the single human orthologue of the prototypical rodent NKR-P1 receptors. Naturally, human NKR-P1 is expressed on the surface of NK cells, where it serves as inhibitory receptor; and on T and NKT cells functioning as co-stimulatory receptor promoting secretion of IFNγ. Most notably, it is expressed on Th17 and Tc17 lymphocytes where presumably promotes targeting into LLT1 expressing immunologically privileged niches. We tested effect of different protein tags (SUMO, TRX, GST, MsyB) on expression of soluble NKR-P1 in E. coli. Then we optimized the expression construct of soluble NKR-P1 by preparing a library of expression constructs in pOPING vector containing the extracellular lectin-like domain with different length of the putative N-terminal stalk region and tested its expression in Sf9 and HEK293 cells. Finally, a high-level expression of soluble NKR-P1 was achieved by stable expression in suspension-adapted HEK293S GnTI- cells utilizing pOPINGTTneo expression vector. Purified soluble NKR-P1 is homogeneous, deglycosylatable, crystallizable and monomeric in solution, as shown by size-exclusion chromatography, multi-angle light scattering and analytical ultracentrifugation.

LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation.

Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones, light and dark, with coordinated roles, controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells, but its functional role in the GC reaction has not been explored. In this study, we report high expression of LLT1 on GC-associated B cells, early plasmablasts, and GC-derived lymphomas. LLT1 expression was readily induced via BCR, CD40, and CpG stimulation on B cells. Unexpectedly, we found high expression of the LLT1 ligand, CD161, on follicular dendritic cells. Triggering of LLT1 supported B cell activation, CD83 upregulation, and CXCR4 downregulation. Overall, these data suggest that LLT1-CD161 interactions play a novel and important role in B cell maturation within the GC in humans.

Monosodium Urate Crystals Induce Upregulation of NK1.1-Dependent Killing by Macrophages and Support Tumor-Resident NK1.1+ Monocyte/Macrophage Populations in Antitumor Therapy.

Macrophages display phenotypic and functional heterogeneity dependent on the changing inflammatory microenvironment. Under some conditions, macrophages can acquire effector functions commonly associated with NK cells. In the current study, we investigated how the endogenous danger signal monosodium urate (MSU) crystals can alter macrophage functions. We report that naive, primary peritoneal macrophages rapidly upregulate the expression of the NK cell-surface marker NK1.1 in response to MSU crystals but not in response to LPS or other urate crystals. NK1.1 upregulation by macrophages was associated with mechanisms including phagocytosis of crystals, NLRP3 inflammasome activation, and autocrine proinflammatory cytokine signaling. Further analysis demonstrated that MSU crystal-activated macrophages exhibited NK cell-like cytotoxic activity against target cells in a perforin/granzyme B-dependent manner. Furthermore, analysis of tumor hemopoietic cell populations showed that effective, MSU-mediated antitumor activity required coadministration with Mycobacterium smegmatis to induce IL-1ß production and significant accumulation of monocytes and macrophages (but not granulocytes or dendritic cells) expressing elevated levels of NK1.1. Our findings provide evidence that MSU crystal-activated macrophages have the potential to develop tumoricidal NK cell-like functions that may be exploited to boost antitumor activity in vivo.

Recombinant expression, in vitro refolding and characterizing disulfide bonds of a mouse inhibitory C-type lectin-like receptor Nkrp1b.

As a part of the innate immunity, NK (Natural Killer) cells provide an early immune response to different stimuli, e.g. viral infections and tumor growths. However, their functions are more complex; they play an important role in reproduction, alloimmunity, autoimmunity and allergic diseases. NK cell activities require an intricate system of regulation that is ensured by many different receptors on a cell surface which integrate signals from interacting cells and soluble factors. One way to understand NK cell biology is through the structure of NK receptors, which can reveal ligand binding conditions. We present a modified protocol for recombinant expression in Escherichia coli and in vitro refolding of the ligand-binding domain of the inhibitory Nkrp1b (SJL/J) protein. Nkrp1b identity and folding was confirmed using mass spectrometry (accurate mass of the intact protein and evaluation of disulfide bonds) and one-dimensional nuclear magnetic resonance spectroscopy. The intention is to provide the basis for conducting structural studies of the inhibitory Nkrp1b protein, since only the activating Nkrp1a receptor structure is known.

