NKG2D and CD94 bind to multimeric α2,3-linked N-acetylneuraminic acid

Killer lectin-like receptors on natural killer cells mediate cytotoxicity through glycans on target cells including the sialyl Lewis X antigen (sLeX). We investigated whether NK group 2D (NKG2D) and CD94 can bind to sialylated N-linked glycans, using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rNKG2Dlec) and CD94 (rCD94lec). Both rNKG2Dlec and rCD94lec bound to plates coated with high-sLeX-expressing transferrin secreted by HepG2 cells (HepTF). The binding of rNKG2Dlec and rCD94lec to HepTF was markedly suppressed by treatment of HepTF with neuraminidase and in the presence of N-acetylneuraminic acid. Moreover, rNKG2Dlec and rCD94lec bound to α2,3-sialylated human α₁-acid glycoprotein (AGP) but not to α2,6-sialylated AGP. Mutagenesis revealed that ¹⁵²Y of NKG2D and ¹⁴⁴F and ¹⁶⁰N of CD94 were critical for HepTF binding. This is the first report that NKG2D and CD94 bind to α2,3-sialylated but not to α2,6-sialylated multi-antennary N-glycans.     

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94⁺ NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94⁺ NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL⁺ CD56^dim NK cells, in contrast to the efficient responses by CD56^bright NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94⁺ KIR2DL^- NK cells may be uniquely beneficial.     

CD4+NKG2D+ T Cells Exhibit Enhanced Migratory and Encephalitogenic Properties in Neuroinflammation

Migration of encephalitogenic CD4⁺ T lymphocytes across the blood-brain barrier is an essential step in the pathogenesis of multiple sclerosis (MS). We here demonstrate that expression of the co-stimulatory receptor NKG2D defines a subpopulation of CD4⁺ T cells with elevated levels of markers for migration, activation, and cytolytic capacity especially when derived from MS patients. Furthermore, CD4⁺NKG2D⁺ cells produce high levels of proinflammatory IFN-γ and IL-17 upon stimulation. NKG2D promotes the capacity of CD4⁺NKG2D⁺ cells to migrate across endothelial cells in an in vitro model of the blood-brain barrier. CD4⁺NKG2D⁺ T cells are enriched in the cerebrospinal fluid of MS patients, and a significant number of CD4⁺ T cells in MS lesions coexpress NKG2D. We further elucidated the role of CD4⁺NKG2D⁺ T cells in the mouse system. NKG2D blockade restricted central nervous system migration of T lymphocytes in vivo, leading to a significant decrease in the clinical and pathologic severity of experimental autoimmune encephalomyelitis, an animal model of MS. Blockade of NKG2D reduced killing of cultivated mouse oligodendrocytes by activated CD4⁺ T cells. Taken together, we identify CD4⁺NKG2D⁺ cells as a subpopulation of T helper cells with enhanced migratory, encephalitogenic and cytotoxic properties involved in inflammatory CNS lesion development.     

Dissection of the NKG2C NK cell response against Puumala Orthohantavirus

BackgroundInfections with the Puumala orthohantavirus (PUUV) in humans may cause hemorrhagic fever with renal syndrome (HFRS), known as nephropathia epidemica (NE), which is associated with acute renal failure in severe cases. In response to PUUV-infections, a subset of potent antiviral NKG2C⁺ NK cells expand, whose role in virus defence and pathogenesis of NE is unclear. NKG2C⁺ NK cell proliferation is mediated by binding of NKG2C/CD94 to HLA-E on infected cells. The proliferation and activation of NKG2C⁺ NK cells via the NKG2C/HLA-E axis is affected by different NKG2C (NKG2C^wt/del) and HLA-E (HLA-E*0101/0103) alleles, which naturally occur in the human host. Homozygous (NKG2C^del/del) and heterozygous (NKG2C^wt/del) deletions of the NKG2C receptor results in an impaired NKG2C/CD94 mediated proliferation and activation of NKG2C⁺ cells. We therefore analyzed the PUUV-mediated NKG2C⁺ NK cell responses and the impact of different NKG2C and HLA-E alleles in NE patients.Methodology/Principal findingsNKG2C⁺ NK cell expansion and effector functions in PUUV-infected cells were investigated using flow cytometry and it was shown that PUUV-infected endothelial cells led to a NKG2C/CD94 mediated NKG2C⁺ NK cell activation and expansion, dependent on the HLA-G-mediated upregulation of HLA-E. Furthermore, the NKG2C^del and HLA-E*0101/0103 alleles were determined in 130 NE patients and 130 matched controls, and it was shown that in NE patients the NKG2C^wt/del allele was significantly overrepresented, compared to the NKG2C^wt/wt variant (p = 0.01). In addition, in vitro analysis revealed that NKG2C^wt/del NK cells exhibited on overall a lower proliferation (p = 0.002) and lower IFNγ expression (p = 0.004) than NKG2C^wt/wt NK cells.Conclusions/SignificanceOur results corroborate the substantial impact of the NKG2C/HLA-E axis on PUUV-specific NK cell responses. A weak NKG2C⁺ NK cell response, as reflected by NKG2C^wt/del variant, may be associated with a higher risk for a severe hantavirus infections.     

