Circulating Natural Killer Lymphocytes Are Potential Cytotoxic Effectors Against Autologous Malignant Cells in Sezary Syndrome Patients Jean-David Bouaziz,

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Circulating Natural Killer Lymphocytes Are Potential Cytotoxic Effectors Against Autologous Malignant Cells in Sezary Syndrome Patients Jean-David Bouaziz, Nicolas Ortonne, Jérôme Giustiniani, Valérie Schiavon, Delphine Huet, Martine Bagot Journal of Investigative Dermatology Volume 125, Issue 6, Pages 1273-1278 (December 2005) DOI: 10.1111/j.0022-202X.2005.23914.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Susceptibility of Sezary cell lines to natural killer (NK) cell lysis. (A) Three Sezary cell lines, Cou-LS, Ogu, and Pno, were used as targets for allogeneic NK cells. We show the representative results with NK lymphocytes from one healthy donor. Similar results were obtained with four other different normal donors and with each donor, two separate experiments were carried out at different time periods. Percent of specific lysis ranged from 12% (target=Pno) to 27% (target=Cou-LS) at an effector-to-target (E/T) ratio of 50. (B) With the Pno cell line, at an E/T ratio of 50, specific lysis increased to 20% with the anti-KIR (killer cell immunoglobulin-like receptors)3DL2 monoclonal antibody (moAb) (12% with the control moAb). This was due to an antibody -dependent cell-mediated cytotoxicity mechanism and not a direct cell apoptosis, since the moAb (without effector NK cells) did not increase the spontaneous 51Cr release compared with medium alone. A similar enhancement was obtained when activating NK cells with IL-2 or when blocking target cell MHC class I molecules. Journal of Investigative Dermatology 2005 125, 1273-1278DOI: (10.1111/j.0022-202X.2005.23914.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Susceptibility of fresh Sezary cells to natural killer (NK) cell lysis. Whatever the donor, fresh Sezary cells were systematically resistant to the NK cell-mediated cytotoxicity; however, their lysis could be obtained with IL-2-activated NK lymphocytes or when coated with an anti-MHC class I F(ab)′2 antibody. There was no synergic effect of IL-2 combined with an anti-MHC class I F(ab)'2. Journal of Investigative Dermatology 2005 125, 1273-1278DOI: (10.1111/j.0022-202X.2005.23914.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Allogeneic natural killer (NK) lymphocyte-mediated antibody -dependent cell-mediated cytotoxicity (ADCC) with the humanized anti-CD52 monoclonal antibody (moAb) alemtuzumab. (A) Alemtuzumab was able to induce a significant increase of NK cell lysis against tumoral cell lines and fresh Sezary cells (60% and 30%, respectively, at an effector-to-target (E/T) ratio of 50). (B) NK lymphocytes from one healthy donor were separated and tested against autologous PBMC. As expected, spontaneously there was no cytotoxicity but alemtuzumab induced “auto-reactivity” via an ADCC mechanism. The anti-KIR (killer cell immunoglobulin-like receptors)3DL2 antibody did not induce such a lysis. The results are representative of three different experiments with different donors. Journal of Investigative Dermatology 2005 125, 1273-1278DOI: (10.1111/j.0022-202X.2005.23914.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Ability of natural killer (NK) lymphocytes from Sezary syndrome patients to mediate cytotoxicity against K562 and against autologous malignant cells. (A) NK lymphocytes from Sezary syndrome patient Lev were able to kill the K562 cell line spontaneously (specific lysis=18%) and after stimulation with IL-2 (specific lysis=83%). Data are shown at an effector-to-target (E/T) ratio of 30. (B) NK cells from patient Mart (B1) and Ogu (B2) exhibited spontaneous cytotoxicity against their own malignant cells (specific lysis 18% (E/T ratio of 30) and 26% (E/T ratio of 50), respectively). When the tumoral cell line Ogu was coated with an anti-MHC class I F(ab)′2 antibody, the lysis raised to 52%. Similarly, anti-KIR (killer cell immunoglobulin-like receptors)3DL2-coated-autologous malignant target cells enhanced the lysis to 35%. The results were similar in two separate experiments. Journal of Investigative Dermatology 2005 125, 1273-1278DOI: (10.1111/j.0022-202X.2005.23914.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions