Expression of NK Cell Surface Receptors in Breast Cancer Tissue as Predictors of Resistance to Antineoplastic Treatment.

BACKGROUND: Currently, one of the most used strategies for the treatment of newly diagnosed patients with breast cancer is neoadjuvant chemotherapy based on the application of taxanes and anthracyclines. However, despite the high number of patients who develop a complete pathological clinical response, resistance and relapse following this therapy continue to be a clinical challenge. As a component of the innate immune system, the cytotoxic function of Natural Killer (NK) cells plays an important role in the elimination of tumor cells. However, the role of NK cells in resistance to systemic therapy in breast cancer remains unclear. The present project aims to evaluate the gene expression profile of human NK cells in breast cancer tissue resistant to treatment with taxanes-anthracyclines. METHODS: Biopsies from tumor tissues were obtained from patients with breast cancer without prior treatment. Histopathological analysis and ex vivo exposure to antineoplastic chemotherapeutics were carried out. Alamar blue and lactate dehydrogenase release assays were performed for quantitative analysis of tumor viability. Gene expression profiles from tumor tissues without prior exposure to therapeutic drugs were analyzed by gene expression microarrays and verified by polymerase chain reaction. RESULTS: A significant decrease in gene expression of cell-surface receptors related to NK cells was observed in tumor samples resistant to antineoplastic treatment compared with those that were sensitive to treatment. CONCLUSION: A decrease in NK cell infiltration into tumor tissue might be a predictive marker for failure of chemotherapeutic treatment in breast cancer.

Cell Surface Downregulation of NK Cell Ligands by Patient-Derived HIV-1 Vpu and Nef Alleles.

OBJECTIVE: HIV-1 Vpu and Nef proteins downregulate cell surface levels of natural killer (NK) cell ligands but functional consequences of individual downregulation events are unclear. We tested how well-conserved NK cell ligand downregulation is among Vpu and Nef variants isolated from chronic HIV patients. METHODS: Proviral vpu and nef sequences were amplified from 27 chronic HIV patients, subcloned, and tested for their ability to downregulate cell surface receptors. RESULTS: Cell surface downregulation of CD4, CD317/tetherin, and major histocompatibility complex class 1 that exert biological functions other than NK cell activation were well conserved among patient-derived Vpu and Nef variants. Among NK cell ligands, NK-T-B-antigen, poliovirus receptor, and UL16-binding protein were identified as main targets for Vpu and Nef, the downregulation of which by at least 1 viral protein was highly conserved. NK cell ligands displayed specific sensitivity to Vpu (NK-T-B-antigen) or Nef (poliovirus receptor), and downregulation of cell surface UL16-binding protein was identified as a novel and highly conserved activity of HIV-1 Vpu but not Nef. CONCLUSIONS: The conservation of downregulation of major NK cell ligands by either HIV-1 Vpu or Nef suggests an important pathophysiological role of this activity, which may impact the acute but not the chronic phase of HIV infection.

Tissue-Specific Education of Decidual NK Cells.

During human pregnancy, fetal trophoblast cells invade the decidua and remodel maternal spiral arteries to establish adequate nutrition during gestation. Tissue NK cells in the decidua (dNK) express inhibitory NK receptors (iNKR) that recognize allogeneic HLA-C molecules on trophoblast. Where this results in excessive dNK inhibition, the risk of pre-eclampsia or growth restriction is increased. However, the role of maternal, self-HLA-C in regulating dNK responsiveness is unknown. We investigated how the expression and function of five iNKR in dNK is influenced by maternal HLA-C. In dNK isolated from women who have HLA-C alleles that carry a C2 epitope, there is decreased expression frequency of the cognate receptor, KIR2DL1. In contrast, women with HLA-C alleles bearing a C1 epitope have increased frequency of the corresponding receptor, KIR2DL3. Maternal HLA-C had no significant effect on KIR2DL1 or KIR2DL3 in peripheral blood NK cells (pbNK). This resulted in a very different KIR repertoire for dNK capable of binding C1 or C2 epitopes compared with pbNK. We also show that, although maternal KIR2DL1 binding to C2 epitope educates dNK cells to acquire functional competence, the effects of other iNKR on dNK responsiveness are quite different from those in pbNK. This provides a basis for understanding how dNK responses to allogeneic trophoblast affect the outcome of pregnancy. Our findings suggest that the mechanisms that determine the repertoire of iNKR and the effect of self-MHC on NK education may differ in tissue NK cells compared with pbNK.