Interactions between NKG2x immunoreceptors and HLA-E ligands display overlapping affinities and thermodynamics

The NKG2x/CD94 family of C-type lectin-like immunoreceptors (x = A, B, C, E, and H) mediates surveillance of MHC class Ia cell surface expression, often dysregulated during infection or tumorigenesis, by recognizing the MHC class Ib protein HLA-E that specifically presents peptides derived from class Ia leader sequences. In this study, we determine the affinities and interaction thermodynamics between three NKG2x/CD94 receptors (NKG2A, NKG2C, and NKG2E) and complexes of HLA-E with four representative peptides. Inhibitory NKG2A/CD94 and activating NKG2E/CD94 receptors bind HLA-E with indistinguishable affinities, but with significantly higher affinities than the activating NKG2C/CD94 receptor. Despite minor sequence differences, the peptide presented by HLA-E significantly influenced the affinities; HLA-E allelic differences had no effect. These results reveal important constraints on the integration of opposing activating and inhibitory signals driving NK cell effector functions.     

Preferential survival of CD8 T and NK cells expressing high levels of CD94

The Qa-1(b)/Qdm tetramer binds to CD94/NKG2 receptors expressed at high levels on approximately 50% of murine NK cells. Although very few CD8 T cells from naive mice express CD94/NKG2 receptors, approximately 50% of CD8 T cells taken from mice undergoing a secondary response against Listeria monocytogenes (LM) are CD94(high) and bind the tetramer. Although CD94(int) NK cells do not bind the tetramer, CD94(int) CD8 T cells do, and this binding is dependent on the CD8 coreceptor. We found that the extent of apoptosis in CD8 T and NK cells was inversely related to the expression of CD94, with lower levels of apoptosis seen in CD94(high) cells after 1-3 days of culture. The difference in CD8 T cell survival was evident as early as 6 h after culture and persisted until nearly all the CD94(neg/int) cells were apoptotic by 48 h. In contrast, expression of inhibitory Ly-49A,G2,C/I molecules was associated with higher levels of apoptosis. Cross-linking CD94/NKG2 receptors on CD8 T cells from a mouse undergoing an LM infection further reduced the percentage of apoptotic cells on the CD94-expressing populations, while cross-linking Ly-49I had no effect on CD8 T cells expressing Ly-49I. Cross-linking CD3 on CD8 T cells from a mouse undergoing a secondary LM infection increases the extent of apoptosis, but this is prevented by cross-linking CD94/NKG2 receptors at the same time. Similar results were observed with NK cells in that the CD94(high) population displayed less apoptosis than CD94(int) cells after 1-3 days in culture. Therefore, the expression of CD94/NKG2 is correlated with a lower level of apoptosis and may play an important role in the maintenance of CD8 T and NK cells.     

