Pathologic natural killer cell subset redistribution in HIV-1 infection: new insights in pathophysiology and clinical outcomes

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1 Review Pathologic natural killer cell subset redistribution in HIV-1 infection: new insights in pathophysiology and clinical outcomes Enrico Brunetta,*, Kelly L. Hudspeth,*, and Domenico Mavilio*,1 *Laboratory of Clinical and Experimental Immunology, IRCCS, Istituto Clinico Humanitas, Rozzano, Milano, Italy; DI.ME.S., Dipartimento di Medicina Sperimentale, University of Genova, Genova, Italy; and Department of Translational Medicine, University of Milan, Rozzano, Milano, Italy RECEIVED APRIL 19, 2010; REVISED JUNE 17, 2010; ACCEPTED JUNE 30, DOI: /jlb ABSTRACT Several lines of evidence indicate that the interaction of HIV-1 with NK cells markedly affects host immune responses and leads to a defective control of the virus. Until recently, it was generally believed that the absolute number of total circulating NK cells was decreased during the course of chronic and active phases of HIV-1 infection and that this explained, at least in part, the defective NK cell antiviral activities. However, scientific advances made over recent years have changed this concept and have clarified that HIV-1 viremia is associated with a pathologic redistribution rather than an absolute decrease in the number of circulating NK cells. In particular, the expansion of dysfunctional Siglec-7 neg and/or CD56 neg NK cell subsets expressing an aberrant repertoire of activating and inhibitory receptors has been associated with functional impairments of NK cells and with clinical outcomes of HIV-1 disease. Indeed, these pathologic NK cell populations often comprise the majority of NK cells in the presence of high levels of chronic HIV-1 viremia. The reasons for these NK cell aberrancies remain unknown, as freshly purified CD4 neg NK cells are not productively infected by HIV-1. Disclosing the cellular and molecular mechanisms underlying such dysfunctions represents an important challenge of biomedical research, also considering that the presence of a rare KIR3DS1 pos NK cell population represents a protective factor against HIV-1 disease progression. In this review, we will summarize the recent updates regarding NK cell pathophysiology during the course of HIV-1 infection. J. Leukoc. Biol. 88: ; Abbreviations: ADCC antibody-dependent cellular cytotoxicity, ART antiretroviral therapy, CD56 neg NK cells CD56 neg /CD16 pos NK cells, HCMV human CMV, HCV hepatitis C virus, HLA-B Bw4-80I HLA-B Bw4 with an isoleucine at position 80, inkr inhibitory NK cell receptor, KIR killer Ig-like receptor, LTNP long-term nonprogressor, MMP matrix metalloproteinase, NCR natural cytotoxiciy receptor, slan/m-dc8 6-sulfo LacNac pos M-DC8 pos Introduction NK cells, identified in the mid-1970s [1, 2], are a subset of lymphoid effectors of the innate immune system that have important roles in the clearance of virus-infected and tumortransformed cells [3, 4]. They account for up to 15% of peripheral blood lymphocytes and can also be found in many major organs, including liver, intestine, lung, cervix, and placenta, and in peripheral lymphoid organs, such as spleen, lymph nodes, and tonsils [4]. The reason that they were named NK cells is based on their unique ability to lyse tumor cells without prior sensitization to antigen [5]. Indeed, early animal studies showed that although RMA T cell lymphomas grow progressively in syngeneic mice, the RMA-S tumor cell line lacking MHC-I molecules was rejected by host NK cells. These pivotal experiments led to the identification of inkrs and made it possible to demonstrate that the binding of inkrs with self-mhc-i molecules represents the key mechanism that prevents the NK cell-mediated lysis of autologous cells. In contrast, the decreased engagement of inkrs against cellular targets with reduced or absent expressions of MHC-I molecules (i.e., virus-infected or tumor-transformed cells) allows NK cells to kill these dangerous non-self targets (missingself hypothesis) [6, 7]. However, although a defective binding between inkrs and MHC-I molecules enables the NK cell-mediated killing, an activation cellular pathway is also required to trigger the cytolytic process. In this regard, several activating NK cell receptors and coreceptors detect the presence of specific ligands on stressed, infected, or tumor-transformed cells, and these interactions led to the elimination of the targeted cells [8, 9]. Therefore, NK cell activation and target cell recognition represent the final step of a complex process that is based on a dynamic balance between activating and inhibitory signals that are delivered simultaneously through several NK cell receptors following the engagement of their putative ligands expressed by neighboring cells. The acquisition of such mechanisms during evolution has been achieved through a 1. Correspondence: Laboratory of Clinical and Experimental Immunology, IRCCS, Istituto Clinico Humanitas Via Alessandro Manzoni, 113, Rozzano, Milano, Italy. domenico.mavilio@humanitas.it /10/ Society for Leukocyte Biology Volume 88, December 2010 Journal of Leukocyte Biology 1119

2 highly sophisticated process that ensures NK cell-mediated recognition of self [10 14]. In addition to their ability to lyse harmful cells, activated NK cells secrete various cytokines and chemokines that mediate the suppression of viral replication, contribute directly to the prevention of viral entry into host cells, and assist in the amplification of the immune response by recruiting and priming cells of the innate and adaptive immunity [15, 16]. Finally, NK cells also represent an important link between the innate and adaptive immune system by interacting directly with autologous DCs. This process requires NK cell DC cellular contacts and secretion of specific cytokines. The outcome of this crosstalk is the coordination and optimization of correct DC maturation to develop an antigen-specific T cell response [17 23] (Fig. 1). All of the functions reported above make it clear that NK cells represent a fundamental component of innate immunity that protects the host from various viruses and tumors