Circulating IL-6 is associated with disease progression in BRAFwt metastatic melanoma patients receiving anti-PD-1 therapy.

AIMS: Despite efficacy of anti-PD-1 blockade in treatment of metastatic melanoma (MM), many patients achieve rapid disease progression (DP). Therefore, the aim of this study is to better define biomarkers for DP by analysing levels of circulating cytokines TGF-ß, IFN-γ, IL-6, IL-8 and IL-10 in MM patients prior to anti-PD-1 therapy. METHODS: Cytokine levels were evaluated before therapy with pembrolizumab in peripheral blood of BRAF wild-type (wt) MM patients by ELISA method. RESULTS: In this study, we give pretherapy levels for circulating TGF-ß, IFN-γ, IL-6, IL-8 and IL-10 in BRAFwt MM patients and analyse them according to metastasis stage (M1a+M1 b, M1c, M1d groups), lactate dehydrogenase (LDH) level and occurrence of DP. Increased IL-6 level was found in M1d group (central nervous system metastasis), while LDH+patients (LDH ≥460 IU/L) have increased IL-6 and IL-8 values that correlate with LDH level. Also, IL-6 correlates with C reactive protein values. Furthermore, patients with DP have significantly higher IL-6 level compared with non-DP patients. Conversely, the other analysed cytokines are similar in investigated groups of MM patients. By receiver operating characteristics curve analysis, pretherapy IL-6 level was found to be a biomarker for the occurrence of DP with cut-off value of 3.02 pg/mL. Patients in M1d stage are prevalent in the group with IL-6 ≥3.02 pg/mL that is characterised with reduced progression-free survival and higher pretherapy IL-8 and LDH. CONCLUSION: The evidence in this study implies that baseline IL-6 could be a biomarker of DP and poor prognosis in BRAFwt MM patients treated with pembrolizumab.

Circulating cytokine dynamics as potential biomarker of response to anti-PD-1 immunotherapy in BRAFwt MM patients.

BACKGROUND: The biomarkers of immune checkpoint inhibitors (ICIs) efficacy and safety are still urgently needed. As cytokines are easily detected and monitored in circulation, they could be used as potential predictors of response and immune-related adverse events (irAEs) for ICIs therapy. METHODS: The levels of TGF-ß, IFN-γ, IL-6, IL-8, IL-10 were measured in sera and plasma by ELISA method of 30 healthy controls (HC) and 32 BRAF wild type (wt) MM patients before and after every 12 weeks of Pembrolizumab, PD-1 inhibitor, until one year or disease progression (DP). RESULTS: Higher pretherapy levels of circulating TGF-ß, IFN-γ, IL-6, and IL-10 were shown in MM patients compared to HC. In patients with disease control, TGF-ß and IL-6 first decreased during the therapy, while then they started to successively increase reaching the initial values by the end of the follow up. Furthermore, in this group of patients IFN-γ increased, while IL-8 and IL-10 decreased at final points of the follow up. In patients with DP IL-6 increased at the time of progression, while IL-8 decreased when the best response was achieved. In patients with pseudoprogression IL-6 and IL-10 significantly increased compared to the pretreatment values. Melanoma patients with irAEs had increased baseline values of TGF-ß, IFN-γ, IL-6, and IL-10 compared to HC. However, no significant changes in cytokines levels were found in these patients during therapy. CONCLUSIONS: Inflammatory cytokines monitoring in circulation of BRAFwt MM patients could help in the selection of patients who will have the benefit from Pembrolizumab therapy.

Increased circulating monocyte MDSCs positively correlate with serum Interleukin-10 in metastatic melanoma patients.

Numerous immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and inhibitory cytokines identified in melanoma microenvironment have the important role in immune escape. Therefore, in this study we analyzed monocytic (m)MDSCs in peripheral blood of metastatic melanoma (MM) patients. In peripheral blood of 35 MM patients and 30 healthy controls we analyzed percentage of CD14 + HLA-DR- mMDSCs in monocyte gate and the mean fluorescence intensity of Foxp3 in CD25 + CD4 + regulatory T cells by Flow cytometry. Serum levels of transforming growth factor beta, interferon-gamma, interleukin (IL)-6, IL-8, IL-10 are measured by ELISA assays. In this study MM patients have significantly higher percentage of CD14 + HLA-DR- mMDSCs, as well as increased the baseline mMDSC/PBMC subset (NK, T, B cells, monocytes) ratio. Although there is no significant difference in the percentage of mMDSCs between groups of MM patients with different localization of distant metastasis, patients with elevated serum lactate dehydrogenase (LDH) have statistically significant higher percentage of these cells compared to LDH negative patients. Furthermore, in MM patients there is statistically significant positive correlation between values of IL-10 and the percentage of mMDSCs, only. Therefore, therapeutics that target circulating mMDSCs and IL-10 may have a big importance in the improvement of antitumor immunity in MM patients.

