Regional Lymph Nodes Of Melanoma Patients Research Articles

PO-372 IL-2 and IL-15 cytokine in vitro treatments induce NKG2D, CD158a and CD158b receptor expression on T, NKT- like and NK cell lymphocyte subsets from regional lymph nodes of melanoma patients

IntroductionRegional lymph nodes (LN)s are important immunological barriers in spreading of malignant tumours but also represent the most frequent early metastatic site in melanoma. Considering that cytokine therapy has shown substantial toxicity, there is a growing need for further in vitro testing of these agents to enlighten aspects of their regional application in malignancies. The aim of this study was to investigate the effect interleukin (IL)−2 and IL-15, cytokines with similar immune-enhancing effects, on the expression of activating (NKG2D) and inhibitory (CD158a,CD158b) receptors on CD8+ T, NKT-like and NK cell lymphocyte subsets from regional LNs of melanoma patients.Material and methodsMononuclear cells (MNC)s were purified from regional LNs of 35 melanoma patients (clinical stages II-IV) and in vitro cultured for 7 days in cell culture medium RPMI1640 (CM) alone, CM with 200 IU/ml rhIL-2 and CM with 25 ng/ml IL-15. Expression of NKG2D, CD158a and CD158b receptors on lymphocytes, CD8+ T, NKT-like and NK cells was estimated by flow cytometry. Statistical significance between the values obtained with cytokine compared to control CM treatments was evaluated by Wilcoxon signed rank test. NK cell cytotoxicity was evaluated by standard 51Cr release assay.Results and discussionsOur results show that IL-2 and IL-15 in vitro treatments significantly increase expression of activating NKG2D receptor on lymphocytes and their CD8+ T, NKT-like and NK cell subsets. Regarding investigated inhibitory receptors, the significant increase was obtained for CD158a only after IL-15 treatment on lymphocytes and their CD8+ T and NKT- like subsets. However, the expression of CD158b receptor significantly increased after IL-2 cytokine treatment on lymphocytes, CD8+ T and NK cells, and after IL-15 treatment on all investigated lymphocyte subsets.Both cytokines augmented NK cell antitumor cytotoxicity that positively correlated (p<0.01, Spearman signed rank test) with the expression of NKG2D activating receptor on NK cells from regional LNs of melanoma patients, irrespective of LN involvement. Conversely, the expression of inhibitory receptors did not correlate with NK cell cytotoxicity except for CD158b that after control CM treatment showed negative correlation.ConclusionAntitumor potential of immune cells from regional LNs induced with IL-2 and IL-15 cytokine treatments may indicate the importance of this immune cell population in the development of therapeutic strategies that involve local cytokine application.

IL-2 And IL-15 Induced NKG2D, CD158a and CD158b Expression on T, NKT- like and NK Cell Lymphocyte Subsets from Regional Lymph Nodes of Melanoma Patients.

Regional lymph nodes (LN)s represent important immunological barriers in spreading of malignant tumors. However, they are the most frequent early metastatic site in melanoma. Immunomodulatory agents including cytokines have been included in therapy of melanoma and have shown severe side effects and toxicity. In this sense, there is a growing need for bringing these agents to further in vitro testing that may enlighten aspects of their regional application. Therefore, the aim of this study was to investigate the effect of interleukin (IL)-2 and IL-15, the two cytokines with similar immune-enhancing effects, on the expression of activating NKG2D, inhibitory CD158a and CD158b receptors on CD8+ T, NKT-like and NK cell lymphocyte subsets from regional LNs of melanoma patients. In this study, we showed significant effects of IL-2 and IL-15 cytokine treatments on the expression of activating NKG2D and on inhibitory CD158a and CD158b receptors on lymphocytes, CD8+ T, NKT-like and NK cell lymphocyte subsets originating from regional LNs of melanoma patients. Furthermore, IL-2 and IL-15 by inducing the expression of NKG2D activating receptor on innate and on adaptive lymphocyte subsets and by augmenting NK cell antitumor cytotoxicity that correlated with the cytokine-induced NKG2D expression, increased antitumor potential of immune cells in regional LNs of melanoma patients irrespective of LN involvement. These findings indicate the importance of immune cell population from regional LNs of melanoma patients in the development of immune intervention strategies that may if applied locally increase antitumor potential to the level that controls tumor progressions.

Decreased Interferon γ Production in CD3+ and CD3- CD56+ Lymphocyte Subsets in Metastatic Regional Lymph Nodes of Melanoma Patients.