by acting as effector and regulatory cells. Nevertheless, NK cells are unable to control the spread of HIV-1 and are defective in the clearance of autologous, HIV-1-infected CD4 T cells that represent the major reservoir of the virus [24 33]. Indeed, it has been reported that high levels of HIV-1 replication severely impair NK cell-mediated killing of tumor cell targets to decrease NK cell production of antiviral cytokines markedly and to disrupt NK cell interactions with autologous DCs [24, 25, 34, 35]. Only in the recent years has it been clarified that these invalidating NK cell functional defects observed in HIV- 1-infected patients in active and advanced disease stages are associated directly with the emergence of pathologic and aberrant NK cell subsets that are rarely represented in healthy donors [36 38]. This review focuses on the clinical significance of abnormal NK cell phenotypes and functions induced by high levels of HIV-1 plasma viremia. In particular, we expand the argument that the direct correlations between the pathologic redistribution of dysfunctional NK cell subsets and the clinical outcomes of HIV-1 diseases might represent a novel diagnostic tool that can help physicians to better characterize the clinical stages of HIV-1 infection. Finally, the expansion of aberrant NK cell populations in response to viral replication is a pathogenic mechanism that may reveal novel therapeutic approaches against HIV-1 infection. PATHOLOGIC REDISTRIBUTION OF NK CELLS IN THE PERIPHERAL BLOOD OF HIV-1-INFECTED PATIENTS Appearance of a dysfunctional CD56 neg NK cell population In humans, NK cells express CD56, a surface antigen that has also been found on a subset of T lymphocytes and on cells derived from neural, muscle, and embryonic tissues [39]. Another common marker on human NK cells is CD16, an Fc RIII involved in ADCC that is also expressed on a subset of CD3 pos /CD8 pos T cells [40], as well as on slan/m-dc8 cells [41], monocytes/macrophages, mast cells, and neutrophils Lymph Node Migration CD4+ T Cell Non-Self Targets (Tumor Cells - Others) Figure 1. NK cells mediate links between innate and adaptive immunity. Under physiological conditions, NK cells can act as effector cells in the immune surveillance against dangerous tumor-transformed or viral-infected, non-self cellular targets. In addition, NK cells can function as regulatory cells that optimize the links between innate and adaptive immune responses through appropriate interactions with autologous DCs (see Introduction and manuscript text for further details). mdcs, Mature DCs; idcs, immature, DCs. Inflammatory Insult Maturation mdcs Crosstalk idcs Priming mdcs Protection from NK Cell Lysis Cytotoxicity Antibody Dependent Cell Cytotoxicity (ADCC) Self/Non-Self Targets NK Cell Inflammatory Insult Decreased Negative HLA-E NCRs-L HLA-A/B/C NKG2A NCRs KIRs Bacteria - Virus - Parasite Antibodies CD56 CD16 Siglec-7 Regular Pathways Abnormal Pathways B Cell 1120 Journal of Leukocyte Biology Volume 88, December

3 Brunetta et al. NK cell subset redistribution in HIV-1 infection [39]. Circulating CD14 neg /CD19 neg /CD3 neg NK cell subsets can be identified on the basis of their surface expression of CD56 and CD16. In particular, the largest NK cell subset (up to 90% of all circulating NK cells), which expresses a CD56 dim /CD16 pos phenotype, exerts a cytolytic function and can also produce high levels of proinflammatory and antiviral cytokines upon stimulation with specific stimuli [42, 43]. Only a minority of the NK cell population in the blood (10 15%) is characterized by a CD56 bright /CD16 dim/neg phenotype. These cells display poor cytotoxicity but can produce large amounts of cytokines (Fig. 2) [42]. For years, several studies reported that the percentage and/or absolute number of total NK cells in the peripheral blood are decreased during the course of chronic and active HIV-1 infection [25, 27]. Based on this, it has been hypothesized that high levels of ongoing HIV-1 replication were likely involved in lowering the number of circulating NK cells through several mechanisms, such as an early NK cell death/ apoptosis or a preferential NK cell distribution into peripheral tissues [25, 27]. In many reports though, identification of NK cell populations was based predominantly on the surface expression of the CD56 and CD16 NK surface markers on CD3 neg cells [44 47]. Therefore, the selective loss of the cytolytic CD56 dim /CD16 pos NK cell subset in peripheral blood of chronic and viremic HIV-1-infected individuals has been interpreted often as an absolute depletion of blood NK cells without considering the possibility of an expansion of other pathologic NK cell populations during HIV-1 infection. Although the first description of a CD56 neg NK cell subset with a low lytic activity in HIV-1-infected patients was published in 1995 [34], the expansion of dysfunctional NK cells during the course of HIV-1 infection began to be investigated extensively only after 2002 [36 38]. Since then, novel, multicolor flow cytometry approaches analyzing the CD56 and CD16 surface expression on CD14 neg /CD19 neg /CD3 neg NK cells (Fig. 2) [48] have made it possible to characterize extensively the phenotype and functions of the pathologic CD56 neg NK cell subset that is highly represented in chronic and active phases of HIV-1 infection and that is rarely observed in uninfected donor. This phenomenon, together with the decreased frequencies of CD56 dim /CD16 pos NK cell populations during chronic HIV-1 infection, demonstrates that HIV-1 viremia is associated with a significant and pathologic redistribution of NK cell subsets rather than with an absolute decrement of total NK cells in the peripheral blood [34, 36 38, 48, 49]. To avoid possible cellular contaminations and to be sure that CD14 neg /CD19 neg /CD3 neg /CD56 neg cells were indeed NK cells, an extensive characterization of activating and surface inkrs was performed [36 38, 48]. These analyses made it possible to distinguish NK cells from other CD14 neg and CD16 pos cell subsets with a size similar to that of lymphocytes, such as slan/m-dc8 [41, 50] or CD7 neg /CD56 pos monocyte/dc-like cells [51]. Indeed, neither slan/m-dc8 pos