Natural Killer Cells Control Tumor Growth by Sensing a Growth Factor.

Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRß signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.

RORγt⁺ innate lymphoid cells acquire a proinflammatory program upon engagement of the activating receptor NKp44.

RORγt⁺ innate lymphoid cells (ILCs) are crucial players of innate immune responses and represent a major source of interleukin-22 (IL-22), which has an important role in mucosal homeostasis. The signals required by RORγt⁺ ILCs to express IL-22 and other cytokines have been elucidated only partially. Here we showed that RORγt⁺ ILCs can directly sense the environment by the engagement of the activating receptor NKp44. NKp44 triggering in RORγt⁺ ILCs selectively activated a coordinated proinflammatory program, including tumor necrosis factor (TNF), whereas cytokine stimulation preferentially induced IL-22 expression. However, combined engagement of NKp44 and cytokine receptors resulted in a strong synergistic effect. These data support the concept that NKp44⁺ RORγt⁺ ILCs can be activated without cytokines and are able to switch between IL-22 or TNF production, depending on the triggering stimulus.

Blocking the PCNA/NKp44 Checkpoint to Stimulate NK Cell Responses to Multiple Myeloma.

Multiple Myeloma (MM) is a devastating malignancy that evades immune destruction using multiple mechanisms. The NKp44 receptor interacts with PCNA (Proliferating Cell Nuclear Antigen) and may inhibit NK cells' functions. Here we studied in vitro the expression and function of PCNA on MM cells. First, we show that PCNA is present on the cell membrane of five out of six MM cell lines, using novel anti-PCNA mAb developed to recognize membrane-associated PCNA. Next, we stained primary bone marrow (BM) mononuclear cells from MM patients and showed significant staining of membrane-associated PCNA in the fraction of CD38+CD138+ BM cells that contain the MM cells. Importantly, blocking of the membrane PCNA on MM cells enhanced the activity of NK cells, including IFN-γ-secretion and degranulation. Our results highlight the possible blocking of the NKp44-PCNA immune checkpoint by the mAb 14-25-9 antibody to enhance NK cell responses against MM, providing a novel treatment option.

CD62L Is a Functional and Phenotypic Marker for Circulating Innate Lymphoid Cell Precursors.

Innate lymphoid cells (ILCs) guard epithelial tissue integrity during homeostasis, but can be potent immune effector cells during inflammation. Precursors to all ILC subsets (ILC precursors [ILCP]) have been identified in human peripheral blood (PB). We found that during homeostasis, ILCP in PB of mouse and human expressed homing receptors for secondary lymphoid organs, mainly CD62L. These ILCP entered mouse lymph nodes in a CD62L-dependent way and relied on S1P receptors for their exit. Importantly, CD62L expression was absent on human ILCs expressing NKp44 in tonsils and PB of Crohn disease patients, and relatively fewer CD62L+ ILCP were present in PB of Crohn disease patients. These data are in agreement with selective expression of CD62L on nonactivated ILCP. As such, we conclude that CD62L not only serves as a functional marker of ILCP, but has potential to be used in the clinic as a diagnostic marker in inflammatory disorders.

Innate Lymphoid Cells Have Decreased HLA-DR Expression but Retain Their Responsiveness to TLR Ligands during Sepsis.