As lymphogenic dissemination is very common in melanoma, regional lymph nodes (LN)s represent first immunological barriers to tumor invasion and play a complex role in antitumor immune defense. In this sense, their most prominent role is the presentation of tumor-derived antigens to naïve T cells and generation of cell-mediated adaptive immune response. Since tumor micro-environment affects immune cell function in this study we have evaluated the ability of T cells and NK cells in metastatic (involved) and non-metastatic regional LNs to produce interferon γ (IFNγ), a pleiotropic cytokine that regulates adaptive antitumor immune response. Our results show reduced IFNγ production in both T and NK lymphocyte subsets and decreased prevalence of T cells in metastatic regional LNs of melanoma patients. The decrease of IFNγ production in T cells was more pronounced with increased number of involved regional LNs indicating tumor-induced functional impairment of both T and NK cell lymphocyte subsets in involved regional LNs. Therefore, shown low IFNγ production in metastatic LNs may represent an obstacle in adaptive cell-mediated antitumor immune response and hence may enable tumor progression.

In-vitro activation of natural killer cells from regional lymph nodes of melanoma patients with interleukin-2 and interleukin-15.

Regional lymph nodes (LNs) represent the first barrier in lymphogenic tumor dissemination in melanoma. Natural killer (NK) cells, the effector cell subpopulation of the innate immune system, are in the first line of antitumor immune defense. Therefore, the aim of this study was to investigate the effect of interleukin (IL)-2 and IL-15, two cytokines with similar immune-enhancing effects, on antitumor cytotoxic function and immunophenotype of NK cells from regional LNs of melanoma patients. Mononuclear cells purified from regional LNs of 50 melanoma patients in clinical stage II-IV were treated in vitro for 72 h and 7 days with 200 IU/ml rhIL-2 and 25 ng/ml IL-15 at 37°C in 5% CO2. Both cytokines significantly augmented NK cell cytotoxic activity, transcription of the cytotoxic molecule perforin, and the level of functionally mature perforin in both nonmetastatic and metastatic regional LNs. IL-2 treatment increased the percentage of CD3CD56 NK cells by increasing the CD56 NK cell subset in both nonmetastatic and metastatic LNs, whereas IL-15 treatment did not affect the percentage of NK cells and their subsets. Both cytokines increased on NK cells from nonmetastatic and metastatic LNs the expression of CD69 early activation antigen, the NKG2D activating receptor, as well as CD16 and inhibitory killer-cell immunoglobulin-like receptor CD158b, both inherent to the mature and the cytotoxic NK cell phenotype. In conclusion, our data may indicate the therapeutic potential of the NK cell population from regional LNs either as immunotherapeutic targets or as adoptively transferred after activation with IL-2 or IL-15.

Distribution of several activating and inhibitory receptors on CD3−CD56+ NK cells in regional lymph nodes of melanoma patients

BackgroundNatural killer (NK) cells, as the main effector subpopulation of the innate immune system, play an important role in the control of the rise and spread of malignant tumors. Regional lymph nodes (LN) represent the first immunologic barrier to tumor metastasis. Since there are scarce data on NK cells from regional LN of cancer patients, the aim of this study was to investigate the expression of several activating and inhibitory receptors on the entire NK cell population as well as their CD3−CD56dim and CD3−CD56bright functional NK subsets from regional LN of melanoma patients. Materials and methodsMononuclear cells were isolated from 50 regional LN of melanoma patients. The expression of several receptors on NK cells and their functional subsets was analyzed by flow cytometry. ResultsWe show increased percentages of CD3−CD56+ NK cells in involved LN compared with uninvolved LN, mostly in favor of the CD56dim NK cell subset. NK cells in involved LN express similar levels of activating receptor NKG2D, while the level of another activating receptor, CD16, is increased compared with uninvolved LN. Regarding the expression of inhibitory NK cell receptors, we show increased CD158b, but similar low CD158a, inhibitory killer Ig-like cell receptor expression in involved LN compared with uninvolved LN. Furthermore, NK cells in involved compared with uninvolved LN displayed increased CD69 early activation antigen expression. ConclusionsOur results indicate that with tumor infiltration into regional LN of melanoma patients, NK cells, mostly of the CD56dim subset, are recruited into draining LN. The invading NK cells show counterbalance of the increased expression of CD16 activating receptor and increased CD158b inhibitory killer Ig-like cell receptor.