cells nor CD7 neg /CD56 pos monocyte/dc-like cells express significant levels of NK cell surface receptors, such as KIRs, NKp30, NKp46, NKG2A, and others, and they express myeloid-associated markers, such as CD13 and CD33 [41, 50, 51]. In contrast, normal 1000 Total PBMCs Healthy Donor % SSC-H Monocytes # Cells NK Cells CD56 +(bright) /CD FSC-H Lymphocytes CD3 Fitc 10 4 T cells T and NK+B cells CD14 PE CY7 NK + B cells CD19 APC CY NK and B Cells B Cells NK cells CD56 Pc5 CD16 APC 94% CD56 +(dim) /CD16 + CD56 - /CD16 + CD56 Pc5 CD56 Pc5 Siglec-7 PE Figure 2. Characterization of NK cell subsets within PBMCs. Within the lymphocyte gate of total PBMCs from a representative example for a healthy donor, NK cell subsets are separated from CD14 pos (monocyte), CD3 pos (T lymphocytes), and CD19 pos (B lymphocytes) and then characterized for CD56, CD16 (right, upper dot-plot graph), and Siglec-7 (right, lower histogram graph) surface expression. SSC/FSC-H, Side-/forwardscatter-height; APC, allophycocyanin. Volume 88, December 2010 Journal of Leukocyte Biology 1121

4 CD56 pos and pathologic CD56 neg NK cell subsets expressing several activating and inkrs do not express myeloid-associated receptors [35 38, 48]. These phenotypic differences allow for the identification of pathologic NK cell subsets lacking typical surface markers such as CD56. The sequential deregulation of the NK cell subset distribution starts during the early phases of HIV-1 infection. In fact, during acute HIV-1 infection, the absolute number of total circulating NK cells is increased significantly when compared with that of uninfected individuals. This phenomenon is associated with high frequencies of CD56 dim /CD16 pos cytolytic NK cell subsets and an early depletion of the CD56 bright /CD16 neg NK cell populations without a significant expansion of the CD56 neg NK cell population [38]. When HIV-1 infection becomes chronic, high levels of viral replication induce a significant depletion of CD56 dim /CD16 pos NK cell subsets with a parallel increase of functionally impaired CD56 neg NK cell subsets. The suppression of HIV-1 viremia for 24 months or longer in patients having undergone ART in cross-sectional studies restores CD56 expression on NK cells to levels similar to those of cells from uninfected individuals [37]. Considering that CD56 neg NK cells interfere severely with several innate and adaptive immune responses against HIV-1 [24 26, 32, 33], the kinetics of CD56 distribution during the course of HIV-1 infection remained an important issue to clarify. In this regard, it has been reported that chronic and ART-naïve, HIV-1- infected patients with a low or undetectable plasma viremia that does not progress toward AIDS (LTNPs) do not display an abnormal expansion of circulating CD56 neg NK cells, thus confirming that high HIV-1 viral load is associated with the expansion of this subset. In addition, we found that regardless of the high levels of ongoing viral replication occurring in the initial phases of HIV-1 infection, the surface levels of CD56 on NK cells from early HIV-1-infected patients are similar to that of healthy donors. These data confirm that only chronic insults given by HIV-1 viremia correlate with the appearance at high frequencies of CD56 neg NK cells [38, 48, 52]. In line with the slow, progressive emergence of CD56 neg NK cells in HIV-1-infected, viremic patients, the suppression of viral load results in a slow and progressive restoration of CD56 on the NK cell surface. Indeed, our longitudinal analyses published recently show that a minimum of 18 months of undetectable viremia is necessary to observe a significant decrease of CD56 neg NK cell subsets in chronic HIV-1-infected viremic patients having undergone ART [48]. Taken together, these data demonstrate that the down-modulation of CD56 on NK cells, as well as its recovery following ART, is associated with high levels of chronic viral replication or prolonged suppression of HIV-1 viremia, respectively. Several studies have reported that the number of NK cells expressing inkrs is conserved or increased significantly in HIV-1-infected, viremic individuals [36, 37, 46, 47, 53], and this accounts for an increased receptor-specific inhibition of NK cytolytic function in vitro against the Fc R pos P815 target cell line in redirected killing assays [36, 37]. Of note, the increased expression of inkrs during HIV-1 infection has not been reported consistently, and some studies showed similar levels of inkr expression in HIV-1-infected and uninfected individuals [46, 47, 53]. These discrepancies are most likely a result of the heterogeneity of several factors: the disease states of the individuals with HIV viremia, the types of samples that were used (i.e., whole blood vs. purified NK cells), the flow cytometric approach, and the fact that the majority of these studies did not take into account the pathologic expansion of the dysfunctional CD56 neg NK cell expressing the highest surface levels of inkrs [24, 37]. Moreover, analyses of expression of activating receptors on NK cells from HIV viremic individuals revealed that all 3 NCRs NKp44, NKp46, and NKp30 are expressed at significantly lower levels when compared with HIV-negative controls [36, 54]. Therefore, despite the persistent and aberrant activation of NK cells as a result of their chronic exposure to HIV-infected cells [55], NK cells have a NCR low phenotype that affects NK cell-mediated immune surveillance severely against those opportunistic infections and tumors that normally occur in the late stages of HIV infection [24 26]. All of these phenotypic and functional NK cell abnormalities are pronounced, particularly on the CD56 neg NK cell population, in which its dramatic expansion at high frequencies in viremic and chronic HIV-1-infected individuals accounts for the functional defects in NK cell-mediated cytotoxicity [28, 36, 38], in the interactions with autologous DCs [35], and in the secretion of important antiviral cytokines such as IFN-, TNF-, and GM-CSF [37]. Regarding the presence of pathologic NK cell subsets during the course of other viral diseases, it has been reported recently that chronic HCV infection is also associated with