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// Kelly C.G. Manfrere 1 , Marina P. Torrealba 1 , Denis R. Miyashiro 2 , Natalli Z. Pereira 1 , Fabio S.Y. Yoshikawa 1 , Luana de M. Oliveira 1 , Jade Cury-Martins 2 , Alberto J.S. Duarte 1 , Jose A. Sanches 2, * and Maria N. Sato 1, * 1 Laboratory of Medical Investigation, LIM-56, Department of Dermatology, Tropical Medicine Institute of Sao Paulo, University of Sao Paulo Medical School, Sao Paulo, Brazil 2 Cutaneous Lymphoma Clinic, Hospital das Clinicas, Department of Dermatology, University of Sao Paulo Medical School, Sao Paulo, Brazil * These authors share the mentorship, critical revision and supervision of this study Correspondence to: Maria N. Sato, email: marisato@usp.br Keywords: Sezary syndrome, natural killer cells, Toll-like receptor 7/8, memory NK cells, differentially expressed genes Received: July 06, 2017 Accepted: August 27, 2017 Published: September 18, 2017 ABSTRACT Sezary syndrome (SS), an aggressive and leukemic form of cutaneous T-cell lymphoma, usually results in shortened survival. Improving innate immunity in SS by targeting natural killer (NK) cells with Toll-like receptor (TLR) agonists could be an interesting modulatory strategy. We evaluated the NK cell populations in SS patients assessing activating and inhibitory receptors expression and profiled the differential expression of TLR signaling pathway genes in unstimulated NK cells and after TLR7/8 stimulation. We observed preserved CD56 bright NK cells and a low percentage of CD56 dim NK cells in the peripheral blood of SS patients compared to those in the healthy control group. Both NK cell populations showed down-modulation of NKG2C and NKG2D expression, which was associated with high serum levels of the soluble form of NKG2D ligands. In contrast, an expansion of “memory” CD57+ NKG2C+ NK cells and high cytomegalovirus antibody titers were detected in SS patients. Profiling of the TLR signaling genes in NK cells from SS patients showed an abundance of differentially expressed genes (DEGs) in NK cells in the unstimulated condition, with mostly up-regulation of NFκB/JNK p38 pathway genes, but there was down-regulation of type I (IFN-α/β) and II (IFN-γ) interferon and IL-12A. After activation of NK cells with TLR7/8 agonist, the down-regulated genes correlated with the IFN response, and IL-12 became up-regulated, together with other antitumor factors. NK cell activation with a dual agonist for TLR7 and TLR8 is able to induce the expression of IFN-γ and type I IFN, which can improve immunity in SS patients.

// Kelly C. G. Manfrere 1 , Marina P. Torrealba 1 , Denis R. Miyashiro 2 , Luanda M. S. Oliveira 1 , Gabriel C. de Carvalho 1 , Josenilson F. Lima 1 , Anna Claudia C. C. Branco 1 , Natalli Z. Pereira 1 , Juliana Pereira 3 , Jose A. Sanches, Jr 2,* and Maria N. Sato 1,* 1 Department of Dermatology, Laboratory of Medical Investigation, LIM 56, Tropical Medicine Institute of Sao Paulo, University of Sao Paulo Medical School, Brazil 2 Department of Dermatology, Cutaneous Lymphoma Clinic, Hospital das Clinicas, University of Sao Paulo, Medical School, Brazil 3 Department of Hematology, University of Sao Paulo Medical School, Brazil * These authors share the mentorship, critical revision and supervision of this study Correspondence to: Maria N. Sato, email: // Keywords : Se,zary syndrome, innate immunity, Toll-like receptor agonists, cytokines, type I interferon, Immunology and Microbiology Section, Immune response, Immunity Received : July 28, 2016 Accepted : October 14, 2016 Published : October 21, 2016 Abstract Sezary syndrome (SS) carries a poor prognosis, and infections represent the most frequent cause of