Activin A as a mediator of NK-dendritic cell functional interactions.

The interaction of NK cells with dendritic cells (DCs) results in reciprocal cell activation through the interaction of membrane proteins and the release of soluble factors. In this article, we report that in NK-DC cocultures, among a set of 84 cytokines investigated, activin A was the second highest induced gene, with CXCL8 being the most upregulated one. Activin A is a member of the TGF-ß superfamily and was previously shown to possess both proinflammatory and anti-inflammatory activities. In NK-DC cocultures, the induction of activin A required cell contact and was dependent on the presence of proinflammatory cytokines (i.e., IFN-γ, TNF-α, and GM-CSF), as well as on NK cell-mediated DC killing. CD1(+) DCs were the main activin A producer cells among myeloid blood DC subsets. In NK-DC cocultures, inhibition of activin A by follistatin, a natural inhibitory protein, or by a specific blocking Ab, resulted in the upregulation of proinflammatory cytokine release (i.e., IL-6, IL-8, TNF-α) by DCs and in the increase of DC maturation. In conclusion, our study reports that activin A, produced during NK-DC interactions, represents a relevant negative feedback mechanism that might function to prevent excessive immune activation by DCs.

Heat shock protein 90 is critical for regulation of phenotype and functional activity of human T lymphocytes and NK cells.

The 90-kDa heat shock protein (Hsp90) has become an important therapeutic target with ongoing evaluation in a number of malignancies. Although Hsp90 inhibitors have a high therapeutic index with limited effects on normal cells, they have been described to inhibit dendritic cell function. However, its effect on human immune effector cells may have significant clinical implications, but remains unexplored. In this study, we have evaluated the effects of Hsp90 inhibition on human T lymphocyte and NK cells, including their Ag expression, activation, proliferation, and functional activities. These studies demonstrate that Hsp90 inhibition irreversibly downregulates cell surface expression of critical Ags (CD3, CD4, CD8), the costimulatory molecule (CD28, CD40L), and αß receptors on T lymphocytes, as well as activating receptors (CD2, CD11a, CD94, NKp30, NKp44, NKp46, KARp50.3) on NK cells. Hsp90 inhibition significantly reduced CD4 protein expression on T lymphocytes at both the cell surface and intracellular level, which was shown to be associated with aberrant regulation of Src-kinase p56(Lck). Downregulation of the Ags triggered by Hsp90 inhibition on CD3(+) T lymphocytes, both in CD4(+) and CD8(+) T cell subsets, was associated with a disruption in their cellular activation, proliferation, and/or IFN-γ production, when the inhibition occurred either in activated or inactivated cells. In addition, downregulation of key activating receptors on NK cells following Hsp90 inhibition resulted in decreased cytotoxicity against tumor cells. Therefore, these observations demonstrate the need to closely monitor immune function in patients being treated with a Hsp90 inhibitor and may provide a potential therapeutic application in autoimmune diseases.

Reduced release and binding of perforin at the immunological synapse underlies the age-related decline in natural killer cell cytotoxicity.

Physiological aging is accompanied by a marked reduction in natural killer (NK) cell cytotoxicity (NKCC) at the single cell level, but the underlying mechanisms are unknown. To address this issue, we isolated NK cells from healthy young (≤ 35 years) and old (≤ 60 years) subjects and examined the effect of age on events fundamental to the process of NKCC. Simultaneous assessment of NKCC and NK cell-target cell conjugate formation revealed a marked age-associated decline in NK cell killing but comparable conjugate formation, indicating a post-target cell binding defect was responsible for impaired NKCC. Despite a reduction in the proportion of NK cells expressing the activatory receptor NKp46, NK cells from old donors were not hyporesponsive to stimulation, as no age-associated difference was observed in the expression of the early activation marker CD69 following target cell coculture. Furthermore, intracellular levels of the key cytotoxic effector molecules perforin and granzyme B, and the fusion of secretory lysosomes with the NK cell membrane were also similar between the two groups. However, when we examined the binding of the pore-forming protein perforin to the surface of its target cell, an event that correlated strongly with target cell lysis, we found the percentage of perforin positive target cells was lower following coculture with NK cells from old subjects. Underlying this reduction in binding was an age-associated impairment in perforin secretion, which was associated with defective polarization of lytic granules towards the immunological synapse. We propose that reduced perforin secretion underlies the reduction in NKCC that accompanies physiological aging.