Detection of humoral and cellular immune response to anti-SARS-CoV-2 BNT162b2 vaccine in breastfeeding women and naïve and previously infected individuals.

This study explored humoral and cellular responses to anti-SARS-CoV-2 BNT162b2 mRNA vaccine in breastfeeding women and naïve and seropositive individuals in the first six months after vaccination.Sixty-one volunteers vaccinated with two doses of the BNT162b2 mRNA vaccine were enrolled in the study. In-house developed ELISA was used for the quantification of SARS-CoV-2 RBD-specific antibodies. Cell surface marker expression and intracellular IFN-γ analysis were carried out by flow cytometry. The concentrations of IFN-γ, IL-6 and TNF were determined by ELISA. A significant rise in anti-RBD IgG antibody levels was observed 14 days after the first vaccine dose (p < 0.0001) in serum and milk. The expression of CD28 on CD4+ T cells was significantly higher compared to baseline (p < 0.05). There was a significant increase (p ≤ 0.05) in B cell lymphocyte subset after revaccination, and increased percentage of CD80+ B cells. The expression of IFN-γ in peripheral blood lymphocytes, CD3+ T cells and serum was significantly increased (p < 0.05). No significant difference in immune response was observed between breastfeeding women and other study participants. The anti-SARS-CoV-2 BNT162b2 mRNA vaccine-induced measurable and durable immune response in breastfeeding women and in naïve and previously infected individuals.

Dictionary Learning: A Novel Approach to Detecting Binary Black Holes in the Presence of Galactic Noise with LISA.

The noise produced by the inspiral of millions of white dwarf binaries in the Milky Way may pose a threat to one of the main goals of the space-based LISA mission: the detection of massive black hole binary mergers. We present a novel study for reconstruction of merger waveforms in the presence of Galactic confusion noise using dictionary learning. We discuss the limitations of untangling signals from binaries with total mass from 10^{2} M_{⊙} to 10^{4} M_{⊙}. Our method proves extremely successful for binaries with total mass greater than ∼3×10^{3} M_{⊙} up to redshift 3 in conservative scenarios, and up to redshift 7.5 in optimistic scenarios. In addition, consistently good waveform reconstruction of merger events is found if the signal-to-noise ratio is approximately 5 or greater.

Does pre-existing immunity determine the course of SARS-CoV-2 infection in health-care workers? Single-center experience.

OBJECTIVES: To assess the severity of symptoms, duration of infection and viral loads of health-care workers (HCWs) who tested positive for Coronavirus disease 2019 (COVID-19) during Omicron's prevalence, in regard to vaccination and previous infection. METHODS: During 2 weeks of highest rate of COVID-19 cases in Bosnia and Herzegovina, the positive nasopharyngeal swabs were analysed in 141 HCWs by reverse transcription quantitative PCR, targeting four different genes: RdRp, E, N and nsp14. Uniformed questionnaire was used to collect relevant sociodemographic and epidemiological data from HCWs divided into four groups: unvaccinated/not previously infected (group 1); unvaccinated/previously infected (group 2); vaccinated/not previously infected (group 3); and vaccinated/previously infected (group 4). RESULTS: We observed that occurrence of fever and smell or taste loss were more frequent in group 1 (86.4% and 25%) and group 3 (76.9% and 19.2%), in comparison to group 2 (64.4% and 6.7%) and group 4 (69.2% and 3.8%), (p = 0.023 and p = 0.003). Although statistically not significant, group 2 (61.9%), group 3 (65.4%), and group 4 (70.8%) experienced negativization within 7 days of positive RT-qPCR test, whereas 51.2% of HCWs from group 1 tested negative later on. There is no significant difference between all four groups regarding Ct values of analysed genes. CONCLUSION: During Omicron's prevalence, the vaccination had less substantial effect on symptomatic disease among HCWs, while fever and loss of smell or taste were considerably less likely to occur upon reinfection. Since viral loads and negativization periods do not seem to significantly vary, irrespective of pre-existing immunity, systemic vaccination and mask-wearing should still be considered among HCWs.

Role of Histone Deacetylase 6 and Histone Deacetylase 6 Inhibition in Colorectal Cancer.

Histone deacetylase 6 (HDAC6), by deacetylation of multiple substrates and association with interacting proteins, regulates many physiological processes that are involved in cancer development and invasiveness such as cell proliferation, apoptosis, motility, epithelial to mesenchymal transition, and angiogenesis. Due to its ability to remove misfolded proteins, induce autophagy, and regulate unfolded protein response, HDAC6 plays a protective role in responses to stress and enables tumor cell survival. The scope of this review is to discuss the roles of HDCA6 and its implications for the therapy of colorectal cancer (CRC). As HDAC6 is overexpressed in CRC, correlates with poor disease prognosis, and is not essential for normal mammalian development, it represents a good therapeutic target. Selective inhibition of HDAC6 impairs growth and progression without inducing major adverse events in experimental animals. In CRC, HDAC6 inhibitors have shown the potential to reduce tumor progression and enhance the therapeutic effect of other drugs. As HDAC6 is involved in the regulation of immune responses, HDAC6 inhibitors have shown the potential to improve antitumor immunity by increasing the immunogenicity of tumor cells, augmenting immune cell activity, and alleviating immunosuppression in the tumor microenvironment. Therefore, HDAC6 inhibitors may represent promising candidates to improve the effect of and overcome resistance to immunotherapy.

Increased circulating TGF-ß1 is associated with impairment in NK cell effector functions in metastatic melanoma patients.

Transforming growth factor beta (TGF-ß) plays a complex role in carcinogenesis. In 30 melanoma patients and 20 healthy controls (HC) we analysed functional and phenotypic characteristics of NK cells by Flow cytometry, gene expression of TGF-ß1 in peripheral blood mononuclear cells by qPCR and serum and supernatant level of free TGF-ß1 by ELISA. Melanoma patients had significantly higher serum level of circulatingTGF-ß1 compared to HC, especially those with metastasis into the central nervous system (subclass M1d) and high LDH serum values. Melanoma patients compared to HC had significantly higher level of TGF-ß1 gene in PBMC. TGF-ß1 serum values negatively correlate with NK cell activity analysed by CD107a (degranulation marker), IFN-γ, NKG2D, and NKp46 in patients. Study shows the association of high level of TGF-ß1 with NK cell inhibition in patients represents the main mechanism of tumour immune evasion. Targeting TGF-ß may become an important cancer treatment for improving antitumor immunity.

Cross-Talk Between Tumor Cells Undergoing Epithelial to Mesenchymal Transition and Natural Killer Cells in Tumor Microenvironment in Colorectal Cancer.

Tumor cells undergoing epithelial to mesenchymal transition (EMT) and immune cells in tumor microenvironment (TME) reciprocally influence each other. Immune cells, by supplying TME with bioactive molecules including cytokines, chemokines, enzymes, metabolites, and by physical interactions with tumor cells via their receptors, represent an important factor that affects EMT. Chronical inflammation in TME favorizes tumor growth and invasiveness and stimulates synthesis of EMT promoting transcription factors. Natural killer (NK) cells, owing to their unique ability to exert cytotoxic function independent of major histocompatibility (MHC)-mediated antigen presentation, play a significant role in the control of metastasis in colorectal cancer (CRC). Although, the cross-talk between immune cells and tumor cells in general favors the induction of EMT and inhibition of antitumor immune responses, there are some changes in the immunogenicity of tumor cells during EMT of CRC cells that increase their susceptibility to NK cell cytotoxic lysis. However, suppressive TME downmodulates the expression of activating NK cell receptors, decreases the expression of activating and increases the expression of inhibitory NK cell ligands on tumor cells, and impairs NK cell metabolism that altogether negatively affects the overall NK cell function. Furthermore, process of EMT is often associated with increased expression of programmed cell death ligand (PD-L) and expression of immune checkpoint molecules PD-1, TIGIT, and TIM3 on functionally exhausted NK cells in TME in CRC. In this review we discuss modalities of cross-talk between tumor cells and NK cells, with regard of EMT-driven changes.

