Polyfunctional natural killer cells with a low activation profile in response to Toll-like receptor 3 activation in HIV-1-exposed seronegative subjects

www.nature.com/scientificreports OPEN Received: 30 June 2016 Accepted: 7 March 2017 Published: xx xx xxxx Polyfunctional natural killer cells with a low activation profile in response to Toll-like receptor 3 activation in HIV-1-exposed seronegative subjects Josenilson F. Lima, Luanda M. S. Oliveira, Nátalli Z. Pereira, Alberto J. S. Duarte & Maria N. Sato Natural killer (NK) cells are the main mediator of the cytotoxic response in innate immunity and may be involved in resistance to HIV-1 infection in exposed seronegative (ESN) individuals. Toll-like receptor (TLR) signalling is crucial for NK cell activation. Here, we investigated the polyfunctional NK cell response to TLR3 activation in serodiscordant couples. ESN subjects showed increased IFN-γ and CD107a expression in both NK subsets, CD56bright and CD56dim cells, in response to stimulation with a TLR3 agonist, while expression was impaired in the HIV-1-infected partners. TLR3-induced expression of IFN-γ, TNF and CD107a by polyfunctional CD56bright NK cells was more pronounced in ESN individuals than that in healthy controls. Activated NK cells, as determined by CD38 expression, were increased only in the HIV-1-infected partners, with reduced IFN-γ and CD107a expression. Moreover, CD38+ NK cells of the HIV-1-infected partners were associated with increased expression of inhibitory molecules, such as NKG2A, PD-1 and Tim-3, while NK cells from ESN subjects showed decreased NKG2A expression. Altogether, these findings indicate that NK cells of ESN individuals were highly responsive to TLR3 activation and had a polyfunctional NK cell phenotype, while the impaired TLR3 response in HIV-1-infected partners was associated with an inhibitory/exhaustion NK cell phenotype. Some individuals remain HIV-1-seronegative despite repeated unprotected exposure to the virus1. These individuals are usually defined as HIV-1-exposed seronegative (ESN) subjects in study cohorts with different exposure profiles: sex workers, children born to HIV-seropositive mothers, intravenous drug users, health care workers accidentally exposed to HIV, and homosexual or heterosexual subjects who have a history of unprotected sex with their seropositive partners2. Resistance to HIV-1 infection in ESN individuals has been associated with the presence of antigen-specific immune responses as well as with components of innate immunity3. Increased natural killer (NK) cell activity has been correlated with protection from infection in several high-risk cohorts of ESN subjects, a finding that suggests the involvement of the innate immune response in resistance to HIV-1 infection4. NK cells are considered the founding member of group 1 innate lymphoid cells (ILC1), which show immunological characteristics of lymphoid developmental origin, the absence of clonally rearranged antigen receptors, and an activation profile that requires T-bet and leads to rapid cytokine production, including IFN-γ5, 6. Two major subtypes of functionally distinct NK cells include CD56brightCD16− (CD56bright) cells, which represent approximately 10% of circulating NK cells and are predominantly involved in cytokine and chemokine secretion, and CD56dimCD16+ cells7, which represent the remaining 90% of circulating NK cells and are responsible for recognition and lysis of target cells8, 9. The L-selectin molecule (CD62L) mediates homing of leukocytes to lymphoid organs, and CD56dimCD62L+ cells represent a unique subset of mature, polyfunctional NK cells that affect the magnitude of the local NK cell response to murine viral infection7, 10. These polyfunctional cells have the ability Laboratory of Dermatology and Immunodeficiencies, LIM-56, Department of Dermatology, Institute of Tropical Medicine, University of São Paulo, São Paulo, Brazil. Correspondence and requests for materials should be addressed to M.N.S. (email: ) Scientific Reports | 7: 524 | DOI:10.1038/s41598-017-00637-3 1 www.nature.com/scientificreports/ to produce IFN-γ after cytokine stimulation, proliferate in vivo during viral infection, and kill target cells upon engagement of activating receptors. The NK cells of ESN drug users showed high cytolytic potential and produced IFN-γ, TNF-α, and β-chemokines when in contact with the K562 cell line11. In addition, NK cells from the ESN subjects produced high levels of IFN-γ in response to activation with phorbol myristate acetate and ionomycin12. The absence of human leukocyte antigen (HLA) binding to inhibitory killer-cell immunoglobulin-like receptors (KIRs), including KIR2DL2, KIR2DL3 and KIR3DL1, leads to a reduced activation threshold of NK cells from ESN individuals and has been associated with resistance to HIV-1 infection13. In contrast, chronic HIV-1 infection alters the population distribution and functional capacity of NK cells. The chronic activation receptor CD38 is an ectoenzyme expressed on CD8+ T cells and is associated with progression to AIDS in chronically HIV-1-infected patients, even in those treated with antiretroviral therapy (ART)14, 15. Moreover, NK cell activation through CD38 is increased in HIV-1-infected subjects progressing to AIDS, but not in elite and viremic controllers, and is associated with viremia and disease progression markers in both HIV-1 and HIV-2 infections16, 17. Whether the NK CD38+ cells in HIV-1 infection are associated with altered expression of inhibitory/exhaustion molecules, such as NKG2A, PD-1, and Tim-3, is unclear. Few cytotoxic NK cells and high NKG2A expression have been observed in patients with late-stage HIV infection18. The expression of Tim-3, a type I transmembrane protein, has been implicated both in activation and inhibition of immune responses19, 20, in the induction of apoptosis of Tim-3–bearing cells through interactions with galectin-921 and suppression of cell-mediated cytotoxicity22. NK cells possess receptors allowing them to sense and respond to viral and bacterial patterns, including Toll-like receptors (TLRs)23. Viral nucleic acids, such as viral DNA, dsRNA, and ssRNA, can activate nucleic acid-sensing TLRs, including TLR3, TLR7, TLR8 and TLR9, to prevent viral invasion of cells via induction of type I IFN and production of antiviral factors24. TLR stimulation in ESN individuals induced a robust release of immunologic factors that can influence adaptive antiviral immune responses25. A TLR3 agonist, polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic mimetic of viral RNA, significantly augmented NK cell-mediated cytotoxicity in healthy individuals26. A common TLR3 polymorphism in ESN individuals suggested the potential use of TLR3 triggering in HIV-1 immunotherapy27. In this study, we evaluated the TLR3-induced activation of NK cells and assessed CD62L and CD38 expression in NK cell subsets to verify the polyfunctional response of NK cells in ESN individuals and their HIV-1-infected partners. Moreover, we examined whether CD38+CD62L+ NK cells were less responsive to TLR3 activation and could be (...truncated)