Gene deregulation and chronic activation in natural killer cells deficient in the transcription factor ETS1.

Multiple transcription factors guide the development of mature functional natural killer (NK) cells, yet little is known about their function. We used global gene expression and genome-wide binding analyses combined with developmental and functional studies to unveil three roles for the ETS1 transcription factor in NK cells. ETS1 functions at the earliest stages of NK cell development to promote expression of critical transcriptional regulators including T-BET and ID2, NK cell receptors (NKRs) including NKp46, Ly49H, and Ly49D, and signaling molecules essential for NKR function. As a consequence, Ets1(-/-) NK cells fail to degranulate after stimulation through activating NKRs. Nonetheless, these cells are hyperresponsive to cytokines and have characteristics of chronic stimulation including increased expression of inhibitory NKRs and multiple activation-associated genes. Therefore, ETS1 regulates a broad gene expression program in NK cells that promotes target cell recognition while limiting cytokine-driven activation.

Effect of azithromycin on natural killer cell function.

Azithromycin (AZM), a macrolide antibiotic for treating mycoplasma infections, may exhibit anti-inflammatory activity aside from its antimicrobial effect, providing additional therapeutic benefit. Natural killer (NK) cells, a first-line innate immune defense against microbial invasions, paradoxically exert a detrimental effect in protecting mycoplasma infection. Little was known regarding the effect of AZM on NK cells. In the present study, we investigated the ability of azithromycin to influence natural killer (NK) cell function with regard to activation, apoptosis and cytotoxic function. AZM had little effect on NK receptor expression and cytotoxic function of NK-92 cells. However, AZM did show a dose-dependent suppression on IL-15-induced CD69 expression of primary NK cells. AZM inhibited the cytotoxicity against K562 cells of resting and IL-15 activated primary NK cells possibly through down-regulation of perforin expression, especially on CD16(+)CD56(+) NK subsets. AZM exerted a dose-dependent inhibition of IFN-gamma and TNF-alpha production from NK-92 cells, but did not affect the cytokine production of IL-15 activated primary NK cells. Taken together, AZM down-regulates NK cytotoxicity and cytokine production and may provide therapeutic benefits aside from its antimicrobial activity.

Expression of natural killer cell activating receptors in patients with chronic lymphocytic leukaemia.

Recent advances in chronic lymphocytic leukaemia (CLL) treatment, more particularly through upfront use of anti-CD20 monoclonal antibodies, have prolonged patient progression-free survival. Nonetheless, apart from allogeneic stem cell transplantation, no curative treatment is available. One possible explanation for the lack of cure in CLL could be a defective immune anti-tumour response. As the result of abnormal HLA class I molecule expression, CLL cells escape from specific T-lymphocyte immunity but should be the target for the innate natural killer (NK) cell-mediated immune response. Defective NK cytotoxicity as the result of decreased expression of the natural cytotoxicity receptors (NCRs) NKp30/NCR3, NKp44/NCR2 and NKp46/NCR1 has been described in haematological malignancies such as acute myeloid leukaemia. This prompted us to focus our attention on NCR expression on NK cells from patients with CLL. Although we failed to detect any difference between CLL patients and healthy age-matched controls, a precise analysis of clinical data showed a correlation between decreased NCR expression and poor prognosis factors such as low haemoglobin level, high (>30×10(9) per litre) lymphocyte count or elevated C-reactive protein. Together, these observations support the rationale for restoration of normal NK cell functions in patients with CLL, putatively through the use of immune therapy protocols that already have demonstrated some benefit in acute myeloid leukaemia such as interleukin-2 plus histamine dihydrochloride.