Implications for First-Order Cosmological Phase Transitions from the Third LIGO-Virgo Observing Run.

We place constraints on the normalized energy density in gravitational waves from first-order strong phase transitions using data from Advanced LIGO and Virgo's first, second, and third observing runs. First, adopting a broken power law model, we place 95% confidence level upper limits simultaneously on the gravitational-wave energy density at 25 Hz from unresolved compact binary mergers, Ω_{CBC}<6.1×10^{-9}, and strong first-order phase transitions, Ω_{BPL}<4.4×10^{-9}. The inclusion of the former is necessary since we expect this astrophysical signal to be the foreground of any detected spectrum. We then consider two more complex phenomenological models, limiting at 25 Hz the gravitational-wave background due to bubble collisions to Ω_{pt}<5.0×10^{-9} and the background due to sound waves to Ω_{pt}<5.8×10^{-9} at 95% confidence level for phase transitions occurring at temperatures above 10^{8} GeV.

Interleukin-4-induced natural killer cell antitumor activity in metastatic melanoma patients.

NK cells are important effectors of innate immunity that mount the first line of defense toward tumor growth. Interleukin-4 (IL-4) has recently been shown to regulate NK cell function, although its role in the regulation of NK cell function in cancer patients has not been clarified. The aim of this study was to investigate the effect of IL-4 on the function and the receptor characteristics of CD16-defined NK cells and their cytotoxic CD16bright and regulatory CD16dim subsets. Peripheral blood lymphocytes obtained from 36 metastatic melanoma (MM) patients treated for 18 h with 10 ng/mL IL-4 were evaluated for NK cell cytotoxicity using the radioactive 51chromium release assay. Expression of the activating receptors NKG2D and CD161, as well as the inhibitory receptors CD158a and CD158b, was analyzed on CD3-CD16+ NK cells and their subsets by flow cytometry. IL-4 induced significant in vitro enhancement of NK cell activity, as well as increased expression of the CD107a degranulation marker, by CD3-CD16dim NK cells. NKG2D expression was also increased on CD3-CD16+ cells by IL-4 with no alteration of the expression of CD161 and inhibitory KIR receptors. Although in vitro treatment with IL-4 increased both the expression of NKG2D and the cytotoxicity of NK cells, it had no detectable effect on the transcription of the TGF-ß gene in NK cells of MM patients. The IL-4-induced NK cell cytotoxicity and increased activating NKG2D receptor expression may indicate an important antitumor effect of IL-4 with a potential application for immunotherapy of MM patients.

Evaluation of cytokine expression and circulating immune cell subsets as potential parameters of acute radiation toxicity in prostate cancer patients.

One of the challenges of radiation oncology in the era of personalized medicine is identification of biomarkers associated with individual radiosensitivity. The aim of research was to evaluate the possible clinical value of the associations between clinical, physical, and biological factors, and risk for development of acute radiotoxicity in patients with prostate cancer. The study involved forty four patients treated with three-dimensional conformal radiotherapy. The concentrations of IL-1ß, IL-2, IL-6, IFN-γ and TGF-ß1 were assessed before radiotherapy, after 5th, 15th and 25th radiotherapy fractions, at the end, and 1 month after the end of radiotherapy. Cytokine gene expression was determined in peripheral blood mononuclear cells. The univariate analysis of circulating cytokine levels during radiotherapy showed that increased serum concentrations of IL-6 were significantly associated with higher grade of acute genitourinary toxicity. The multivariate analysis demonstrated that increased level of IL-6 during the radiotherapy was significantly associated with higher grade of acute genitourinary toxicity across treatment. TGF-ß expression levels significantly decreased during course of radiotherapy. Research indicates that changes in circulating cytokine levels might be important parameter of radiotoxicity in patients with prostate cancer. These findings suggest that future studies based on multi-parameter examination are necessary for prediction of individual radiosensitivity.

The role of cytokines in the regulation of NK cells in the tumor environment.