high frequencies of dysfunctional CD56 neg cells, whose cytolytic capacity is impaired significantly compared with that of the conventional CD56 pos NK cells. This expansion is greater in patients who failed to clear HCV in response to pegylated IFN- and ribavirin. Moreover, the same study shows that those therapynaïve, HCV-infected patients with the highest levels of CD56 neg NK cells were at least able to clear HCV in response to treatment. However, CD56 neg NK cells retain the capacity to produce MIP-1 during HCV infection, and this has been proposed as a possible mechanism involved in the establishment of chronic liver inflammation [56]. Taken together, these data indicate that the expansion of a dysfunctional CD56 neg NK cell population occurs, not only during the course of HIV-1 ingection but also in other chronic viral diseases (i.e., HCV infection). Furthermore, similar to HIV-1 infections, high frequencies of CD56 neg NK cells correlate with clinical outcomes of HCV disease and with responses to antiviral therapies. Further investigations are needed to understand if other infectious or immune-mediated disorders (i.e., autoimmune diseases) sharing a chronic and aberrant activation of the immune system are associated with the emergence of CD56 neg NK cells or any other dysfunctional NK cell populations. Siglec-7 as an early marker of dysfunctional NK cell subsets Although several groups have reported aberrancies of the NK cell receptor repertoire during active phases of HIV-1 infections, since the late-1990s, a cellular marker highly sensitive to viral replication that can be associated with NK cell abnormalities starting from the initial stages of HIV-1 infection has been 1122 Journal of Leukocyte Biology Volume 88, December

5 Brunetta et al. NK cell subset redistribution in HIV-1 infection identified only recently. Indeed, we reported recently that Siglec-7 represents a novel and sensitive marker that identifies and tracks NK cell phenotypic changes and impaired functions during different stages of HIV-1 infection [48]. In this study, we show that the early, decreased expression of Siglec-7 on NK cells in HIV-1-infected individuals precedes the down-regulation of CD56, which occurs mostly in patients in chronic stages of infection and with high levels of ongoing viral replication. The different kinetics of Siglec-7 versus CD56 down-modulation resulted in a reduction of normal Siglec-7 pos /CD56 pos NK cells and allowed the detection of 2 new pathologic Siglec- 7 neg /CD56 pos and Siglec-7 neg /CD56 neg NK cell subsets. Siglec- 7 neg /CD56 pos NK cells are preferentially expanded in early stages of infection, and the Siglec-7 neg /CD56 neg phenotype was detectable only in NK cells from chronic, viremic, HIV-1- infected patients. The low or undetectable levels of viral replication in LTNPs do not affect the expression of Siglec-7 and CD56 on NK cells. Indeed, the distribution of peripheral blood NK cell subsets in LTNPs is comparable with that observed in healthy, uninfected individuals, thus indicating that these pathologic NK cell phenotypes cannot be observed in the absence of high HIV-1 viremia (Fig. 3). These phenotypic abnormalities on NK cells were associated directly with progressive and distinct impairments of NK cell cytolytic activities and cytokine production. The maximal loss of NK cell function was reached with the expansion of the Siglec-7 neg / CD56 neg subset in chronic, viremic, HIV-1-infected patients. Finally, the possibility of following longitudinally chronic, viremic, HIV-1-infected patients, who suppressed HIV-1 replication successfully to undetectable viremia following ART, allowed us to identify exactly the kinetics of NK cell restoration of Siglec-7 and CD56 expression. In particular, the different kinetics of Siglec-7 and CD56 recovery on NK cell surface in the presence of a successful viral suppression in patients having undergone ART results in a progressive switch from Siglec- 7 neg /CD56 neg to Siglec-7 pos /CD56 neg NK cells within the first 18 months of therapy. A complete restoration of the Siglec- 7 pos /CD56 pos NK cell phenotype can be observed only after 24 months of ART. The identification of these 2 dysfunctional NK cell subsets confirms further that the expansion of aberrant populations is an important mechanism by which HIV-1 impairs NK cell antiviral functions, thus affecting the quality of host immune responses. We did not observe any significant differences in the amount of total NK cells between uninfected and HIV-1-infected individuals when we analyzed the frequencies of the entire NK cell population, which includes normal Siglec-7 pos /CD56 pos and pathologic Siglec-7 neg /CD56 pos and Siglec-7 neg /CD56 neg cells. These data confirm further that high levels of HIV-1 replication are associated with a pathological redistribution of NK cell subsets rather than a reduction in the absolute numbers of circulating NK cells. The importance of these new findings is also represented by the fact that the specific and peculiar distribution of NK cell subsets at different stages of HIV-1 infection correlates perfectly with the status of immune competence of NK cells. This scientific disclosure can be proposed as a new, potential clinical tool to help physicians track the effectiveness of treatment and to characterize the clinical stages of HIV-1-infected patients associated with their status of immunodeficiency. Multicentric and larger clinical trials are needed to validate this hypothesis and to propose Siglec-7 (together with CD56) as a novel biomarker to use in HIV-1 infection. NKG2A/NKG2C ratio in HIV-1-infected patients coinfected with HCMV Among the many abnormalities in the surface levels of the NK cell receptors from HIV-1-infected viremic patients, a decreased expression and function of NKG2A has also been reported [36, 37]. NKG2A is a c-type lectin receptor coupled with CD94 to form an inhibitory heterodimer that recognizes 10 4 Healthy Donor CD56 + / Siglec-7 + : 92% CD56 + / Siglec-7 - : 3% CD56 - / Siglec-7 - : 4% 10 4 Early HIV-1 Infected Viremic Patient CD56 + / Siglec-7 - : 38% CD56 + / Siglec-7 - : 50% CD56 - / Siglec-7 - : 9% 10 4 Chronic HIV-1 Infected Viremic Patient CD56 + / Siglec-7 - : 24% CD56 + / Siglec-7 - : 20% CD56 - / Siglec-7 - : 51% 10 4 Chronic HIV-1 Infected LTNP CD56 + / Siglec-7 - : 91% CD56 + / Siglec-7 - : 7% CD56 - / Siglec-7 - : 0.5% CD56 Pc5 Siglec-7 PE Figure 3. Pathologic redistribution of NK cell subsets in HIV-1 infection on the basis of CD56 and Siglec-7 cell surface expression. Flow cytometric dot-plot graphs showing the distribution of fresh Siglec-7 pos /CD56 pos (green), Siglec-7 neg /CD56 pos (blue), and Siglec-7 neg /CD56 neg (red) NK cell subsets from a representative healthy donor (first column), early viremic (second column), chronic-viremic (third column), and LTNP (fourth column) HIV-1-infected patient. Volume 88, December 2010 Journal of Leukocyte Biology 1123