Sepsis, one of the leading causes of death in intensive care units, is caused by a dysregulated host response to infection that leads to life-threatening organ dysfunction. The proinflammatory and anti-inflammatory responses activated by the infecting microorganism become systemic, and the sustained anti-inflammatory response induces a state of immunosuppression that is characterized by decreased expression of HLA-DR on monocytes, T cell apoptosis, and reduced production of TNF-α by monocytes and macrophages in response to TLR ligands. Innate lymphoid cells (ILCs) are lymphocytes that lack Ag-specific receptors and lineage-specific markers; they express HLA-DR and are activated by cytokines and by direct recognition of microbial molecules. In this study, we evaluated if ILCs are affected by the anti-inflammatory response during sepsis. We found that the number of peripheral blood ILCs was decreased in septic patients compared with healthy volunteers; this decrease was caused by a reduction in ILC1 and ILC3 and is associated with apoptosis, because ILCs from septic patients expressed active caspase 3. ILCs from septic patients had decreased HLA-DR expression but increased expression of the activating receptors NKp46 and NKp44; they also showed a sustained expression of CD127 (IL-7R α-chain) and retained their capacity to produce TNF-α in response to TLR ligands. These results indicate that during sepsis, ILCs have decreased HLA-DR expression and die via apoptosis, similar to monocytes and T cells, respectively. However, other effector functions of ILCs (activation through NKp46 and NKp44, TNF-α production) may remain unaffected by the immunosuppressive environment prevailing in septic patients.

Characterization of circulating T-, NK-, and NKT cell subsets in patients with colorectal cancer: the peripheral blood immune cell profile.

OBJECTIVE: As the development and progression of colorectal cancer (CRC) are known to be affected by the immune system, cell subsets such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are considered interesting targets for immunotherapy and clinical biomarker research. Until now, the role of systemic immune profiles in tumor progression remains unclear. In this study, we aimed to characterize the immunophenotype of circulating T cells, NK cells, and NKT-like cells in patients with CRC, and to subsequently correlate these immunophenotypes to clinical follow-up data. METHODS: Using multiparameter flow cytometry, the subset distribution and immunophenotype of T cells (CD3+CD56-), CD56dim NK cells (CD3-CD56dim), CD56bright NK cells (CD3-CD56bright), and NKT-like (CD3+CD56+) cells were investigated in peripheral blood mononuclear cell (PBMC) samples from 71 CRC patients and 19 healthy donors. RESULTS: CRC patients showed profound differences in immune cell subset distribution and their immunophenotype compared to healthy donors, as characterized by increased percentage of regulatory T cells, and reduced expression level of the natural cytotoxicity receptors NKp44 and NKp46 on both CD56dim NK cells and NKT-like cells. Finally, we showed in a multivariate analysis that above-median percentage of CD16+ NKT-like cells was independently associated with shorter disease-free survival in CRC patients. CONCLUSION: The altered phenotype of circulating immune cell subsets in CRC and its association with clinical outcome highlight the potential use of PBMC subsets as prognostic biomarkers in CRC, thereby contributing to better insight into the role of systemic immune profiles in tumor progression.

Severe hypersensitivity reactions to biological drugs in children with rheumatic diseases.

BACKGROUND: Hypersensitivity reactions (HSR) to biologic drugs (BD) may limit their use in children with rheumatic diseases. We aimed to analyze the incidence and clinical characteristics of immediate type I (IgE/non-IgE) hypersensitivity reactions to BD and the risk factors for these reactions. METHODS: Children with rheumatic diseases using BD who were evaluated in the pediatric allergy department for possible drug hypersensitivity reaction (DHR) due to BD or any other drug were included in the study. RESULTS: One hundred and twenty-eight children (49.2% boys; 14.6 years [9.9-16.9 years] with juvenile idiopathic arthritis [58%], familial Mediterranean fever [14%], vasculitis [14%], and other diseases [14%]) had used eight different BD with 32 494 infusions/injections. Fifteen patients were evaluated for DHR [injection-site reactions [n = 4], adverse events [n = 2], drug hypersensitivity other than BD [n = 3], and immediate BD hypersensitivity [n = 6]). The incidence of immediate BD HSR was 4.7%, with a clinical presentation of anaphylaxis in 3.9% (tocilizumab [n = 3], rituximab [n = 2], positive skin test with culprit BD [n = 3]). Among patients with BD HSR, the median follow-up was longer (84.5 vs 54 months, P = .048), and renal (33.3% vs 4.1%, P = .002), hematologic involvement (16.7% vs 0, P < .001), and active disease (83.3% vs 13.9%, P < .001) were more common. Logistic regression analysis revealed that renal involvement, more than 14 hospitalizations per lifetime, and more than two different BD used were associated with BD hypersensitivity. CONCLUSION: The frequency of severe immediate HSR due to BD was shown to be 3.9% in children with rheumatic diseases. Children with active rheumatic disease and who have exposure to multiple BD should be monitored for BD HSR, particularly during intravenous BD infusions.

