In vitro increased natural killer cell activity of metastatic melanoma patients with interferon-α alone as opposed to its combination with 13-cis retinoic acid is associated with modulation of NKG2D and CD161 activating receptor expression.

PURPOSE: Considering tumor-induced suppression of natural killer (NK) cell activity the aim of this study was to investigate the in vitro effect of a standard immunotherapeutic cytokine, interferon (IFN)α, and a less investigated agent, 13-cis retinoic acid (RA) on the functional and receptor characteristics of CD16-defined NK cells and their functionally diverse dim and bright subsets in patients with metastatic melanoma (MM). METHODS: Peripheral blood lymphocytes (PBL) of patients with clinical stage IV MM were stimulated in vitro for 18 h in RPMI 1640 culture medium (CM) alone, CM supplemented with IFN-α (250 U7sol;ml), RA (10-6M) and their combination. NK cell activity was determined using standard 4 h radioactive cytotoxicity assay, while the expression of activating (NKG2D, CD1617rpar; and inhibitory (CD158a, CD158b) NK cell receptors on CD3-CD16+ NK cells and their functional bright and dim subsets were analyzed by flow cytometry. RESULTS: NK cell cytotoxic activity was increased after in vitro treatment with IFN-α alone and in combination with RA, while only IFN-α induced increase in NKG2D and CD161 activating NK cell receptor expression. Contrary to this, RA treatment increased the expression of inhibitory KIR CD158b. IFN-α-obtained increase in CD161 expression was due to its induction on both NK cell subsets, while for NKG2D only on CD16bright subset. CONCLUSION: The favorable enhancement of NK cell activity of MM patients obtained with IFN-α is associated with upregulation of activating NKG2D and CD161 receptors, while the lack of RA-associated upregulation is probably due to the shown increased expression of inhibitory KIR receptor CD158b after in vitro treatment with this agent.

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.

CD69 on CD56+ NK cells and response to chemoimmunotherapy in metastatic melanoma.

BACKGROUND: The few chemoimmunotherapy trials that together with dacarbazine (DTIC) and interferon-alpha 2a (IFNalpha), include retinoic acid (RA), did not include detailed immunological evaluation of functional and phenotypic natural killer (NK) cell characteristics, and have shown contradictory clinical results. MATERIALS AND METHODS: Malignant melanoma (MM) patients undergoing phase II-randomized chemoimmunotherapy trials were treated with DTIC, IFNalpha (Hoffmann-La Roche) (group A, n = 31), and with DTIC, IFNalpha and 13-cis-RA (Isotretinoin, Hoffmann-La Roche, Basel, Switzerland) (group B, n = 29). Patients and 42 healthy controls were evaluated by FACS flow analyses for CD3/CD56/CD69 positive cells, NK cytotoxicity in fresh peripheral blood lymphocytes (PBL) and for interferon regulatory factor-1 mRNA expression by reverse transcriptase polymerase chain reaction in treated PBL. RESULTS: The addition of RA to a DTIC-IFN regime did not bring any therapeutical benefit in terms of response or survival. Immunological follow-up on days 1, 6 and 27 of each therapy cycle shows a significant increase in NK cell activity in both groups, only on day 6 of the first cycle, while CD69+CD56+ expression increased significantly on day 6 of each therapy cycle, in both groups. Evaluation of the dynamics of expression of IRF-1 of in vitro treated PBL, shows its strong and prompt up-regulation by IFNalpha and synergistic effect of IFNalpha and RA combination. CONCLUSION: The dynamics of the increase in CD69 early activation antigen expression on CD56+ NK cells is systematic and serial with the increase being significantly higher on day six of the first cycle in group B patients with clinical response, compared to those without, indicating possible predictive value of CD69 expression for clinical response to chemoimmunotherapy.