6 HLA-E on target cells [57]. In cross-sectional studies, suppression of HIV-1 viremia to undetectable levels in patients who underwent ART for 2 years or longer restored the expression of NKG2A on NK cells [36, 58]. The loss of NKG2A pos NK cells in chronic, viremic, HIV-1-infected individuals has also been associated with a dramatic expansion of NKG2C pos NK cells, whose frequency is still elevated even after prolonged viral suppression in patients having undergone ART [58]. The CD94-NKG2C heterodimer recognizes HLA-E with similar specificity to the CD94-NKG2A complex but triggers an activating NK cell pathway [59]. In this context, it has also been reported that the association between high levels of NKG2C pos NK cells and HIV-1 infection vanishes when HCMV serological status is considered in a multivariate regression model, thus suggesting that changes in NKG2C expression on NK cells in HIV-1-positive patients are related to a concomitant comorbidity with HCMV rather than HIV-1 infection alone [60, 61]. Therefore, the identification of the viral trigger associated with the expansion of NK cell subsets expressing high levels of NKG2C is still being debated [62]. A recent report investigating the kinetics of NKG2A and NKG2C expression on NK cells during the course of HIV-1 infection confirmed that HCMV infection represents the main trigger inducing the expansion of higher frequencies NKG2C pos NK cells [63]. Indeed, the fraction of NK cells expressing NKG2C in all donors who tested seronegative for IgG anti-hcmv was low or undetectable, regardless of their HIV-1 status. Moreover, this study also analyzed the NKG2A/NKG2C ratio on NK cells from HIV-1- uninfected, healthy donors and showed that the fraction of NKG2A pos NK cells always exceeds that of NKG2C pos cells, regardless of HCMV serological status. Therefore, the NKG2A/ NKG2C ratio on NK cells from healthy donors is constantly 1. A completely different picture is detected on NK cells from chronic, viremic, HIV-1-infected patients, which always showed a NKG2A/NKG2C ratio 1. As all HIV-1-infected, chronic, viremic patients analyzed in this study tested positive for anti-hcmv IgG, the inversion of the NKG2A/NKG2C ratio was associated with the high frequencies of circulating NKG2C pos NK cells and with the low levels of circulating NKG2A pos NK cells. In contrast, the absence of chronic HIV-1 replication in early-infected patients and the low or undetectable levels of HIV-1 viremia in LTNPs were not associated with the NK cell expression of NKG2A. Hence, the frequencies of NKG2A pos NK cells in LTNPs and in early HIV-1-infected patients are always higher compared with those of NKG2C pos NK cells, thus making the NKG2A/NKG2C ratio on NK cells constantly 1 [63]. Finally, longitudinal analyses on a cohort of chronic, viremic, HIV-1-infected patients demonstrated that the normalization of the NKG2A/NKG2C ratio to values 1 occurs only after 24 months of successful ART. This phenomenon is associated mainly with the slow recovery of NKG2A expression on NK cells, which despite the still-elevated, high frequencies of NKG2C pos NK in response to the chronic suppression of HIV-1 replication, normalized the NKG2A/NKG2C ratio after 2 years of successful treatment. Again, the pathologic distribution of NKG2A and NKG2C during the course of HIV-1 infection did not affect the absolute number and percentage of total NK cells [63]. In summary, these data demonstrate that high frequencies of NKG2C pos NK cells, together with a decreased NK cell expression of NKG2A, pathologically reverse the NKG2A/NKG2C ratio only on NK cells from chronic, viremic, HIV-1-infected individuals in advanced stages of disease and with a concomitant HCMV infection. This NK cell phenotypic feature renders this cohort of patients distinguishable from LTNPs and early, HIV-1-infected patients. The present characterization of the NKG2A/NKG2C ratio on NK cells also emphasizes the importance of considering HCMV infection in the context of the immunodeficiency and of the opportunistic diseases occurring in patients with AIDS. Indeed, the presence of a pathologic inversion of the NKG2A/NKG2C ratio on NK cells is associated with a concomitant HCMV infection and other opportunistic diseases in the presence of a clinical history of chronic HIV-1 replication. In this context, it has also been reported that HIV-1-infected individuals have higher anti-hcmv antibody titers compared with HIV-1-uninfected individuals who are HCMV-seropositive, which is indicative of HCMV reactivation [64]. Therefore, changes of physiologic NKG2A/NKG2C ratios on NK cells might be proposed as a novel biomarker that can help physicians follow the progression of HIV-1 disease and identify coinfections with HCMV. Moreover, the normalization of this proposed biomarker in response to a successful treatment can also be associated with effectiveness of chronic suppression of HIV-1 replication by ART. Larger clinical trials are needed to validate this hypothesis. DIRECT INTERACTIONS BETWEEN HIV-1 AND NK CELL SUBSETS The mechanism(s) inducing such impressive NK cells aberrancies in phenotypes and function in the course of HIV-1 infection are still unclear. One of the first hypotheses that have been tested to disclose this important issue was to investigate the direct infection of NK cells by HIV-1. Nevertheless, whether NK cells are naturally infected by HIV-1 in vivo is still controversial. CD4, a key receptor for HIV-1 infection, is