Natural killer (NK) cells are innate lymphoid cells that are important effectors in the first line of defense toward transformed cells. This is mediated both by direct cytotoxic mechanisms and by production of immunoregulatory cytokines. Recent evidence has shown that NK cells also display memory, similar to the cells of the adaptive immune system. Cytokines are pivotal for the maturation, activation and survival of NK cells. Interleukins (IL)-2, IL-12, IL-15, IL-18, IL-21 and type I interferons positively regulate NK cell function, either independently or in cooperation, whereas other cytokines, such as IL-23 and IL-27, may enhance or suppress NK cell function depending on the context. In the tumor microenvironment, TGFß, IL-10 and IL-6 suppress NK cell activity not only directly, but also indirectly, by affecting immunosuppressive cells and by antagonizing the effect of stimulatory cytokines, thereby dampening the antitumor response of NK cells and promoting subsequent tumor evasion and progression. Increased understanding of the NK cell response to cytokines has provided a better understanding of their impaired function in tumors which may aid in the development of novel immunotherapeutic strategies to enhance NK cell responses in cancer patients.

Effect of cytokines on NK cell activity and activating receptor expression in high-risk cutaneous melanoma patients.

OBJECTIVE: Stage II melanoma patients have high risk for regional and distant metastases and may benefit from novel therapeutic strategies. To clarify the role of NK cells in Stage II melanoma, we characterized the cytotoxic activity of NK cells and the expression of various activating and inhibitory receptors in high-risk cutaneous melanoma patients (Stages IIB and IIC) compared to low-risk patients (Stage IA). MATERIALS AND METHODS: Native and cytokine-treated peripheral blood mononuclear cells were used for functional and phenotypical analyses. RESULTS: Compared to Stage IA-B patients, Stage IIB-C patients showed significantly decreased NK cell activity, as well as decreased expression of the activating NKG2D and CD161 receptors, most likely due to increased serum levels of the immunosuppressive cytokine TGF-ß1 in these patients. Interestingly, treatment of periperal blood mononuclear cells with IFN-α, IL-2, IL-12 or the combination of IL-12 and IL-18 significantly induced NK cell activity for both groups of melanoma patients. However, only low-risk patients had a significant increase in the expression of the NKG2D receptor after in vitro treatment with IFN-α, as well as an significant increase in the expression of CD161 after treatment with IFN-α or IL-12. Although IL-2 induced the expression of NKG2D in both groups of patients, this increase was significantly lower in high-risk melanoma. CONCLUSION: NK cell parameters may be useful as biomarkers of disease progression in localized melanoma patients. Our results further suggest that the use of NK cell-activating cytokines in combination with inhibitors of immunosuppressive factors like TGF-ß1 could be a therapeutic option for the treatment of high-risk cutaneous melanoma patients.

Evaluation of the Functional Capacity of NK Cells of Melanoma Patients in an In Vitro Model of NK Cell Contact with K562 and FemX Tumor Cell Lines.

NK cells of metastatic melanoma (MM) patients display impaired function, making them incapable to mount an effective antitumor response. In this study, we evaluated immunophenotypic characteristics and functional capacity of CD3-CD16+ NK cells of MM patients in an in vitro model based on NK cell contact with an NK sensitive, K562, and a tumor-specific, melanoma FemX tumor cell line. Although our results indicate similar NK cell antitumor cytotoxic potential of MM patients in contact with both cell lines based on the expression of CD107a degranulation marker, there is a discrepancy in NK cell IFNγ production, as it is not significantly induced by FemX tumor cells, found to be, contrary to K562, HLA class I positive. Furthermore, we show NKG2D receptor downregulation by K562 tumor cell line, only. This may result from the obtained higher gene expression of TGFß and VEGFA growth factors in K562 tumor cells that can negatively regulate NKG2D expression. Additionally, aside from postcontact downmodulation of activating CD16 receptor, there are no significant changes in the expression of CD161, CD158a, and CD158b NK cell receptors. Therefore, the applied in vitro model shows that, compared to the full NK cell functional capacity of MM patients displayed in a tumor-sensitive setting represented by contact with K562 cells, tumor-specific melanoma setting provided by FemX tumor cells leads to reduced NK functional potential. The obtained insight into NK cell capacity may be of use for evaluation of the state of disease and can help in selecting effective immunotherapeutic agents for MM patients.