Fate Decision Between Group 3 Innate Lymphoid and Conventional NK Cell Lineages by Notch Signaling in Human Circulating Hematopoietic Progenitors.

The role of Notch signaling in human innate lymphoid cell (ILC) differentiation is unclear, although IL-7 and IL-15 promote differentiation of natural cytotoxicity receptor (NCR) NKp44+ group 3 ILCs (NCR+ILC3s) and conventional NK (cNK) cells from CD34+ hematopoietic progenitor cells (HPCs) ex vivo. In this study, we analyzed the functions of Notch in the differentiation of NCR+ILC3s and cNK cells from human HPC subpopulations circulating in peripheral blood by limiting dilution and clonal assays using high-throughput flow cytometry. We demonstrated that Notch signaling in combination with IL-7 induced NCR+ILC3 differentiation, but conversely suppressed IL-15-dependent cNK cell generation in CD45RA+Flt-3-c-Kitlow, a novel innate lymphocyte-committed HPC subpopulation. In contrast, Notch signaling induced CD45RA-Flt-3+c-Kithigh multipotent HPCs to generate CD34+CD7+CD62Lhigh, the earliest thymic progenitor-like cells, which preserved high cNK/T cell potential, but lost NCR+ILC3 potential. These findings implicate the countervailing functions of Notch signaling in the fate decision between NCR+ILC3 and cNK cell lineages at different maturational stages of human HPCs. Inhibition of Notch functions by Abs specific for either the Notch1 or Notch2 negative regulatory region suggested that both Notch1 and Notch2 signals were involved in the fate decision of innate lymphocyte-committed HPCs and in the generation of earliest thymic progenitor-like cells from multipotent HPCs. Furthermore, the synergistic interaction between Notch and IL-7 in NCR+ILC3 commitment was primarily explicable by the induction of IL-7 receptor expression in the innate lymphocyte-committed HPCs by Notch stimulation, suggesting the pivotal role of Notch in the transcriptional control required for human NCR+ILC3 commitment.

Modulation the expression of natural killer cell activating receptor (NKp44) in the peripheral blood of diffuse large B-cell lymphoma patients and the correlation with clinic pathological features.

NK cell activation is one strategy to improve the immunotherapy of non-Hodgkin's lymphoma. So, we aimed to investigate expression of Natural killer cell activating receptor NKp44 in patients with diffuse large B-cell lymphoma (DLBCL) and its correlation with clinic pathological data. In this study, 30 new cases with DLBCL in addition to 20 healthy control were involved. All were submitted to full history, clinical examination, histopathology, Routine laboratory investigations including CBC, LDH, ß2microgloubine and bone marrow examination. Cell culture of peripheral blood mononuclear cells and expression of CD56 and NKp44 by flowcytometry was done. We demonstrated increased NK cell populations (CD 56 +ve NKp44 -ve, CD 56 -veNKp44 +ve, total CD 56 +ve) and NKp44 MFI after in-vitro activation in both healthy control and DLBCL cases except for CD 56 +ve NKp44 +ve which significantly increased in patients not in healthy control (p=0.005, 0.601) respectively. No significant difference between the DLBCL and healthy control regarding all NK cell populations without PHA stimulation. However, the culture with PHA in DLBCL showed significant increase in NK cell populations than the healthy control (CD 56 +ve NKp44 +ve 12.37±7.52vs 6.80±4.07, p=0.008), (Total CD 56 +ve 18.80±8.74vs 12.66±5.17, p=0.017), (MFI of NKp44 10.95±6.18vs 5.58±1.70, p=0.001). Regarding the association with clinic pathologic features, increased expression of NKp44 was associated with lower values of LDH and earlier stages of DLBCL (p<0.05). So, activating receptor NKp44 can be modulated by in-vitro activation, hence improvement of its function as an approach of immunotherapy of DLBCL.