generally expressed on Th cells and monocytes/macrophages as well as on subsets of DCs, / T cells, mast cells/basophils, neutrophils, and NKT cells [42, 65 68]. Fresh NK cells purified ex vivo from human donor blood lack cell-surface expression of CD4 [36, 69]. They do, however, express the chemokine coreceptors for HIV-1, CXCR4, and CCR5 [70], but these receptors alone are unlikely to allow productive infection of NK cell by HIV-1 in vivo. Indeed, freshly isolated blood NK cells from HIV-1-infected patients do not carry HIV-1 proviral DNA [36]. In contrast, another report identified a small subset of CD3 neg /CD56 pos /CD16 pos cells expressing CD4 and HIV-1 coreceptors CCR5 and CXCR4. In this study, Valentin et al. [71] argue that this NK cell subset remains persistently infected even in HIV-1-positive patients with undetectable viral load after 1 or 2 years of highly effective ART. Although these cells were not fully characterized for the expression of specific NK cell receptors, such as NCRs, the productive infection of this NK cell population by HIV-1 has been proposed as an another reservoir for virus persistence in addition to the predominant one represented by latently infected CD4 T cells Journal of Leukocyte Biology Volume 88, December

7 Brunetta et al. NK cell subset redistribution in HIV-1 infection One of the reasons for this discrepancy might be linked to the activation state of NK cells and to their ability to express CD4 only after a significant proinflammatory insult. In fact, triggering the TCR complex in vitro leads to the de novo expression of CD4 on CD8 pos T cells and makes these cells susceptible to HIV-1 infection [72 74]. Similarly, a maximal activation in vitro of freshly purified NK cells with several concurrent stimuli induces a significant de novo expression of CD4. Under these experimental conditions, NK cells become susceptible to infection if cocultured with HIV-1-infected T cells [69]. High levels of chronic HIV-1 viremia are certainly associated with an aberrant activation of NK cells [55, 70]. Although CD4 neg NK cells, purified freshly ex vivo from peripheral blood of HIV-1-infected patients in active phases of disease, have been reported not to be infected by HIV-1, it remains to be determined whether NK cells can modulate CD4 expression in response to a maximal insult given by the virus chronically circulating in the bloodstream. Moreover, given the fact that NK cells freshly isolated from secondary lymphoid organs have been shown to express CD4 [75], it is also possible that this small subset of CD4 pos NK cells is susceptible to HIV-1 infection. Regardless of the capacity of HIV-1 to infect NK cells productively through the CD4- and chemokine receptor-dependent mechanisms, it has been reported that the HIV-1 gp120 viral envelope is able to disrupt NK cell-mediated cytolytic activity [76]. This phenomenon is a result of a direct binding of gp120 with an activated form of the 4 7 integrin on NK cells, an interaction that leads to the phosphorylation of the p38 MAPK [77]. This binding also takes place on T cells, and in particular, it has been described that 4 7 high CD4 pos T cells are more susceptible to be infected productively than 4 7 low-neg CD4 pos T cells, in part, because this subset is enriched with metabolically active CD4 pos T cells. The specific affinity of gp120 for the 4 7 receptor provides a mechanism for HIV-1 to target activated cells that are critical for efficient virus propagation and dissemination [78]. Whether 4 7 pos NK cell subsets can be infected productively by HIV-1 through a similar process has not yet been disclosed. Nevertheless, it has been demonstrated already that the binding of gp120 with 4 7 on NK cells markedly impairs their lytic functions and activation state as well as the NK cell-mediated secretion of antiviral cytokines and increases the rate of apoptotic events [76]. The previously reported, decreased NK cell expression of Siglec-7 in patients with high levels with HIV-1 plasma viremia can also disclose new insights regarding the interactions between NK cells and HIV-1. Siglec-7 belongs to a family of 14 sialic acid-binding, Ig-like lectins and is constitutively expressed on all NK cells and monocytes [79, 80]. Siglec-7, as well as many other members of its family, is masked at the cell surface as a result of cis interactions with abundantly expressed, lowaffinity sialic acid ligands on the cell surface. Following exposure of cells to sialidase or in some cases, following cellular activation, which cleaves the cis-interacting, low-affinity ligands, Siglec-7 becomes unmasked and is free to interact in trans with highly glycosylated ligands [80]. Siglec-7 has an unusual binding preference for 2,8-linked disialic acids, such as those displayed by ganglioside GD3. In line with the inhibitory function of this sialoadhesin [79] reported previously, NK cell-mediated killing of GD3 synthase-transfected P815 cells was highly reduced following sialidase treatment of NK cells [81]. These findings suggest that the binding of Siglec-7 with sialic acids might have important implications in modulating NK cell cytolytic activity. Until recently, Siglecs have been studied mostly in the context of adhesion and cellular signaling, but there are growing evidences suggesting their role in endocytosis [82]. This is of particular relevance for cells of the innate immune system, where phagocytic clearance of apoptotic cells and pathogens is critical. In this context, it has been shown recently that Siglec-7 on NK cells is able to recognize sialylated glycans expressed on Campylobacter jejuni lipooligasaccharides [83], but neither the signaling pathway involved in this recognition nor the potential NK cell endocytosis of this pathogen was investigated. HIV-1 envelope is a highly glycosylated protein [84]. The fact that Siglec-7 is decreased rapidly on NK cells in the presence of high levels of virus suggests that interactions between Siglec-7 and HIV-1 might occur on NK cells, as it has been demonstrated recently for Siglec-1 on monocytes [85]. In this regard, similar to the HIV-1 envelope binding to CD209 (DC sign) on DCs [86], the supposed binding between HIV-1 envelope and