Cutting edge: CD94/NKG2 is expressed on Th1 but not Th2 cells and costimulates Th1 effector functions

Th1 and Th2 cells can be phenotypically distinguished by very few cell surface markers. To identify cell surface molecules that are specifically expressed on Th1 cells, we have generated a panel of mAbs that specifically bind the surfaces of murine Th1 but not Th2 cells. One of these Abs identified the NK cell receptor CD94 as a molecule also specifically expressed on the surface of Th1 cells. As in NK cells, CD94 is expressed on Th1 cells together with members of the NKG2 family of molecules, including NKG2A, C, and E. Cross-linking these receptors on differentiated Th1 cells in vitro costimulates proliferation and cytokine production with a potency similar to that obtained by cross-linking CD28. We propose that CD94/NKG2 heterodimers may costimulate effector functions of differentiated Th1 cells.     

TNK cells (NKG2D<Superscript>+</Superscript> CD8<Superscript>+</Superscript> or CD4<Superscript>+</Superscript> T lymphocytes) in the control of human tumors

Innate and adaptive immune responses have many interactions that are regulated by the balance of signals initiated by a variety of activatory and inhibitory receptors. Among these, the NKG2D molecule was identified as expressed by T lymphocytes, including most CD8⁺ cells and a minority of CD4⁺ cells, designated TNK cells in this paper. Tumor cells may overexpress the stress-inducible NKG2D ligands (NKG2DLs: MICA/B, ULBPs) and the NKG2D signaling has been shown to be involved in lymphocyte-mediated anti-tumor activity. Aberrant expression of NKG2DLs by cancer cells, such as the release of soluble form of NKG2DLs, can lead to the impairment of these immune responses. Here, we discuss the significance of NKG2D in TNK-mediated anti-tumor activity. Our studies demonstrate that NKG2D⁺ T cells (TNK) are commonly recruited at the tumor site in melanoma patients where they may exert anti-tumor activity by engaging both TCR and NKG2D. Moreover, NKG2D and TCR triggering was also observed by peripheral blood derived T lymphocyte- or T cell clone-mediated tumor recognition, both in melanoma and colorectal cancer (CRC) patients. Notably, heterogeneous expression of NKG2DLs was found in melanoma and CRC cells, with a decrease of these molecules along with tumor progression. Therefore, through the mechanisms that govern NKG2D engagement in anti-tumor activity and the expression of NKG2DLs by tumor cells that still need to be dissected, we showed that NKG2D expressing TNK cells are a relevant T cell subtype for immunosurveillance of tumors and we propose that new immunotherapeutic interventions for cancer patients should be aimed also at enhancing NKG2DLs expression by tumor cells. This paper is a focused research review based on a presentation given at the sixth annual meeting of the Association for Immunotherapy of Cancer (CIMT), held in Mainz, Germany, 15–16 May 2008.     

Human IgG1 antibodies antagonizing activating receptor NKG2D on natural killer cells

NKG2D is a surface receptor expressed on NK cells but also on CD8⁺ T cells, γδ T cells, and auto-reactive CD4⁺/CD28⁻ T cells of patients with rheumatoid arthritis. Various studies suggested that NKG2D plays a critical role in autoimmune diseases, e.g., in diabetes, celiac disease and rheumatoid arthritis (RA), rendering the activating receptor a potential target for antibody-based therapies. Here, we describe the generation and characteristics of a panel of human, high-affinity anti-NKG2D IgG1 monoclonal antibodies (mAbs) derived by phage display. The lead molecule mAb E4 bound with an affinity (K_D) of 2.7 ± 1.4 × 10⁻¹¹ M to soluble and membrane-bound human NKG2D, and cross-reacted with NKG2D from cynomolgus macaque, indicating potential suitability for studies in a relevant primate model. MAb E4 potently antagonized the cytolytic activity of NKL cells against BaF/3-MICA cells expressing NKG2D ligand, and blocked the NKG2D ligand-induced secretion of TNFα, IFNγ and GM-CSF, as well as surface expression of CRTAM by NK cells cultured on immobilized MICA or ULBP-1 ligands. The antibody did not show a detectable loss of binding to NKG2D after seven days in human serum at 37°C, and resisted thermal inactivation up to 70°C. Based on these results, anti-human NKG2D mAb E4 provides an ideal candidate for development of a novel therapeutic agent antagonizing a key receptor of NK and cytotoxic T cells with implications in autoimmune diseases.Key words: NKG2D, NK cell, T cell, monoclonal antibody, human IgG1, humanization, phage display, autoimmune disease     