Attenuated in vitro effects of IFN-α, IL-2 and IL-12 on functional and receptor characteristics of peripheral blood lymphocytes in metastatic melanoma patients.

Considering tumor-induced suppression of lymphocytes the aim of this study was to investigate in vitro effects of IFN-α, IL-2, IL-12 and IL-18 as immunomodulating agents on the functional and receptor characteristics of peripheral blood lymphocytes (PBL) in metastatic melanoma (MM) patients compared to healthy controls (HC). In HC IFN-α, IL-2 and IL-12 enhanced mRNA level of perforin by inducing pSTAT-1 and pSTAT-5 signaling molecules. Additionally, the expression of NKG2D activating receptor and its DAP10 signaling molecule was upregulated by IL-2. Contrary to this, in MM patients only IL-2 by upregulating pSTAT-5 increased perforin-mediated cytotoxicity of lymphocytes. Furthermore, there was significantly negative correlation between the percentage of CD4+CD25bright+CD27+ regulatory T (Treg) cells and NK cell cytotoxicity, as well as the expression of NKG2D receptor on PBL in HC and MM patients. Therefore, the absence of IL-2 effect on the increase of NKG2D/DAP10 level in MM patients could be the consequence of the increased percentage of immunosuppressive CD4+CD25bright+CD27+ cells after this cytokine treatment in patients. However, in MM IL-12 significantly decreases the percentage of these inhibitory cells. Although IL-2 as a single agent has numerous side effects, it remains the important cytokine for PBL activation in melanoma immunotherapy. Additionally, the removal of Treg cells from patient PBL by IL-12 before in vitro stimulation with IL-2, may lead to the generation of more potent cytotoxic lymphocytes against tumor cells. Therefore, lymphocyte based therapy for MM patients should integrate not only the choice of appropriate immunostimulatory cytokine, but also the removal of inhibitory cells from tumor microenvironment.

Beneficial in-vitro effects of interleukin-2, interleukin-12, and their combination on functional and receptor characteristics of natural killer cells in metastatic melanoma patients with normal serum lactate dehydrogenase levels.

Considering tumor-mediated suppression of natural killer (NK) cells, the aim of this study was to investigate the in-vitro effects of interleukin (IL)-2 and IL-12, as immunostimulatory cytokines, on the functional and receptor characteristics of NK cells and their subsets in healthy control (HC) and metastatic melanoma (MM) patients. Peripheral blood mononuclear cells of 27 HC and 35 MM patients were stimulated in vitro with IL-2, IL-12, and their combination for functional and phenotypic analysis. IL-2, IL-12, and primarily their combination, significantly induced NK cell activity, CD107a degranulation marker, and perforin expression in NK cells and their subsets in HC and MM patients. Furthermore, the combination of IL-2 and IL-12 was significantly more efficient than IL-12 alone in the augmentation of NK cell cytotoxicity and CD107a expression. Also, IL-2 and IL-12 reciprocally upregulated each other's receptors, IL-2Rα and IL-12Rß1/ß2, on NK cells and their subsets in MM and HCs. In addition, the priming of NK cells with IL-2 before IL-12 treatment led to an increase in the expression of both IL-12 receptors. In contrast to IL-12, IL-2 increased activating NKG2D and DNAM-1, as well as inhibitory CD158a and CD158b KIRs. In addition, the cytokines investigated exerted a more potent effect on the increase in NK cell activity and the expression of various NK cell receptors in MM patients with normal lactate dehydrogenase (LDH) serum levels. Therefore, serum LDH could represent a predictor of response to cytokine immunotherapy in MM patients. The optimization of combined IL-2/IL-12 therapy is needed to enhance NK cell functions in MM patients stratified by their LDH levels.

Decreased CD161 activating and increased CD158a inhibitory receptor expression on NK cells underlies impaired NK cell cytotoxicity in patients with multiple myeloma.