Increased plasmacytoid dendritic cells and RORγt-expressing immune effectors in cutaneous acute graft-versus-host disease.

The role of PDCs and Th17 cells is not well understood in the pathogenesis of aGVHD. We evaluated PDC and Th17 cells in skin biopsies of 38 patients at diagnosis of aGVHD. The biopsies were tested by immunohistochemistry for the expression of BDCA2, a typical marker of PDCs. We found an increase of BDCA2(+) cells in the skin of the patients with aGVHD. Moreover, we observed a strong expression of the type I IFN-inducible protein Mx1 in the skin of the patients with aGVHD, compared with that of those without it, suggesting that PDCs produce type I IFN. We also analyzed the expression of two Th17 surface markers-CD161 and CCR6-and RORγt, the key transcription factor that orchestrates the differentiation of Th17 cells. Significantly higher numbers of RORγt(+), CD161(+), and CCR6(+) cells were counted in the skin of the patients with aGVHD than in the skin of those who underwent allo-SCT and in whom aGVHD did not develop. This study provides evidence for a role of Th17-mediated responses and a potential new pathophysiological link between PDCs and Th17 in human cutaneous aGVHD.

Enriched CD161high CCR6+ γδ T cells in the cerebrospinal fluid of patients with multiple sclerosis.

OBJECTIVE: To investigate the expression of CD161 (KLRB1) and CCR6 on human γδ T cells in blood and cerebrospinal fluid (CSF) of patients with a clinically isolated syndrome (CIS) and multiple sclerosis (MS) in relapse. DESIGN: Flow cytometry analysis of CD161 and CCR6 expression and intracellular cytokine staining for interleukin 17 and interferon-γ on human γδ T cells in blood and CSF samples. SETTING: Department of Neurology, Klinikum rechts der Isar, Technische Universität München, a tertiary referral center. PATIENTS: Twenty-six patients with CIS/MS in active relapse, 10 patients with other autoimmune disorders, 12 patients with neuroinfectious diseases, and 15 patients with noninflammatory neurological diseases. MAIN OUTCOME MEASURES: Frequencies of CD161high and CCR6+ γδ T cells in blood and CSF samples of patients with CIS/MS in relapse and control patients. RESULTS: γδ T cells were increased in both blood and CSF of patients with CIS/MS in relapse as compared with controls with noninflammatory disease. The fraction of CD161high CCR6+ γδ T cells was significantly higher in the CSF of patients with CIS/MS in relapse than of those with systemic autoimmune disorders or controls with noninflammatory disease. The CD161high CCR6+ double-positive γδ T-cell population was further enriched in the CSF in relation to blood in patients with CIS/MS in relapse but not in patients with infectious disease or the other control groups. The CD161high CCR6+ γδ T-cell population was characterized by its capacity to produce interleukin 17. CONCLUSION: Interleukin 17­producing CD161high CCR6+ γδ T cells might contribute to the compartmentalized inflammatory process in the central nervous system of patients with MS.

NKRP1A+ γδ and αß T cells are preferentially induced in patients with Salmonella infection.

NKRP1A(+) γδ and αß T cells play an important role at the early phase of Salmonella infection in mice. Meanwhile, association between NKRP1A(+) T cells and human Salmonella infection has not been reported. The objective of this study was to investigate the role of the peripheral NKRP1A(+) T cells in immune response to Salmonella infection. Expression of NKRP1A in peripheral γδ and αß T cells and production of interferon (IFN) γ and interleukin (IL)-4 in NKRP1A(+) γδ and αß T cells were analyzed in 28 patients with acute phase Salmonella infection, 23 patients with acute bacterial enterocolitis other than Salmonella infection (disease controls) and 44 normal controls by flow cytometry. The proportion of γδ T cells expressing NKRP1A and that of IFNγ-producing cells in NKRP1A(+) γδ cells were significantly higher in Salmonella group than those in other two groups. Compared with normal controls, the proportion of αß T cells expressing NKRP1A and that of IL-4-producing cells in NKRP1A(+) αß cells were significantly higher in Salmonella group. These data suggested that NKRP1A(+) T cells might play an important role in the early defense mechanism against Salmonella infection.