Functional Dichotomy between NKG2D and CD28-Mediated Co-Stimulation in Human CD8+ T Cells

Both CD28 and NKG2D can function as co-stimulatory receptors in human CD8⁺ T cells. However, their independent functional contributions in distinct CD8⁺ T cell subsets are not well understood. In this study, CD8⁺ T cells in human peripheral blood- and lung-derived lymphocytes were analyzed for CD28 and NKG2D expression and function. We found a higher level of CD28 expression in PBMC-derived naïve (CD45RA⁺CD27⁺) and memory (CD45RA⁻CD27⁺) CD8⁺ T cells (CD28^Hi), while its expression was significantly lower in effector (CD45RA⁺CD27⁻) CD8⁺ T cells (CD28^Lo). Irrespective of the differences in the CD28 levels, NKG2D expression was comparable in all three CD8⁺ T cell subsets. CD28 and NKG2D expressions followed similar patterns in human lung-resident GILGFVFTL/HLA-A2-pentamer positive CD8⁺ T cells. Co-stimulation of CD28^Lo effector T cells via NKG2D significantly increased IFN-γ and TNF-α levels. On the contrary, irrespective of its comparable levels, NKG2D-mediated co-stimulation failed to augment IFN-γ and TNF-α production in CD28^Hi naïve/memory T cells. Additionally, CD28-mediated co-stimulation was obligatory for IL-2 generation and thereby its production was limited only to the CD28^Hi naïve/memory subsets. MICA, a ligand for NKG2D was abundantly expressed in the tracheal epithelial cells, validating the use of NKG2D as the major co-stimulatory receptor by tissue-resident CD8⁺ effector T cells. Based on these findings, we conclude that NKG2D may provide an expanded level of co-stimulation to tissue-residing effector CD8⁺ T cells. Thus, incorporation of co-stimulation via NKG2D in addition to CD28 is essential to activate tumor or tissue-infiltrating effector CD8⁺ T cells. However, boosting a recall immune response via memory CD8⁺ T cells or vaccination to stimulate naïve CD8⁺ T cells would require CD28-mediated co-stimulation.     

Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E.

The lytic function of human natural killer (NK) cells is markedly influenced by recognition of class I major histocompatibility complex (MHC) molecules, a process mediated by several types of activating and inhibitory receptors expressed on the NK cell. One of the most important of these mechanisms of regulation is the recognition of the non-classical class I MHC molecule HLA-E, in complex with nonamer peptides derived from the signal sequences of certain class I MHC molecules, by heterodimers of the C-type lectin-like proteins CD94 and NKG2. Using soluble, recombinant HLA-E molecules assembled with peptides derived from different leader sequences and soluble CD94/NKG2-A and CD94/NKG2-C proteins, the binding of these receptor-ligand pairs has been analysed. We show first that these interactions have very fast association and dissociation rate constants, secondly, that the inhibitory CD94/NKG2-A receptor has a higher binding affinity for HLA-E than the activating CD94/NKG2-C receptor and, finally, that recognition of HLA-E by both CD94/NKG2-A and CD94/NKG2-C is peptide dependent. There appears to be a strong, direct correlation between the binding affinity of the peptide-HLA-E complexes for the CD94/NKG2 receptors and the triggering of a response by the NK cell. These data may help to understand the balance of signals that control cytotoxicity by NK cells.     

Distribution of Several Activating and Inhibitory Receptors on CD3<Superscript>−</Superscript>CD16<Superscript>+</Superscript> NK Cells and Their Correlation with NK Cell Function in Healthy Individuals