Haemagglutinin-neuraminidase from HPIV3 mediates human NK regulation of T cell proliferation via NKp44 and NKp46.

HPIV3 is a respiratory virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. Currently there is no effective vaccine or anti-viral therapy for this virus. Studies have suggested that poor T cell proliferation following HPIV3 infection is responsible for impaired immunological memory associated with this virus. We have previously demonstrated that NK cells mediate regulation of T cell proliferation during HPIV3 infection. Here we add to these studies by demonstrating that the regulation of T cell proliferation during HPIV3 infection is mediated via NK receptors NKp44 and NKp46 and involves the surface glycoprotein haemagglutinin-neuraminidase but not the fusion protein of the virus. These studies extend our knowledge of the regulatory repertoire of NK cells and provide mechanistic insights which may explain reoccurring failures of vaccines against this virus.

Enrichment of IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets is associated with reduction of NKp44+ ILC3s in the terminal ileum of Crohn's disease patients.

Crohn's disease (CD) is a chronic inflammatory condition of the human gastrointestinal tract whose aetiology remains largely unknown. Dysregulated adaptive immune responses and defective innate immunity both contribute to this process. In this study, we demonstrated that the interleukin (IL)-17A+ interferon (IFN)-γ+ and IL-22+ IFN-γ+ T cell subsets accumulated specifically in the inflamed terminal ileum of CD patients. These cells had higher expression of Ki-67 and were active cytokine producers. In addition, their proportions within both the IL-17A-producer and IL-22-producer populations were increased significantly. These data suggest that IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets might represent the pathogenic T helper type 17 (Th17) population in the context of intestinal inflammation for CD patients. In the innate immunity compartment we detected a dramatic alteration of both phenotype and function of the intestinal innate lymphoid cells (ILCs), that play an important role in the maintenance of mucosal homeostasis. In the inflamed gut the frequency of the NKp44- CD117- ILC1s subset was increased significantly, while the frequency of NKp44+ ILC3s was reduced. Furthermore, the frequency of human leucocyte antigen D-related (HLA-DR)-expressing-NKp44+ ILC3s was also reduced significantly. Interestingly, the decrease in the NKp44+ ILC3s population was associated with an increase of pathogenic IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets in the adaptive compartment. This might suggest a potential link between NKp44+ ILC3s and the IL-17A+ IFN-γ+ and IL-22+ IFN-γ+ T cell subsets in the terminal ileum of CD patients.

Regulation of natural cytotoxicity receptors by heparan sulfate proteoglycans in -cis: A lesson from NKp44.

NKp44 (NCR2) is a distinct member of natural cytotoxicity receptors (NCRs) family that can induce cytokine production and cytolytic activity in human NK cells. Heparan sulfate proteoglycans (HSPGs) are differentially expressed in various normal and cancerous tissues. HSPGs were reported to serve as ligands/co-ligands for NKp44 and other NCRs. However, HSPG expression is not restricted to either group and can be found also in NK cells. Our current study reveals that NKp44 function can be modulated through interactions with HSPGs on NK cells themselves in -cis rather than on target cells in -trans. The intimate interaction of NKp44 and the NK cell-associated HSPG syndecan-4 (SDC4) in -cis can directly regulate membrane distribution of NKp44 and constitutively dampens the triggering of the receptor. We further demonstrate, that the disruption of NKp44 and SDC4 interaction releases the receptor to engage with its ligands in -trans and therefore enhances NKp44 activation potential and NK cell functional response.

Hypercytotoxicity and rapid loss of NKp44+ innate lymphoid cells during acute SIV infection.

