Investigation of NK cell function and their modulation in different malignancies.

NK cells have become a subject of investigation not only in the field of tumor immunology and infectious diseases, but also within all aspects of immunology, such as transplantation, autoimmunity, and hypersensitivity. Our early studies aside from investigating NK cell activity in experimental animals and humans included studies of perforin expression and modulation in this lymphocyte subset. As NK cell activity is modified by their environment, we showed clinical stage-dependent impairment of their activity and in vitro effect of different sera, Th1 cytokines, and their combination in breast cancer, Hodgkin's disease, and non-Hodgkin's lymphoma patients, especially with respect to metabolic and cell membrane changes of peripheral blood lymphocytes evaluated by spontaneous release of the enzyme lactate dehydrogenase (LDH) that led to the correction of the LDH enzyme release assay for natural cytotoxicity. By long-term immuno-monitoring of patients with malignancies, we also showed the kinetics of NK cell modulation during chemo-immunotherapy. In our more recent studies, we give data of NK function and novel families of NK cell receptor expression in healthy individuals that may be of help in NK cell profiling, by giving referent values of basic and cytokine-induced expression of some NK cell receptors either in evaluation of disease or in immuno-monitoring during cytokine therapy of patients with malignancies. Moreover, we give novel aspects of modulation of NK cell activity by cytokines approved for immunotherapy, IFN and IL-2, in melanoma and other malignancies with respect to alterations in new activating (NKG2D and CD161) and inhibitory (CD158a and CD158b) receptor characteristics and signaling molecules in CD16- and CD56-defined NK cells and their small immunoregulatory and large cytotoxic subsets in peripheral blood and lymph nodes, as NK cell-mediated killing of tumor cells depends on the balance between stimulatory and inhibitory signaling.

Distribution of several activating and inhibitory receptors on CD3-CD16+ 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.

Biomarkers of suppressed natural killer (NK) cell function in metastatic melanoma: decreased NKG2D and increased CD158a receptors on CD3-CD16+ NK cells.

In metastatic melanoma (MM) patients we evaluated natural killer (NK)-cell activity, distribution of several NK receptors and their correlation with NK function. Peripheral blood lymphocytes (PBL) of MM patients and controls were analysed for NK activity and expression of activating NKG2D, CD161 and KIR, CD158a and CD158b receptors on CD3-CD16+ NK cells. MM patients not only had significantly decreased NK activity and NK-cell interferon (IFN)-gamma production, a redistribution of NK-cell subsets with an increase in CD16(dim) and a reduction in CD16(bright) NK subsets. There was a decreased CD161 and NKG2D and an increased CD158a NK-cell expression in MM patients, with a positive correlation between NKG2D expression and NK cytotoxicity and an inverse correlation between CD158b expression and NK-cell cytotoxicity in patients. Furthermore, patients' CD3-CD16(bright) NK subset showed lower expression of CD161 and CD158a. Therefore, NKG2D, CD158a and CD158b expression in MM patients may represent several clinically useful 'biomarkers' of suppressed NK function.

Low expression of CD161 and NKG2D activating NK receptor is associated with impaired NK cell cytotoxicity in metastatic melanoma patients.

Natural killer (NK) cells play a role in the innate and adaptive antitumor immune responses. The activity of NK cells is regulated by functionally opposing, activating and inhibitory receptors whose balance ultimately determines whether target cells will be susceptible to NK cell mediated lysis. As melanoma is an immunogenic tumor, the effect of immunomodulating agents is consistently investigated. In this study in 79 metastatic melanoma (MM) patients and 52 controls NK activity, expression of activating NKG2D and CD161 receptors and KIR receptors, CD158a and CD158b, on freshly isolated PBL and NK cells were evaluated. Native NK cell activity of melanoma patients in clinical stage I-III and MM patients was determined against NK sensitive K562, NK resistant Daudi, human melanoma FemX, HeLa and HL 60 target tumor cell lines. In addition, predictive pretherapy immunomodulating effect after 18 h in vitro treatments of PBL of MM patients with rh IL-2, IFN-alpha (IFN), 13-cis retinoic acid (RA) and combination IFN-alpha and RA was evaluated with respect to NK cell lyses against K562 and FemX cell lines. In this study we show for the first time that low expression of CD161 and activating NKG2D receptors, without increased expression of KIR receptors CD158a and CD158b, as well as a decrease in the cytotoxic, CD16(bright) NK cell subset, is associated with a significant impairment in NK cell activity in MM patients. Furthermore, the predictive pretherapy finding that IL-2, IFN, IFN and RA, unlike RA alone, can enhance NK cell activity of MM patients against FemX melanoma tumor cell line can be of help in the design and development of therapeutic regimens, considering that it has recently been shown that low-dose combination of different immunomodulators represents the most promising approach in the therapy of MM.

IL-2-mediated augmentation of NK-cell activity and activation antigen expression on NK- and T-cell subsets in patients with metastatic melanoma treated with interferon-alpha and DTIC.

Considering that well-defined and comprehensive immunological monitoring is the basis for the evaluation of the obtained immunmodulatory effects, we evaluated NK-cell activity, the number of CD3+ CD4+, CD3+ CD8+ T cells and CD16+ CD56+ NK cells, as well as the expression of activation antigens, CD69, CD38 and HLA-DR on CD56+ NK cells, CD8+ and CD3+ T cells, simultaneously with IL-2 and TNF-alpha production, during chemoimmunotherapy with dacarbazine (DTIC) and interferon-alpha (IFN-alpha) in 39 patients with metastatic melanoma. In the first cycle of therapy, there was a significant rise in NK-cell activity, CD4+ T helper cell number, CD4/CD8 T-cell ratio, and the expression of activation antigens CD69 and CD38, on NK and T cells, respectively. However, in the following cycles there was a significant increase only in activation antigens without an increase in the percent or activity of NK cells. The early, but transient, immunopotentiation, present only in the first cycle of combined DTIC and IFN-alpha therapy, suggests that, in spite of increased IL-2 level, associated with augmented NK-cell activity, this therapy has a limited effect probably owing to the adverse effect of persistently high level of TNF-alpha in metastatic disease.