The aim of this study was to estimate the distribution and density of a representative set of activating and inhibitory receptors on gated natural killer (NK) cells, as well as on their bright and dim subsets, and to correlate the receptor expression with NK cell activity for healthy individuals on CD3⁻CD16⁺ NK cells. We show that in 43 healthy controls NK cell activity against K562 target cells was 37.34% (E:T, 80:1) by standard chromium release assay. The expression of receptors on NK cells and their subsets was analyzed by flow cytometry. The cytotoxic CD3⁻CD16^bright NK subset constituted 78.97%, while the regulatory CD3⁻CD16^dim NK subset constituted 21.03% of NK cells. We show the distribution of NKG2D, CD161, CD158a, and CD158b receptors on CD3⁻CD16⁺ NK cells in peripheral blood lymphocytes (PBLs), on gated NK cells, and on the CD3⁻CD16^bright and CD3⁻CD16^dim subsets. Contrary to CD158a and CD158b killer immunoglobulin-like receptors (KIRs), there is a significant positive correlation of NKG2D and CD161 expression with NK cytotoxicity. We show the kinetics of change in CD3⁻CD16⁺NK/K562 conjugate composition, together with the stronger target binding capacity of CD16^bright NK cells. Furthermore, we show that after coculture of PBLs with K562 the expression of CD107a, a degranulation marker, on CD3⁻CD16⁺NK cells and subsets is time dependent and significantly higher on the cytotoxic CD3⁻CD16^bright NK subset. The novel data obtained regarding expression of NK cell activating and inhibitory receptors for healthy individuals may aid in detecting changes that are associated with various diseases.     

Orderly and nonstochastic acquisition of CD94/NKG2 receptors by developing NK cells derived from embryonic stem cells in vitro

In mice there are two families of MHC class I-specific receptors, namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently, how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study, we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers, but not Ly49 molecules, and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis, we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order, with their order of acquisition being first CD94; subsequently NKG2D, NKG2A, and NKG2E; and finally, NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells, and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells, suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic, orderly, and cumulative.     

Dynamic shift from CD85j/ILT-2 to NKG2D NK receptor expression pattern on human decidual NK during the first trimester of pregnancy

During the first trimester of human pregnancy, Natural Killer (NK) cells of the maternal uterine mucosa (e.g. decidua) have a unique phenotype and are involved in crucial physiological processes during pregnancy. We investigated whether modifications of the NK receptor repertoire occur during the first trimester of pregnancy. We found significantly decreased expression of KIR2DL1/S1 and KIR2DL2/L3/S2 receptors, NKp30 and NKp44 activatory receptors, and the CD85j (ILT-2) inhibitory receptor. We also observed significantly increased expression of the NKG2D activatory receptor at the decidual NK cell surface. By flow cytometry, we further highlighted an evolution of NK subsets between 8 and 12 weeks of gestation, with a shift from the KIR2DL1/S1⁺/KIR2DL2/L3/S2⁺ subset towards the double negative subset, coupled with a decrease of the CD85j⁺/NKG2D⁻ subset in favour of the CD85j⁻/NKG2D⁺ subset. Furthermore, cell surface expression of NK receptor ligands, including CD85j and NKG2D ligands, has been characterized by flow cytometry on decidual immune CD14⁺ and CD3⁺ cells. HLA-G, the high affinity ligand of CD85j, was detected on both cell types. In contrast, NKG2D ligands ULBP-2 ULBP-3 and MICA/B were not expressed on CD14⁺ and CD3⁺ cells, however a variable expression of ULBP-1 was observed. The ligand expression of KIR2DL1/S1 and KIR2DL2/L3/S2 was also analyzed: the HLA-C molecule was expressed at a low level on some CD14⁺ cells whereas it was not detected on CD3⁺ cell surface. NK receptor ligands are known to be also expressed on the invading placental trophoblast cells. Thus, the phenotypic evolutions of decidual NK cells described in this present study may preserve their activation/inhibition balance during the first trimester of pregnancy.     

Binding of sialyl Lewis X antigen to lectin-like receptors on NK cells induces cytotoxicity and tyrosine phosphorylation of a 17-kDa protein