HIV/SIV infections break down the integrity of the gastrointestinal mucosa and lead to chronic immune activation and associated disease progression. Innate lymphoid cells (ILCs), distinguishable by high expression of NKp44 and RORγt, play key roles in mucosal defense and homeostasis, but are depleted from gastrointestinal (GI) tract large bowel during chronic SIV infection. However, less is known about the kinetics of ILC loss, or if it occurs systemically. In acute SIV infection, we found a massive, up to 8-fold, loss of NKp44+ILCs in all mucosae as early as day 6 post-infection, which was sustained through chronic disease. Interestingly, no loss of ILCs was observed in mucosa-draining lymph nodes. In contrast, classical NK cells were not depleted either from gut or draining lymph nodes. Both ILCs and NK cells exhibited significantly increased levels of apoptosis as measured by increased Annexin-V expression, but while classical NK cells also showed increased proliferation, ILCs did not. Interestingly, ILCs, which are normally noncytolytic, dramatically upregulated cytotoxic functions in acute and chronic infection and acquired a polyfunctional phenotype secreting IFN-γ, MIP1-ß, and TNF-α, but decreased production of the prototypical cytokine, IL-17. Classical NK cells had less dramatic functional change, but upregulated perforin expression and increased cytotoxic potential. Finally, we show that numerical and functional loss of ILCs was due to increased apoptosis and ROR γt suppression induced by inflammatory cytokines in the gut milieu. Herein we demonstrate the first evidence for acute, systemic, and permanent loss of mucosal ILCs during SIV infection associated with reduction of IL-17. The massive reduction of ILCs involves apoptosis without compensatory de novo development/proliferation, but the full mechanism of depletion and the impact of functional change so early in infection remain unclear.

The differential frequency of Lineage(-)CRTH2(-)CD45(+)NKp44(-)CD117(-)CD127(+)ILC subset in the inflamed terminal ileum of patients with Crohn's disease.

Deregulation of various components of the immune system has been reported in the inflamed gut of Crohn's disease (CD) patients. Innate lymphoid cells (ILCs) are novel innate effector lymphocytes which can rapidly respond to danger signals, from invading pathogens or tissue damage, to maintain homeostasis, especially along the mucosal surfaces. The purpose of this study is to compare composition of the intestinal ILCs subsets of CD patients with differential inflammatory conditions of the terminal ileum, which are marked by distinct histological appearances and mucosal profiles of cytokines. We observed alterations in the frequency of Lineage(-)CRTH2(-)CD45(+)NKp44(-)CD117(-)CD127(+)ILC subset in the inflamed terminal ileum.

Dysregulation of regulatory CD56(bright) NK cells/T cells interactions in multiple sclerosis.

Recent evidence has shown that CD56(bright) NK cells, a subset of NK cells abundant in lymph nodes, may have an immunoregulatory function. In multiple sclerosis (MS), expansion of CD56(bright) NK cells has been associated to successful response to different treatments and to remission of disease during pregnancy; how whether they exert immunoregulation in physiologic conditions and whether this is impaired in MS is not known. We dissected the immunoregulatory role of CD56(bright) NK cells function in healthy subjects (HS) and compared it with that of untreated MS subjects or patients with clinically isolated syndrome suggestive of MS (CIS). We found that CD56(bright) NK cells from HS acquire, upon inflammatory cues, the capability of suppressing autologous CD4+T cell proliferation through direct cytotoxicity requiring engagement of natural cytotoxicity receptors (NCRs) and secretion of granzyme B. CD56(bright) NK cells from patients with MS/CIS did not differ in frequency and share a similar phenotype but displayed a significantly lower ability to inhibit autologous T cell proliferation. This impairment was not related to deficient expression of NCRs or granzyme B by CD56(bright) NK cells, but to increased HLA-E expression on T cells from MS/CIS subjects, which could enhance the inhibitory effect mediated by NKG2A that is homogeneously expressed on CD56(bright) NK cells. The defect in controlling autologous T cells by CD56(bright) NK cells in MS/CIS might contribute to the excess of autoimmune response that is associated to disease development.