Investigation of NK cell function and their modulation in different malignancies.

NK cells have become a subject of investigation not only in the field of tumor immunology and infectious diseases, but also within all aspects of immunology, such as transplantation, autoimmunity, and hypersensitivity. Our early studies aside from investigating NK cell activity in experimental animals and humans included studies of perforin expression and modulation in this lymphocyte subset. As NK cell activity is modified by their environment, we showed clinical stage-dependent impairment of their activity and in vitro effect of different sera, Th1 cytokines, and their combination in breast cancer, Hodgkin's disease, and non-Hodgkin's lymphoma patients, especially with respect to metabolic and cell membrane changes of peripheral blood lymphocytes evaluated by spontaneous release of the enzyme lactate dehydrogenase (LDH) that led to the correction of the LDH enzyme release assay for natural cytotoxicity. By long-term immuno-monitoring of patients with malignancies, we also showed the kinetics of NK cell modulation during chemo-immunotherapy. In our more recent studies, we give data of NK function and novel families of NK cell receptor expression in healthy individuals that may be of help in NK cell profiling, by giving referent values of basic and cytokine-induced expression of some NK cell receptors either in evaluation of disease or in immuno-monitoring during cytokine therapy of patients with malignancies. Moreover, we give novel aspects of modulation of NK cell activity by cytokines approved for immunotherapy, IFN and IL-2, in melanoma and other malignancies with respect to alterations in new activating (NKG2D and CD161) and inhibitory (CD158a and CD158b) receptor characteristics and signaling molecules in CD16- and CD56-defined NK cells and their small immunoregulatory and large cytotoxic subsets in peripheral blood and lymph nodes, as NK cell-mediated killing of tumor cells depends on the balance between stimulatory and inhibitory signaling.

Higher frequencies of CD161+ circulating T lymphocytes in allergic rhinitis patients compared to healthy donors.

BACKGROUND: Th17 is a subset of T-helper lymphocytes that produce proinflammatory cytokines, mainly IL-17. Serum IL-17 is increased in allergic patients and relates to clinical severity. Recently, it has been reported that CD161 is a highly upregulated gene in Th17 clones and all IL-17-producing cells are contained in CD161(+) T cells. This study aimed at comparing the frequency of peripheral CD161(+) T cells in patients with allergic rhinitis (AR) and in healthy controls and at relating CD161 expression with symptom severity. METHODS: Forty-four patients with AR and 29 healthy non-allergic subjects were evaluated. CD161 expression was evaluated on CD3(+), CD4(+) and CD8(+) cells by double immunofluorescence staining and fluorescence activated cell sorter analysis. Symptom severity was assessed by the Visual Analogue Scale. RESULTS: Allergic patients showed a significantly higher frequency of CD3(+)CD161(+), CD4(+)CD161(+) and CD8(+)CD161(+) cells than healthy non-allergic subjects (p < 0.0001). Moreover, the expression of CD161 cells was significantly related to clinical severity. CONCLUSIONS: This study provides evidence that a higher frequency of CD161(+) T cells is present in the peripheral blood of AR patients.

Neutralizing IL-6 reduces human arterial allograft rejection by allowing emergence of CD161+ CD4+ regulatory T cells.

Perioperative injuries to an allograft exacerbate graft rejection, which in humans is primarily mediated by effector memory T cells. IL-6 transcripts in human coronary artery segments rapidly increase posttransplantation into immunodeficient mouse hosts compared with those of pretransplant specimens and fall dramatically by 30 d. Adoptive transfer of human PBMCs allogeneic to the artery 2 d postoperatively results in T cell infiltrates and intimal expansion 4 wk later. Ab neutralization of human IL-6 reduces the magnitude of intimal expansion and total T cell infiltration but increases the relative expression of CD161 while decreasing other Th17 markers. Coculture of MHC class II-expressing human endothelial cells (ECs) with allogeneic CD4(+) memory T cells results in T cell activation and EC secretion of IL-6. Neutralizing IL-6 in primary allogeneic T cell-EC cocultures results in enhanced T cell proliferation of CD161(+) CD4(+) T cells, reduces total T cell proliferation upon restimulation in secondary cultures (an effect dependent on CD161(+) T cells), increases expression of FOXP3 in CD161(+) T cells, and generates T cells that suppress proliferation of freshly isolated T cells. These data suggest that IL-6 released from injured allograft vessels enhances allogeneic T cell infiltration and intimal expansion in a model of human allograft rejection by inhibiting an increase in CD161(+) regulatory T cells.