AIM: As innate immune cells natural killer (NK), NK-like T and CTLγδ are important in antitumour response in multiple myeloma (MM), the aim of this study was to investigate some functional and phenotypical characteristics of these cells in MM. METHODS: 29 patients with MM prior to therapy, in clinical stage I-III and 15 healthy controls (HCs) were investigated. Percent of immune cells in peripheral blood, NK cell activity, expression of activating (CD161) and inhibitory (CD158a, CD158b) NK cell receptors on CD3-CD16+ NK cells were evaluated using 51-chromium-release assay and by flow cytometry. Production of interleukin (IL) 2 and tumour necrosis factor (TNF)α was analysed in supernatants from in vitro activated peripheral blood mononuclear cells. RESULTS: In patients with MM the percent of NK cells and their two subsets did not differ from controls, while NK-like T and CTLγδ cells were significantly decreased. Significant impairment of NK cell cytotoxicity, CD107a expression and interferon γ intracellular level was also shown. There was a significant decrease in CD161 and an increase in CD158a receptor expression on NK cells in these patients. Also IL-2 production was lowest in clinical stage III. However, TNF-α production did not differ between patients and HCs. CONCLUSIONS: Altered expression of CD161 activating and CD158a KIR inhibitory receptor is responsible for impaired antitumour activity of NK cells in MM patients. These new biomarkers may be helpful for patient selection for immunotherapy with cytokines, and novel KIR blocking monoclonal antibodies that enhance NK cell antimyeloma activity and provide clinical benefit.

Decreased expression of pSTAT, IRF-1 and DAP10 signalling molecules in peripheral blood lymphocytes of patients with metastatic melanoma.

AIMS: As numerous signalling molecules regulate effector functions of peripheral blood lymphocytes (PBLs) that have an important anti-tumour activity, the aim of this study was to analyse their level in patients with metastatic melanoma (MM) compared with healthy controls (HCs). METHODS: Peripheral blood mononuclear cells (PBMCs) of 36 MMs and 28 HCs were analysed for the level of perforin, interferon-regulating transcription factor-1 (IRF-1), DAP10 and Src homology 2 domain-containing tyrosine phosphatase-1 by reverse transcriptase PCR, level of phosphorylated signal transducers and activators of transcription (pSTAT)-1, pSTAT-4, pSTAT-5 by western blot and interferon (IFN)-γ production by ELISA. The expression of activating NKG2D and inhibitory killer immunoglobulin-like receptors (KIR), CD158a and CD158b, on PBL, CD3-CD56+ natural killer (NK) cells and CD3+CD8+ cytotoxic T lymphocytes (CTLs), as well as the percentage of CD14+HLA-DR- cells in PBMC were estimated by flow cytometry. RESULTS: Patients with MM, compared with HCs, had significantly lower level of cytotoxic molecule perforin and decreased IFN-γ production, as well as lower level of pSTAT-1, pSTAT-4, pSTAT-5 and IRF-1 signalling molecules in PBMC. Furthermore, MM had decreased expression of activating NKG2D receptor on PBL and NK cells and low level of its DAP10 signalling molecule contrary to no changes in KIR expression on all investigated cells. These results could be associated with increased percentage of immunosuppressive CD14+HLA-DR- myeloid-derived suppressor cells detected in patients with MM. CONCLUSIONS: The altered signalling molecules of PBL could represent biomarkers of impaired cytotoxic and immunoregulatory function of these cells, indicating melanoma-associated immunosuppression that facilitates tumour progression.

Natural killer cell receptors: alterations and therapeutic targeting in malignancies.

Numerous newly identified activating and inhibitory NK cell receptors and their engagement by cognate ligands on target tumor cells regulate NK cell antitumor activity. Alterations in NK cell receptor expression and signaling underlie diminished cytotoxic NK cell function. Cytokines, IFN-α, IL-2, IL-12, IL-15 and IL-18, applied systemically and for ex vivo activation and expansion of NK cells have improved NK cell antitumor activity by increasing the expression of NK cell activating receptors and by inducing cytotoxic effector molecules. Moreover, it has been recognized that classical and novel pharmacological agents upregulate cognate ligands for activating receptors on tumor cells and provide better NK cell antitumor response. Some other immunotherapeutic approaches in cancer in the setting of donor-recipient KIR/HLA mismatch have evolved with the aim to potentiate NK cell activity in allogeneic hematopoietic stem cell transplantation that lead to beneficial graft vs. tumor effect. Therefore, better understanding of NK cell activating and inhibitory receptor biology is needed to assist in developing novel approaches to effectively manipulate NK cells and create effective NK cell-based immunotherapy for treatment of cancer patients.