Natural killer cells in HIV controller patients express an activated effector phenotype and do not up-regulate NKp44 on IL-2 stimulation.

Control of HIV replication in elite controller (EC) and long-term nonprogressor (LTNP) patients has been associated with efficient CD8(+)cytotoxic T-lymphocyte function. However, innate immunity may play a role in HIV control. We studied the expression of natural cytotoxicity receptors (NKp46, NKp30, and NKp44) and their induction over a short time frame (2-4 d) on activation of natural killer (NK) cells in 31 HIV controller patients (15 ECs, 16 LTNPs). In EC/LTNP, induction of NKp46 expression was normal but short (2 d), and NKp30 was induced to lower levels vs. healthy donors. Notably, in antiretroviral-treated aviremic progressor patients (TAPPs), no induction of NKp46 or NKp30 expression occurred. More importantly, EC/LTNP failed to induce expression of NKp44, a receptor efficiently induced in activated NK cells in TAPPs. The specific lack of NKp44 expression resulted in sharply decreased capability of killing target cells by NKp44, whereas TAPPs had conserved NKp44-mediated lysis. Importantly, conserved NK cell responses, accompanied by a selective defect in the NKp44-activating pathway, may result in lack of killing of uninfected CD4(+)NKp44Ligand(+) cells when induced by HIVgp41 peptide-S3, representing a relevant mechanism of CD4(+) depletion. In addition, peripheral NK cells from EC/LTNP had increased NKG2D expression, significant HLA-DR up-regulation, and a mature (NKG2A-CD57(+)killer cell Ig-like receptor(+)CD85j(+)) phenotype, with cytolytic function also against immature dendritic cells. Thus, NK cells in EC/LTNP can maintain substantially unchanged functional capabilities, whereas the lack of NKp44 induction may be related to CD4 maintenance, representing a hallmark of these patients.

Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy.

The signaling lymphocytic activation molecule (SLAM) receptor-associated adaptor Ewing's sarcoma-associated transcript-2 (EAT-2) is primarily expressed in innate immune cells including dendritic cells (DCs), macrophages and NK cells. A recent human HIV vaccine study confirmed that EAT-2 expression was associated with the enhanced immunogenicity induced by the MRKAd5/HIV vaccine. We previously harnessed the capability of EAT-2 to modulate signaling mediated by SLAM receptors and demonstrated that by incorporating EAT-2 expression into vaccines, one could enhance innate and adaptive immune responses in mice, even in the face of pre-existing immunity to the vaccine vectors. Herein, we investigated the innate immune responses of human cells exposed to EAT-2-over-expressing vaccines. Our results demonstrate that EAT-2 over-expression can significantly alter the kinetics of critical pro-inflammatory cytokine and chemokine responses elaborated by human PBMCs. In addition, enhanced DC maturation and increased monocyte phagocytosis were observed in EAT-2-transduced human cells. We also found that EAT-2 over-expression improved antigen presentation by human cells. Moreover, EAT-2 over-expression increased the anti-tumor activity of human NK cells against K562 tumor cell targets. Many of these responses were extinguished with use of an EAT-2 variant carrying a mutant SH2 domain (R31Q), suggesting a critical role for the interaction between EAT-2 and SLAM receptors in mediating these responses. In conclusion, these results provide evidence that EAT-2 interacts with key components of multiple arms of the human innate immune system, and that this role highlights the potential for targeting EAT-2 functions so as to improve a number of human immunotherapeutic approaches, including vaccine development.

Balance between activating NKG2D, DNAM-1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast-like synoviocytes (FLS) are central components of the aggressive, tumour-like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA-FLS and NK cells. We used cultured RA-FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA-FLS/NK cell cross-talk. We show that RA-FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM-1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA-FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA-E expressed on RA-FLS further enhanced Nishi cell degranulation in co-culture with RA-FLS. Using cultured RA-FLS and the human NK cell line Nishi as an in vitro model system of RA-FLS/NK cell cross-talk, our results suggest that cell-mediated cytotoxicity of RA-FLS may be one mechanism by which NK cells influence local joint inflammation in RA.

N6-isopentenyladenosine affects cytotoxic activity and cytokines production by IL-2 activated NK cells and exerts topical anti-inflammatory activity in mice.

N6-isopentenyladenosine (iPA) is a modified adenosine with an isopentenyl moiety derived from the mevalonate pathway which displays pleiotropic biological effects, including anti-tumor and anti-angiogenic activity. Previous evidence revealed a biphasic effect of iPA on phytohemagglutinin-stimulated lymphocytes, being pro-proliferative at low doses and anti-proliferative at high doses. Analogously, we have recently shown that low iPA concentrations (<1µM) increased the immune response of natural killer (NK) cells against cancer targets. In the present study, we evaluated the effect of iPA at high concentration (10µM) on IL-2-activated NK cells. iPA, inhibited NK cell proliferation and cytotoxicity against their conventional tumor target, human K562 cells. This inhibition was associated with decreased expression and functionality of NK cell activating receptors NKp44 and NKG2D as well as impaired cyto/chemokines secretion (RANTES, MIP-1α, TNF-α and IFN-γ). ERK/MAPK and STAT5 activation in IL-2-activated NK cells were inhibited by iPA. The results obtained in vitro were validated in vivo in the inflammatory murine model of croton oil-induced ear dermatitis. The topical application of iPA significantly reduced mouse ear oedema, thus suggesting anti-inflammatory properties of this molecule. These results show the ability of iPA to exert anti-inflammatory effects both in vitro and in vivo directly targeting NK cells, providing a novel pharmacological tool in those diseases characterized by a deregulated immune-response, such as cancer or inflammatory conditions.

[Role and mechanism of NKp44+NK cells in the proliferation and inflammation of synovium of rheumatoid arthritis patients].

OBJECTIVE: To explore the effects of NKp44+NK cells from rheumatoid arthritis (RA) patients on the proliferation and monocyte chemotactic protein 1 production of fibroblast-like synoviocytes (FLS). METHODS: The proportions of natural killer (NK) p44 NK cells in peripheral blood (PB) of 50 RA patients and 50 healthy individuals were detected by flow cytometry. Synovial fluid (SF) samples from 30 RA patients were also detected. NKp44+NK cells in RA SF were sorted by flow cytometry for 5 times. The supernatant level of interleukin (IL)-22 was measured by enzyme-linked immunosorbent assay (ELISA). The proliferation of FLS after an addition of culture supernatant of NKp44+NK cells was detected by methyl thiazolyl tetrazolium (MTT) at 24, 48 and 72 h. Monocyte chemotactic protein (MCP)-1 production of RA FLS after an addition of rhIL-22 was detected by ELISA. RESULTS: The proportion of NKp44+NK cells in PB of RA patients was significantly higher than that of normal controls while the proportion of NKp44+NK cells in SF of RA patients was higher than that in PB of matched RA patients (1.270% vs 0, 15.190% vs 2.425%, P < 0.01). The supernatant level of IL-22 in NKp44+NK cell culture was (1 603 ± 332) ng/L. Rapid proliferation of RA FLS was observed at 24, 48, 72 h after an addition of culture supernatant (P < 0.01). IL-22 antibody obviously inhibited the proliferation of RA FLS induced by NKp44+NK cells. MCP-1 production of RA FLS was detected at 72 h after an addition of rhIL-22 (P < 0.01). CONCLUSION: NKp44+NK cells can promote the proliferation and MCP-1 production of RA FLS through the production of IL-22 so as to play an important role in the synovial proliferation and inflammation of RA.