Induction of lectin-like transcript 1 (LLT1) protein cell surface expression by pathogens and interferon-γ contributes to modulate immune responses.

CD161 is a C-type lectin-like receptor expressed on human natural killer (NK) cells and subsets of T cells. CD161 has been described as an inhibitory receptor that regulates NK cell-mediated cytotoxicity and IFN-γ production. Its role on T cells has remained unclear. Studies have shown that triggering of CD161 enhances NK T cell proliferation and T cell-IFN-γ production while inhibiting TNF-α production by CD8(+) T cells. Lectin-like transcript 1 (LLT1), the ligand of CD161, was found to be expressed on Toll-like receptor (TLR)-activated plasmacytoid and monocyte-derived dendritic cells (DC) and on activated B cells. Using newly developed anti-LLT1 mAbs, we show that LLT1 is not expressed on the surface of circulating B and T lymphocytes, NK cells, monocytes, and dendritic cells but that LLT1 is up-regulated upon activation. Not only TLR-stimulated dendritic cells and B cells but also T cell receptor-activated T cells and activated NK cells up-regulate LLT1. Interestingly, IFN-γ increases LLT1 expression level on antigen-presenting cells. LLT1 is also induced on B cells upon viral infection such as Epstein-Barr virus or HIV infection and in inflamed tonsils. Finally, expression of LLT1 on B cells inhibits NK cell function but costimulates T cell proliferation or IFN-γ production, and coengagement of CD161 with CD3 increases IL-17 secretion. Altogether, our results point toward a role for LLT1/CD161 in modulating immune responses to pathogens.

A critical role for dendritic cells in the formation of lymphatic vessels within tertiary lymphoid structures.

Ectopic, or tertiary, lymphoid aggregates often form in chronically inflamed areas. Lymphatic vessels, as well as high endothelial venules, form within these lymphoid aggregates, but the mechanisms underlying their development are poorly understood. Overexpression of the chemokine CCL21 in the thyroid of transgenic mice leads to formation of lymphoid aggregates containing topologically segregated T and B lymphocytes, dendritic cells (DCs), and specialized vasculature, including Lyve-1(+)/Prox-1(+) lymphatic vessels. In this article, we show that adoptive transfer of mature CD4(+) T cells into animals expressing CCL21 in a RAG-deficient background promotes the influx of host NK cells and DCs into the thyroid and the formation of new lymphatic vessels within 10 d. This process is dependent on the expression of lymphotoxin ligands by host cells, but not by the transferred CD4(+) T cells. Ablation of host DCs, but not NK cells, reduces the formation of new lymphatic vessels in the thyroid. Taken together, these data suggest a critical role for CD11c(+) DCs in the induction of lymphangiogenesis in tertiary lymphoid structures.

Involvement of NK 1.1-positive γδT cells in interleukin-18 plus interleukin-2-induced interstitial lung disease.

Interstitial lung disease (ILD) is induced by various factors in humans. However, the exact mechanism of ILD remains elusive. This study sought to determine the role of natural killer (NK) 1.1(+) γδT cells in ILD. The injection of IL-18 plus IL-2 (IL-18/IL-2) into C57BL6 (B6) mice induced acute ILD that resembled early-stage human ILD. An accumulation of NK1.1(+) γδT cells similar to NK cells was evident in the lungs. The T Cell Receptor (TCR) Vγ and Vδ repertoires of NK1.1(+) γδT cells indicated polyclonal expansion. The expression of IL-2 receptor ß (Rß) and IL-18Rß in NK1.1(+) γδT cells was higher than in NK1.1(-) γδT cells. IL-18/IL-2 stimulated the proliferation of NK1.1(+) γδT cells, but not NK1.1(-) γδT cells. The IL-18/IL-2-stimulated NK1.1(+) γδT cells produced higher concentrations of IFN-γ than did NK1.1(-) γδT cells. Moreover, NK1.1(+) γδT and NK1.1(-) γδT cells constituted completely different cell populations. The IL-18/IL-2-induced ILD was milder in TCRδ(-/-) and IFN-γ(-/-) mice, compared with B6 mice. Furthermore, cell-transfer experiments demonstrated that NK1.1(+) γδT cells could induce the expansion of NK cells and IFN-γ mRNA in the lung by IL-18/IL-2. Our results suggest that NK1.1(+) γδT cells function as inflammatory mediators in the early phase of IL-18/IL-2-induced ILD.