Siglec-7 could trigger an endocytic process, possibly explaining the decreased Siglec-7 expression on NK cells starting from the initial phases of HIV-1 infection. The direct contact between HIV-1 envelope and this sugarbinding lectin could also trigger the inhibitory Siglec-7 pathway in NK cells, thus affecting their cytolytic potential. Further studies are needed to validate this hypothesis and disclose the mechanism(s) underlying the reduction of this sialoadhesin on NK cells in the presence of high levels of HIV-1 replication. Moreover, the identification of a sialoadhesin as a novel NK cell marker highly sensitive to HIV-1 viremia opens new scientific perspectives about the potential role of Siglec-7 as a regulator of NK cell activities during the course of HIV-1 replication. KIR3DS1 POS NK CELL SUBSET IS ASSOCIATED WITH A POSITIVE CLINICAL OUTCOME OF HIV-1 INFECTION It has been known for many years that the expression of certain HLA-I alleles is associated with a better clinical outcome of HIV-1 disease. However, most of these studies focused on virus-specific CD8 pos T cells [87, 88]. Although the effect of HIV-1 viremia alters the conventional NK cell phenotypic profile and markedly impairs several NK cell functions, a parallel field of research explored the possible existence of NK cellrelated immune correlates of protection from HIV-1 infection. In this regard, epidemiological studies reported that several host genetic factors, such as the association between a 32-bp deletion in CCR5 and the increased resistance to infection or the protective role exerted by several HLA-I alleles, are strongly associated with a better clinical outcome of HIV-1 disease [87 91]. These investigations were focused on HIV-1-specific CD8 pos T cells and highlighted their critical role in the control of viral replication. Only recently has it been demon- Volume 88, December 2010 Journal of Leukocyte Biology 1125

8 strated that the expression of an activating form of the KIR3D NK cell receptor (KIR3DS1) in conjunction with its putative ligand, HLA-B Bw4-80I, is associated with a significant control of HIV replication and with a slower progression to AIDS [92]. In the absence of the KIR3DS1 allele, HLA-B Bw4-80I allele expression did not protect against disease progression. Furthermore, occurrence of the KIR3DS1 allele in the absence of the HLA-B Bw4-80I allele was associated with a rapid progression to AIDS among HIV-infected individuals, suggesting that an epistatic association between the 2 loci is necessary for protection [93]. These epidemiologic analyses have been supported recently and confirmed functionally by experimental data showing that the slower disease progression is caused by the ability of KIR3DS1 pos NK cell subsets to strongly inhibit HIV-1 replication in autologous CD4 pos T cells expressing HLA-B Bw4-80I. Moreover, the presence of KIR3DS1 pos NK cell subsets is also associated with higher NK cell-mediated secretion of IFN- and cytotoxicity, starting from the early phases of HIV-1 infection [94]. Altogether, these epidemiologic and experimental evidences demonstrate that particular NK cell subsets expressing KIRDS1 can also exert a protective role in HIV-1 pathogenesis through a mechanism that might be useful in the future to develop new antiviral, therapeutic strategies. In an additional study, the protective role of KIR3DL1, the inhibitory counterpart to KIR3DS1, and various HLA-Bw4 alleles [95] has also been reported. This is quite surprising given the protective role of the KIR3DS1, which is an activating receptor. One given hypothesis to explain this phenomenon is that the expression of inhibitory receptors by NK cells grants them a license to kill [96, 97]. Indeed, it has been described that KIR3DL1 pos NK cells from donors express 2 copies of HLA-Bw4 genes, rather than only 1 or no copies, thus making NK cells more active in response to tumor cell targets [98]. ADCC ADCC is an adaptive immune response mediated largely by NK cells through the CD16 receptor (FC RIII), which binds the Fc portion of IgG antibodies triggering the lysis of targeted cells. HIV-1 stimulations should theoretically generate specific and/or neutralizing antibodies helpful for an effective ADCC against infected cells carrying viral antigens on their surface. It has been reported that the titers of env-specific, ADCC-mediated antibodies decrease in the sera of HIV-1-infected individuals as the infection progresses toward AIDS [99]. Moreover, cells that mediate ADCC also become functionally compromised starting from early stages of HIV infection, thus depriving the host of the potential benefits of this effector function exerted by the immune system. A significant inverse correlation between the degree of impairment of NK cell cytotoxicity and the levels of HIV plasma viremia has also been shown, making it clear that chronic viral replication negatively affects ADCC [100]. Furthermore, a recent study showed that impaired ADCC function mediated by NK cells is abrogated by inhibitors of MMPs, which have been shown to induce the sloughing of CD16 from the surface of NK cells [101]. In this regard, it has been reported that the levels of MMPs in the serum of patients with HIV-1 infection are increased [102]. Altogether, these data suggest that despite the potential benefits of ADCC in the context of HIV-1 infection, the virus largely evades this mechanism. Finally, another recent work analyzed ADCC antibodies from a cohort of untreated, HIV-1-infected patients and found that serum of infected donors contained HIV-1 antibodies that could induce NK cell-mediated IFN- production and CD107a degranulation as a measure of ADCC cytoxicity [103]. Interestingly, this study found that NK cells from HIV-1-infected donors produced more IFN- and expressed higher levels of CD107a compared with those of healthy donors, despite reports of highly dysfunctional NK cell subsets present in HIV-1- infected donors [24 27, 32, 33]. However, this report did not correlate aberrant NK cell populations with ADCC. Another interesting finding in this study is that there was a poor correlation between ADCC-mediated killing and IFN- production by NK cells, which suggests differential