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.

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.

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.

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.

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.

Novel aspects of in vitro IL-2 or IFN-α enhanced NK cytotoxicity of healthy individuals based on NKG2D and CD161 NK cell receptor induction.

As IL-2 and IFN-α modulate NK cell activity it was of interest to investigate the expression of newly defined NK cell receptors and augmented NK cell activity in healthy individuals after cytokine in vitro treatment. Peripheral blood lymphocytes (PBL) obtained from 31 healthy volunteers treated for 18 h with 200 IU/ml IL-2 and 250 IU/ml IFN-α were evaluated for NK cell cytotoxicity. Expression of NKG2D, CD161, CD158a, CD158b receptors was analyzed on CD3⁻CD16+ NK cells, cytotoxic CD16(bright) and regulatory CD16(dim) subsets by FACS flow. The found induced significant in vitro enhancement of NK cell activity by both cytokines is supported by specific cytokine induction in PBL of pSTAT1 and pSTAT5, determined by Western blotting, as well as induction of IRF-1 transcription. Both cytokines induce significant up-regulation of NKG2D expression while only IFN-α induced significant up-regulation of CD161, with no alteration in KIR expression by either cytokine on CD3⁻CD16+ NK cells. Investigated cytokines did not induce change in NK cell bright and dim subset distribution. Moreover, we find that, not only cytokine receptor induction on the CD3⁻CD16+ NK cells, but also simultaneous increase in their percentage and/or density on CD16(bright) and CD16(dim) subsets, represent good indicators of receptor cytokine-susceptibility. As the role of NK cells has been shown in the loss of tolerance, infection and cancer, the data obtained in this study may be of help in NK cell profiling, by giving referent values of cytokine-induced novel NK cell receptor expression either in evaluation of these diseases or in immunomonitoring during cytokine immunotherapy.

Aberrant expression of NK cell receptors in Epstein-Barr virus-positive gammadelta T-cell lymphoproliferative disorders.

To clarify the difference between gene expression signature of Epstein-Barr virus (EBV)-positive gammadelta T-cell lymphoproliferative disorders (LPD) and those in normal gammadelta T-cells, we set out to determine gene expression profiling using three gammadelta T-cell lines derived from patients with EBV-positive gammadelta T-cell LPD. Gene expression profiling using an Affymetrix Genechip revealed a unique gene expression pattern of NK cell receptors in gammadelta T-LPD cell lines. Genes encoding killer cell lectin-like receptors (KLR), such as KLRG1 and KLRB1 (CD161), were generally low in gammadelta T-LPD cell lines, while killer cell immunoglobulin-like receptors varied among cell lines. In addition, expression of CD161 surface antigen was found to be remarkably decreased in gammadelta T-LPD cell lines by flow cytometric analysis. We conclude that gammadelta T-cell expansion in EBV-positive gammadelta T-LPD is in part due to inappropriate expression of the NK cell receptors which related to impaired function cytotoxic T cells.

Tumor necrosis factor-alpha enhances IL-15-induced natural killer cell differentiation.

The differentiation of natural killer (NK) cells is regulated by various factors including soluble growth factors and transcription factors. Here, we have demonstrated that tumor necrosis factor-alpha (TNF-alpha) is a positive regulator of NK cell differentiation. TNF-alpha augmented the IL-15-induced expression of NK1.1 and CD122 in mature NK cells, and TNF-alpha alone also induced NK cell maturation as well as IL-15. TNF-alpha also increased IFN-gamma production in NK cells in the presence of IL-15. Meanwhile, mRNA expression of several transcription factors, including T-bet and GATA-3, was increased by the addition of TNF-alpha and IL-15. In addition, TNF-alpha increased nuclear factor-kappa B (NF-kappaB) activity in NK cells and inhibition of NF-kappaB impeded TNF-alpha-enhanced NK cell maturation. Overall, these data suggest that TNF-alpha significantly increased IL-15-driven NK cell differentiation by increasing the expression of transcription factors that play crucial roles in NK cell maturation and inducing the NF-kappaB activity.

Inhibition of tumor growth by NK1.1+ cells and CD8+ T cells activated by IL-15 through receptor beta/common gamma signaling in trans.

IL-15 is an important cytokine involved in the survival and function of CD8(+) T cells and NK cells. IL-15 can be presented by IL-15Ralpha (IL-15RA) to bind with the shared IL-2/IL-15Rbeta and common gamma-chains, which activate signaling pathways on NK cells and CD8(+) T cells. In the present study, we characterized the function of trans-presented IL-15 on NK cells and CD8(+) T cells using TC-1 tumor cells transduced with a retrovirus encoding IL-15 linked to IL-15RA (IL-15/IL-15RA). We demonstrated that the expression of IL-15/IL-15RA on TC-1 cells led to increased percentages of tumor-infiltrating NK cells, NKT cells, and CD8(+) T cells, resulting in the inhibition of tumor growth in challenged mice. Additionally, in vivo Ab depletion experiments demonstrated that NK1.1(+) cells and CD8(+) T cells were important in this inhibition of tumor growth. Furthermore, this accumulation of immune cells and inhibition of tumor growth was abolished by a single amino acid mutation in the common gamma-chain binding site on IL-15. We also observed that IL-15/IL-15RA-transduced TC-1 cells led to the activation of STAT5 in NK and CD8(+) T cells in trans, which was abolished in the mutated IL-15/IL-15RA-transduced TC-1 cells. Taken together, our data suggest that common gamma-chain binding-dependent activation of the shared IL-15/IL-2Rbeta/common gamma signaling pathway may play an important role in the activation of NK cells and CD8(+) T cells, resulting in IL-15/IL-15RA trans-presentation-mediated inhibition of tumor growth.