CD161(high)CD8+T cells bear pathogenetic potential in multiple sclerosis.

To identify differentially expressed genes in multiple sclerosis, microarrays were used in a stringent experimental setting-leukapheresis from disease-discordant monozygotic twins and gene expression profiling in CD4(+) and CD8(+) T-cell subsets. Disease-related differences emerged only in the CD8(+) T-cell subset. The five differentially expressed genes identified included killer cell lectin-like receptor subfamily B, member 1, also known as natural killer receptor protein 1a/CD161, presented by the International Multiple Sclerosis Genetics Consortium as one of the non-MHC candidate loci. Flow cytometric analysis on peripheral blood of healthy donors and patients with multiple sclerosis and rheumatoid arthritis confirmed an upregulation of CD161 at the protein level, showing also a significant excess of CD161(high)CD8(+) T cells in multiple sclerosis. This subset prevalently included chemokine (C-C motif) receptor 6(+), cytokine-producing, effector-memory T cells with proinflammatory profiles. It also included all circulating interleukin-17(+)CD8(+) T cells. In the CD161(high)CD8(+) subset, interleukin-12 facilitated proliferation and interferon-γ production, with CD161 acting as a co-stimulatory receptor. CD161(+)CD8(+)CD3(+) T cells producing interferon-γ were part of intralesional immune infiltrates and ectopic B cell follicles in autopsy multiple sclerosis brains. Variations of CD161 expression on CD8(+) T cells identify a subset of lymphocytes with proinflammatory characteristics that have not been previously reported in multiple sclerosis and are likely to contribute to disease immunopathology.

Cutting edge: crucial role of IL-1 and IL-23 in the innate IL-17 response of peripheral lymph node NK1.1- invariant NKT cells to bacteria.

We have shown previously that peripheral lymph node-resident retinoic acid receptor-related orphan receptor γt(+) NK1.1(-) invariant NKT (iNKT) cells produce IL-17A independently of IL-6. In this study, we show that the concomitant presence of IL-1 and IL-23 is crucial to induce a rapid and sustained IL-17A/F and IL-22 response by these cells that requires TCR-CD1d interaction and partly relies on IL-23-mediated upregulation of IL-23R and IL-1R1 expression. We further show that IL-1 and IL-23 produced by pathogen-associated molecular pattern-stimulated dendritic cells induce this response from NK1.1(-) iNKT cells in vitro, involving mainly TLR2/4-signaling pathways. Finally, we found that IL-17A production by these cells occurs very early and transiently in vivo in response to heat-killed bacteria. Overall, our study indicates that peripheral lymph node NK1.1(-) iNKT cells could be a source of innate Th17-related cytokines during bacterial infections and supports the hypothesis that they are able to provide an efficient first line of defense against bacterial invasion.

The transient nature of the Th17 phenotype.

CD4(+) Th lymphocytes represent a heterogeneous population of cells that play an essential role in adaptive immunity. In addition to type 1 (Th1) and type 2 (Th2) cells, a third subset of CD4(+) Th effector cells has recently been discovered, named type 17 (Th17) because of its unique ability to produce interleukin (IL)-17. Initial studies in mice suggested that Th17 cells are the pathogenic cells in autoimmune disorders, whereas Th1 cells are protective. Studies in humans have demonstrated the plasticity of Th17 cells and their ability to convert to Th1 cells. This Th17 to Th1 cell plasticity has also been confirmed in mice and, furthermore, it was found that Th17 cells appear to be pathogenic only when they shift to Th1 cells. A study in this issue of the European Journal of Immunology uses an IL-17 fate mapping mouse strain, which permits the identification of the cells that have been IL-17 producers, to provide definitive evidence that Th17 cells, either generated in vitro or in vivo, represent a transient phenotype that tend to convert into IFN-γ-producing cells. Our Commentary discusses this interesting point in light of previous data suggesting the same concept.