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.

SHIP influences signals from CD48 and MHC class I ligands that regulate NK cell homeostasis, effector function, and repertoire formation.

Previously, we showed that 2B4 is a dominant inhibitory receptor in SHIP-deficient NK cells that prevents efficient cytolysis of complex targets. We show in this study that 2B4 deficiency restores homeostatic control and cytolytic function to SHIP-deficient NK cells. However, 2B4(-/-)SHIP(-/-) NK cells still exhibit a profound disruption of their NK receptor repertoire and are compromised for induction of IFN-gamma by several NK-activating receptors, including NKp46, NK.1.1, and NKG2D. In addition, we find that 2B4(-/-) NK cells have an extensively disrupted repertoire, including a supernormal frequency of NKp46(+) NK cells. Consequently IFN-gamma is induced on a much higher percentage of 2B4(-/-) NK cells following engagement of NKp46. We also find that both SHIP and 2B4 are required to prevent expression of Ly49B, a myeloid lineage MHC class I receptor not normally expressed by the NK lineage. Finally, when SHIP-deficient NK cells are on an H-2(d) background, they exhibit supernormal levels of Ly49A and possess normal cytolytic function against MHC-matched tumor targets and enhanced cytolysis of MHC mismatched tumor targets. However, despite normal or elevated cytolytic function, H2d SHIP(-/-) NK cells exhibit poor induction of IFN-gamma like their H2b(+) or 2B4(-/-) counterparts, demonstrating a uniform requirement for SHIP in induction of IFN-gamma downstream of key NK activating receptors. These findings reveal a complex interplay of SHIP, 2B4, and MHC in the regulation of homeostasis, effector function, and repertoire formation in the NK cell lineage.

CD8+ NK cells are predominant in chimpanzees, characterized by high NCR expression and cytokine production, and preserved in chronic HIV-1 infection.

HIV-1 infection in humans results in an early and progressive NK cell dysfunction and an accumulation of an "anergic" CD56- CD16+ NK subset, which is characterised by low natural cytotoxicity receptor expression and low cytokine producing capacity. In contrast to humans, chimpanzee NK cells do not display a distinguishable CD56(bright) and CD56(dim) subset but, as shown here, could be subdivided into functionally different CD8+ and CD8- subsets. The CD8+ NK cells expressed significantly higher levels of triggering receptors including NKp46 and, upon in vitro activation, produced more IFN-gamma, TNF-alpha and CD107 than their CD8- counterparts. In addition, chimpanzee CD8- NK cells had relatively high levels of HLA-DR expression, suggestive of an activated state. Killing inhibitory receptors were expressed only at low levels; however, upon in vitro stimulation, they were up-regulated in CD8+ but not in CD8- NK cells and were functionally capable of inhibiting NKp30-triggered killing. In contrast to HIV-1-infected humans, infected chimpanzees maintained their dominant CD8+ NK cell population, with high expression of natural cytotoxicity receptors.

IL-2-driven regulation of NK cell receptors with regard to the distribution of CD16+ and CD16- subpopulations and in vivo influence after haploidentical NK cell infusion.

To characterize natural killer (NK) cell subpopulations during activation, we analyzed the NK cell receptor repertoire and functionality of purified clinical scale CD56CD3 donor NK cells during stimulation with 1000 U/mL interleukin (IL)-2 for up to 14 days. In a phase I/II trial, we investigated the efficacy and feasibility of nonidentical NK cell infusion in patients with neuroblastoma after haploidentical stem cell transplantation. After IL-2 stimulation, large differences in the distribution of CD16 and CD16 subpopulations were found in 12 donors. Thereby, surface expression for all natural cytotoxicity receptors (NCRs) and NKG2D increased. In addition, killer cell immunoglobulin-like receptor (KIR) NK cells were overgrown by KIR proportion and the homing receptor CD62L was lost during stimulation. NK cell cytotoxicity against K562 and neuroblastoma cells increased and significantly higher cytokine secretion (eg, interferon-gamma, tumor necrosis factor-beta, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta) was observed after IL-2 stimulation compared with freshly isolated NK cells. However, NK cells of donors showing an initially enhanced cytotoxicity combined with NCR and CD69 expression, seemed to be exhausted and did not favor a stimulation period over 9 days. When IL-2-stimulated NK cells were given to transplant recipients, they induced a decrease of peripheral blood NK, in particular of CD56-NK cells. Our data indicate that IL-2 stimulation increases the expression of activating receptors and emphasizes mechanisms beside KIR/human leukocyte antigen. Furthermore, the results suggest that the expansion period of purified NK cells has to be individualized to optimize NK cell 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.