regulation of these 2 responses. These are compelling findings that highlight the complexities of NK cell responses to HIV-1 and furthermore, could suggest new ways of treating patients by taking advantage of ADCC-mediated mechanisms. IMMUNOMODULATORY HIV-1 TARGETING OF NKR LIGANDS It is well known that HIV-1 viremia induces a CD4 pos T cell depletion that leads to immunodeficiency and correlates with disease progression. However, it has also been reported that the majority of CD4 pos T cells dying during infection are not infected productively with HIV-1 [104]. One possible explanation is that these uninfected CD4 T cells are eliminated through a mechanism not directly linked to viral replication. In this regard, it has been demonstrated in vitro [29] and ex vivo [28] that HIV-1 replication can modulate the expression of ligands for NKp46, NKp30, and NKp44 on uninfected CD4 T cell blasts. In particular, a highly conserved motif of HIV-1 gp41 envelope protein can induce the expression of the NKp44 ligand on uninfected CD4 T cell blasts and render these cells susceptible to NK cell-mediated killing via the NKp44 activation pathway [105]. A recent report showed that immunization against the 3S gp41 peptide prevents the expression of the NKp44 ligand on CD4 pos T cells, thus lowering the depletion of these cells in a model of SHIV-infected primates. These results provide new insight to possibly develop alternative preventive and therapeutic HIV-1 vaccine strategies [106]. However, recent results from the same group showed unexplained differences between R5 and X4 infection [107]. Another interesting interaction between NK cell receptors and their ligands is the one mediated by NKG2D, a major activating receptor. It has been shown in vitro and ex vivo that HIV-1-infected p24 pos CD4 pos T cells down-modulate MHC-I molecules and this phenomenon makes these infected cells more susceptible to NK cell-mediated killing [28, 108]. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4 pos T cell blasts that down Journal of Leukocyte Biology Volume 88, December

9 Brunetta et al. NK cell subset redistribution in HIV-1 infection Lymph Node Migration Spreading of HIV-1 Infection Non-Self Targets (Tumor Cells - Others) CD4+ T Cell Infection Defective Priming Impaired Cytotoxicity Aberrant Maturation Self/Non-Self Targets mdcs Defective Crosstalk Defective ADCC Hyper γ globulinemia NK Cell (Expansion of Siglec-7 neg neg and/or CD56 subsets) B Cell Decreased Negative HLA-E NKG2A Siglec-7 NCRs-L NCRs HLA-A/B/C KIRs HIV-1 Antibodies CD56 Figure 4. Effect on HIV-1 viremia on NK cells and their links with innate and adaptive immunity. HIV-1 viremia significantly alters the normal NK cell phenotype and induces the expansion of dysfunctional Siglec-7neg and/or CD56neg NK cell subsets expressing an aberrant repertoire of activating and inkrs. This phenomenon severely affects the NK cell-mediated lysis of tumor cell targets and autologous and endogenously HIV-1-infected CD4pos T cells. Even the ability of the CD56neg NK cell subset to interact with autologous, HIV-1-infected DCs is highly impaired. The defective NK-DC cross-talk in HIV-1infected, viremic patients is responsible for generating functionally immature DCs that fail to prime a correct antigen-specific adaptive immune response and contribute to spread the infection. High frequencies of dysfunctional NK cell subsets associated with high levels of HIV-1 viremia correlate with clinical outcomes of HIV-1 infection and with responses to ART. CD16 modulate HLA-A and -B alleles is particularly low. This phenomenon is associated with the defective surface expression and engagement of NCRs and with the high frequency of the dysfunctional CD56neg subsets of highly dysfunctional NK cells from HIV-1-infected, viremic patients [28]. The residual NK cell-mediated killing occurs mainly through the activation pathway of NKG2D [28, 29], the only main activating NK cell receptor whose surface expression is not affected by high levels of plasma viremia [36, 37]. In this regard, it has been demonstrated in vitro and ex vivo that the cell-surface expression of specific ligands for NKG2D on primary-infected CD4pos T cells is up-regulated by HIV-1 Vpr. Indeed, Vpr actively triggers the expression of UL16-binding protein molecules on p24pos CD4pos T cells, thus suggesting an immunomodulatory role for this protein. Further investigations are required to possibly approach novel strategies able to improve the NK cell-mediated clearance of HIV-1-positive CD4pos T cells through the regulation of NKG2D ligands on infected targets. Regular Pathways Abnormal Pathways The recent scientific advances about the role of NK cells in the pathophysiology of HIV-1 infection represent important steps forward that helped us to disclose new mechanisms underlying the lack of control of viral spread. In this regard, the redistribution of NK cell subsets during the course of HIV-1 disease delineates an important aspect of pathogenesis that highly affects adaptive and innate immune responses (Fig. 4). These aberrancies of NK cell phenotype in response to high levels of HIV-1 viremia are also associated with functional impairments of NK cells and with clinical outcomes of the disease and may be useful in the future to improve the clinical management of patients and/or to disclose novel concepts in the field of prophylactic and therapeutic vaccinations. AUTHORSHIP All authors contributed equally to the writing of this review. CONCLUDING REMARKS ACKNOWLEDGMENTS Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is still an important challenge of biomedical research. The recent failure of vaccine trials to induce protective B and T cellular responses against HIV-1 highlights the importance of investigating the ways in which HIV-1 escapes the immune response. This work was supported by the Intramural Research Program of Istituto Clinico Humanitas (grant no ), by the European Union (Marie Curie International Reintegration grant no ), and Italian Ministry of Health (Ricerca Finalizzata, grant no. ICH ). E.B. and K.L.H. performed this study as a partial fulfilment of their Ph.D. programs at Volume 88, December 2010 Journal of Leukocyte Biology 1127