In-vitro IL-2 or IFN-α-induced NKG2D and CD161 NK cell receptor expression indicates novel aspects of NK cell activation in metastatic melanoma patients.

In metastatic melanoma (MM) immunomodulating agents, such as interleukin-2 (IL-2) and interferon-α (IFN-α), have shown therapeutic benefit as they enhance antitumor immune response. Considering tumor-induced suppression of natural killer (NK) cell activity, it is of interest to study the affect of these cytokines on the functional and receptor characteristics of CD16-defined NK cells and their dim and bright subsets. Peripheral blood lymphocytes of MM patients in clinical stage IV were treated in vitro for 18 h with IFN-α (250 U/ml) and rhIL-2 (200 U/ml) at 37°C. Both the cytokines induced significant in-vitro enhancement of NK cell activity. NKG2D receptor was induced by IL-2, whereas both the NKG2D and CD161 receptor expression was induced by IFN-α on NK cells and CD16(bright) NK cell subset. However, only IL-2 mediated induction of NKG2D on CD3⁻CD16(+) NK cells correlates with enhanced NK cytotoxicity by this cytokine, whereas, on the cytotoxic CD16(bright) subset NKG2D induction by both cytokines correlates with their induction of NK cell activity. In contrast, the observed induction of these receptors on the regulatory CD16(dim) subset shows no correlation with the obtained augmentation of cytotoxicity. We found substantial specific inducibility of pSTAT1 and pSTAT5, as well as induction of interferon-regulatory transcription factor-1 transcription by investigated cytokines in peripheral blood lymphocytes of MM patients. As NK cell-mediated killing of tumor cells depends on the balance between stimulatory and inhibitory signaling, induction of activating NKG2D receptor by IL-2 and IFN-α, especially in CD16(bright) NK cell subset, gives insight to novel aspects of NK cell activation by these cytokines that are applied in immunotherapy.

The role of etanercept on the expression of markers of T helper 17 cells and their precursors in skin lesions of patients with psoriasis vulgaris.

Very recently, it has been demonstrated that CD161, retinoic acid-related orphan receptor gamma-t (RORgamma-t) and CC-chemokin receptor 6 (CCR6) can be considered good surface markers to detect T helper 17 cells and their precursors, T cell populations that are considered to play an important role in the pathogenesis of psoriasis. In the present study, we evaluate the clinical involvement by calculating the PASI score and the number of CD4+, CD161+, RORgammat+ and CCR6+ cells before and after a 12-week course with etanercept or acitretin in patients with moderate-to-severe, plaque-type psoriasis vulgaris. Ten patients were given etanercept 50 mg twice weekly and 10 patients acitretin 0.4 mg/kg per day, both for 12 weeks. At the baseline and at the end of the treatment PASI was calculated, and skin biopsies were taken to evaluate the expression of CD4, CD161, RORgammat and CCR6 by immunohistochemistry. As controls, 10 patients with atopic dermatitis (AD) were included in the study. After 12 weeks, PASI was significantly lower than at the baseline for both groups. However, etanercept-treated patients showed lower PASI than acitretin-treated ones. While CD4+ cell numbers were similar in both diseases, all the other markers, that are considered more specific for Th17 cells and their precursors, were more expressed in psoriasis than in AD. Furthermore, only etanercept, but not acitretin, was able to significantly reduce CD161+, RORgammat+ and CCR6+ cells in skin lesions of patients with psoriasis. Our study provides further evidence of the role of Th17 pathway in the pathogenesis of psoriasis. Furthermore, our findings suggest that etanercept is able to downregulate the expression of the recently recognized markers of Th17 cells and their precursors CD161, RORgammat and CCR6, while acitretin is not. This activity on the Th17 lineage may contribute to the efficacy of etanercept in the treatment of psoriasis.