BACKGROUND: Natural killer (NK) cells mediate cytotoxicity through cell-surface receptors including lectin-like receptors. We have investigated whether sialyl Lewis X (sLe(X)) antigen, Neu5Acalpha2,3Galbeta1,4(Fucalpha1,3) GlcNAc-R, can bind to the lectin-like receptors on human NK-derived KHYG cells, using transferrin secreted by human hepatoma-derived HepG2 cells (Hep-TF), whose N-glycans are rich in alpha1,3-fucosylated bi-, tri-, and tetra-antennary type complexes, and commercially available human transferrin (Nor-TF), which is comprised of bi-antennary N-glycans without alpha1,3-fucosylation. RESULTS: High sLeX-expressing erythroleukemia-derived K562 cells isolated from fucosyltransferase-3-transfected cells were 2.5-fold more susceptible than wild-type K562 cells to KHYG cells. Fluorescein isothiocyanate (FITC)-labeled Hep-TF bound 1.8-fold more strongly to KHYG cells than did FITC-labeled Nor-TF; the binding was suppressed by treatment with anti-NKG2D, anti-NKG2C, anti-CD94 and anti-CD161 antibodies. FITC-labeled Hep-TF bound more strongly to human monocyte-derived U937 cells transfected with NKG2D and CD94 than to wild-type U937 cells. Moreover, tyrosine phosphorylation of a 17-kDa protein in the KHYG cells was enhanced by incubation on a Hep-TF coated plate and treatment with an anti-NKG2D antibody, but not by a Nor-TF coated plate and an anti-CD94 antibody. CONCLUSION: The interaction of sLe(X) antigen with lectin-like receptors on NK cells induces cytotoxicity that is mediated through a tyrosine-phosphorylated 17-kDa protein.     

The Epidermal Growth Factor-like Domain of CD93 Is a Potent Angiogenic Factor

Human CD93, an epidermal growth factor (EGF)-like domain containing transmembrane protein, is predominantly expressed in the vascular endothelium. Studies have shown that AA4, the homolog of CD93 in mice, may mediate cell migration and angiogenesis in endothelial cells. Soluble CD93 has been detected in the plasma of healthy individuals. However, the role of soluble CD93 in the endothelium remains unclear. Recombinant soluble CD93 proteins with EGF-like domains (rCD93D123, with domains 1, 2, and 3; and rCD93D23, with domains 2 and 3) were generated to determine their functions in angiogenesis. We found that rCD93D23 was more potent than rCD93D123 in stimulating the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Production of matrix-metalloproteinase 2 increased after the HUVECs were treated with rCD93D23. Further, in a tube formation assay, rCD93D23 induced cell differentiation of HUVECs through phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase and extracellular signal-regulated kinases-1/2 signaling. Moreover, rCD93D23 promoted blood vessel formation in a Matrigel-plug assay and an oxygen-induced retinopathy model in vivo. Our findings suggest that the soluble EGF-like domain containing CD93 protein is a novel angiogenic factor acting on the endothelium.     

Differential expression of NK receptors CD94 and NKG2A by T cells in rheumatoid arthritis patients in remission compared to active disease

Objective

TNF inhibitors (TNFi) have revolutionised the treatment of rheumatoid arthritis (RA). Natural killer (NK) cells and Natural Killer Cell Receptor⁺ T (NKT) cells comprise important effector lymphocytes whose activity is tightly regulated through surface NK receptors (NKRs). Dysregulation of NKRs in patients with autoimmune diseases has been shown, however little is known regarding NKRs expression in patients with TNFi-induced remission and in those who maintain remission vs disease flare following TNFi withdrawal.

Methods

Patients with RA were recruited for this study, (i) RA patients in clinical remission following a minimum of one year of TNFi therapy (n = −15); (2) Active RA patients, not currently or ever receiving TNFi (n = 18); and healthy control volunteers (n = 15). Patients in remission were divided into two groups: those who were maintained on TNFi and those who withdrew from TNFi and maintained on DMARDS. All patients underwent full clinical assessment. Peripheral blood mononuclear cells were isolated and NKR (CD94, NKG2A, CD161, CD69, CD57, CD158a, CD158b) expression on T-(CD3⁺CD56⁻), NK-(CD3⁻CD56⁺) and NKT-(CD3⁺CD56⁺) cells was determined by flow cytometry.

Results

Following TNFi withdrawal, percentages and numbers of circulating T cells, NK cells or NKT cell populations were unchanged in patients in remission versus active RA or HCs. Expression of the NKRs CD161, CD57, CD94 and NKG2A was significantly increased on CD3⁺CD56-T cells from patients in remission compared to active RA (p<0.05). CD3⁺CD56-T cell expression of CD94 and NKG2A was significantly increased in patients who remained in remission compared with patients whose disease flared (p<0.05), with no differences observed for CD161 and CD57. CD3⁺CD56⁻ cell expression of NKG2A was inversely related to DAS28 (r = −0.612, p<0.005).