death in SS patients. Toll-like receptors (TLRs) are a family of innate immune receptors that induce protective immune responses against infections. We sought to evaluate the ability of TLR agonists to induce inflammatory cytokine, Th2 cytokine, and type I interferon (IFN-I) production by peripheral blood mononuclear cells (PBMC) of untreated SS patients. We detected impaired IL-6, IL-10 and IL-13 secretion by PBMC induced by the agonists for TLR5, TLR3, TLR7 and TLR9 in SS patients, while it was partially recovered by TLR2/TLR4 and TLR7/8 agonists TNF secretion was restored following stimulation with TLR2/TLR4 agonists. IFN-γ was scarcely produced upon TLR activation in SS cells, albeit TLR 7/8 (CL097) enhanced their secretion at lower levels than the control group. TLR9 agonist efficiently induced IFN-I in SS patients, although this positive regulation was not observed for other cytokines, in direct contrast to the broad activity of CL097. Among the TLR agonists, TLR4 was able to induce pro-inflammatory, IL-10 and Th2 secretion, while TLR7-8 agonist induced the inflammatory cytokines, IFN-I and IFN-γ. These findings reveal a dysfunctional cytokine response upon both extracellular and intracellular TLR activation in SS patients, which was partially restored by TLRs agonists.

// Marina Passos Torrealba 1 , Kelly Cristina Manfrere 1 , Denis R. Miyashiro 2 , Josenilson F. Lima 1 , Luana de M. Oliveira 1 , Natalli Z. Pereira 1 , Jade Cury-Martins 2 , Juliana Pereira 3 , Alberto J.S. Duarte 1 , Maria N. Sato 1, * and Jose A. Sanches 2, * 1 Medical Investigation Laboratory (LIM-56), Tropical Medicine Institute of Sao Paulo, Department of Dermatology, University of Sao Paulo Medical School, Sao Paulo, Brazil 2 Cutaneous Lymphoma Clinic, Hospital das Clinicas, Department of Dermatology, University of Sao Paulo Medical School, Sao Paulo, Brazil 3 Hematology Department, Hospital das Clinicas, University of Sao Paulo Medical School, Sao Paulo, Brazil * These authors share the mentorship, critical revision and supervision of this study Correspondence to: Marina Passos Torrealba, email: marinatorrealba@usp.br Keywords: Sezary syndrome; CD8+ T cells; chronic activation markers; sCD38; sCD127 Received: October 12, 2017 Accepted: December 01, 2017 Published: December 16, 2017 ABSTRACT Sezary syndrome (SS) is a leukemic variant of cutaneous T cell lymphoma (CTCL), and the neoplastic CD4+ T cells of SS patients undergo intense clonal proliferation. Although Sezary cells have been studied extensively, studies on adaptive immunity regarding CD8+T cells are scarce. This study aimed to investigate activation marker expression in CD8+ T cells according to the differentiation stages and IL-7/IL7Rα axis responses of patients with SS. Moreover, this study aimed to verify the soluble forms of CD38, sCD127 and IL-7 in serum. Although the SS patients of our cohort had reduced numbers of CD8+ T cells, they exhibited higher percentages of CD8+CD38+ T cells, mainly effector/memory CD8+ T cells, than the control group. In contrast, down-regulated expression of the activation markers CD127/IL-7R and CD26 was found in the CD8+ T cells of SS patients. High serum levels of sCD38 and sCD127 and scarce serum levels of IL-7 were detected, emphasizing the immune activation status of SS patients. Moreover, CD8+ T cells from SS patients exhibited IL-7 unresponsiveness to STAT5 phosphorylation and Bcl-2 expression, and IL-7 priming partially restored IFNγ production. Our findings showed a chronic activation profile of CD8+ T cells, as an attenuated cytotoxic profile and impaired IL-7 responsiveness was observed, suggesting chronic activation status of CD8+ T cells in SS patients.