Interleukin-15 enhances natural killer cell cytotoxicity in patients with acute myeloid leukemia by upregulating the activating NK cell receptors.

Interleukin-15 (IL-15) has a major role in NK-cell homeostasis. Modulation of the relative frequency and expression intensity of the NK-cell receptors by IL-15 may increase NK cell-mediated cytotoxicity in cancer patients. We investigated the receptor repertoire and measured NK-cell activity in newly diagnosed AML patients and evaluated the ex vivo effects of IL-15. The expression of the activating NK cell receptors was significantly decreased in the AML patients compared to that in NK cells of healthy donors. When NK cells obtained from AML patients were cultured with IL-15, expression of the activating receptors was significantly upregulated compared to pre-culture levels. Concomitantly, cytotoxic activity of NK cells against autologous leukemic blasts increased following IL-15 stimulation. This IL-15 induced increase in activity was blocked by neutralizing antibodies specific for the NK cell activating receptors. These pre-clinical data support the future use of IL-15 for NK cell- based therapies for AML patients.

Low NKp30, NKp46 and NKG2D expression and reduced cytotoxic activity on NK cells in cervical cancer and precursor lesions.

BACKGROUND: Persistent high risk HPV infection can lead to cervical cancer, the second most common malignant tumor in women worldwide. NK cells play a crucial role against tumors and virus-infected cells through a fine balance between activating and inhibitory receptors. Expression of triggering receptors NKp30, NKp44, NKp46 and NKG2D on NK cells correlates with cytolytic activity against tumor cells, but these receptors have not been studied in cervical cancer and precursor lesions. The aim of the present work was to study NKp30, NKp46, NKG2D, NKp80 and 2B4 expression in NK cells from patients with cervical cancer and precursor lesions, in the context of HPV infection. METHODS: NKp30, NKp46, NKG2D, NKp80 and 2B4 expression was analyzed by flow cytometry on NK cells from 59 patients with cervical cancer and squamous intraepithelial lesions. NK cell cytotoxicity was evaluated in a 4 hour CFSE/7-AAD flow cytometry assay. HPV types were identified by PCR assays. RESULTS: We report here for the first time that NK cell-activating receptors NKp30 and NKp46 are significantly down-regulated in cervical cancer and high grade squamous intraepithelial lesion (HGSIL) patients. NCRs down-regulation correlated with low cytolytic activity, HPV-16 infection and clinical stage. NKG2D was also down-regulated in cervical cancer patients. CONCLUSION: Our results suggest that NKp30, NKp46 and NKG2D down-regulation represent an evasion mechanism associated to low NK cell activity, HPV-16 infection and cervical cancer progression.

NK cells recognize and kill human glioblastoma cells with stem cell-like properties.

In this study, cancer cells were isolated from tumor specimens of nine glioblastoma patients. Glioblastoma cells, cultured under suitable culture conditions, displayed markers typical of neural stem cells, were capable of partial multilineage differentiation in vitro, and gave origin to infiltrating tumors when orthotopically injected in NOD/SCID mice. These cells, although resistant to freshly isolated NK cells, were highly susceptible to lysis mediated by both allogeneic and autologous IL-2 (or IL-15)-activated NK cells. Indeed, all stem cell-cultured glioblastoma cells analyzed did not express protective amounts of HLA class I molecules, while expressing various ligands of activating NK receptors that triggered optimal NK cell cytotoxicity. Importantly, glioblastoma stem cells expressed high levels of PVR and Nectin-2, the ligands of DNAM-1-activating NK receptor.