Conclusion

High CD94/NKG2A expression by T cells was demonstrated in remission patients following TNFi therapy compared to active RA, while low CD94/NKG2A were associated with disease flare following withdrawal of therapy.     

Implication of different effector mechanisms by cord blood–derived and peripheral blood–derived cytokine-induced killer cells to kill precursor B acute lymphoblastic leukemia cell lines

Background aimsCytokine-induced killer (CIK) cells ex vivo–expanded from cord blood (CB) or peripheral blood (PB) have been shown to be cytotoxic against autologous and allogeneic tumor cells. We have previously shown that CD56⁺ CIK cells (CD3⁺CD56⁺ and CD3⁻CD56⁺) are capable of killing precursor B-cell acute lymphoblastic leukemia (B-ALL) cell lines. However, the lytic pathways used by CD56⁺ PB and CB-CIK cells to kill B-ALL cell lines have not been studied.MethodsCB and PB-CIK cells were differentiated. CD56⁺ CB- and PB-CIK cells were compared for expression of different phenotypic markers and for the lytic pathways used to kill B-ALL cell lines.ResultsWe found that cytotoxic granule proteins were expressed at higher levels in CD56⁺ PB-CIK than in CD56⁺ CB-CIK cells. However, CD56⁺ CB-CIK cells expressed more tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) compared with CD56⁺ PB-CIK cells. We observed that CD56⁺ CB-CIK cells used both the NKG2D and TRAIL cytotoxic pathways and were more effective at killing REH cells than CD56⁺ PB-CIK cells that used only the NKG2D pathway. In contrast, CD56⁺ PB-CIK cells used both NKG2D and TRAIL pathways to kill NALM6 cells, whereas CD56⁺ CB-CIK cells used only the NKG2D pathway.ConclusionsOur results suggest that both the source of CIK and the type of B-ALL cell line have an impact on the intensity of the cytolytic activity and on the pathway used. These findings may have clinical implications with respect to optimizing therapeutic efficacy, which may be dependent on the source of the CIK cells and on the target tumor cells.     

Expression of inhibitory receptors Ly49E and CD94/NKG2 on fetal thymic and adult epidermal TCR V gamma 3 lymphocytes

Ly49 and CD94/NKG2 inhibitory receptors are predominantly expressed on murine NK cells, but they are also expressed on a subpopulation of peripheral CD8 memory TCR alphabeta lymphocytes. In this study we demonstrate that Ly49E and CD94/NKG2 receptors are expressed on mature TCR Vgamma3(+) cells in the fetal thymus. Expression correlated with a memory phenotype, such as expression of CD44, 2B4, and IL-2Rbeta (CD122), and absence of IL-2Ralpha (CD25) expression. No expression of Ly49A, C, D, G2, or I receptors was observed. This phenotype is similar to that of fetal thymic NK cells. Skin-located Vgamma3 T cells, the progeny of fetal thymic Vgamma3 cells, also expressed CD94/NKG2 and Ly49E but not the other members of the Ly49 family. The development and survival of Ly49E(+) or CD94/NKG2(+) Vgamma3 T lymphocytes was not dependent upon expression of MHC class I molecules. The cytotoxicity of TCR Vgamma3 cells was inhibited when Qdm, the ligand for CD94/NKG2, was presented by Qa1(b)-transfected target cells. Also, upon cross-linking of CD94/NKG2 with mAb 3S9, TCR Vgamma3 thymocytes were prevented from killing FcgammaR(+) P815 target cells. These effects were most pronounced in the CD94/NKG2(high) subpopulation as compared with the CD94/NKG2(low) subpopulation of Vgamma3 cells. Our data demonstrate that Vgamma3 T cells expressing inhibitory Ly49E and CD94/NKG2 receptors are mature and display a memory phenotype, and that CD94/NKG2 functions as an inhibitory receptor on these T lymphocytes.