Some individuals remain seronegative despite repeated exposure to HIV.1–3 These HIV-exposed seronegative (HESN) individuals include sexual partners of HIV-1–infected individuals, children born from HIV-infected mothers, female sex workers in areas where HIV infection is epidemic, intravenous drug users, hemophiliacs who received HIV-1–contaminated blood preparations, and health care workers subjected to accidental exposure to the virus.2–4 Susceptibility to HIV infection is a multifactorial phenomenon involving both genetic factors and innate and adaptive immunity.5,6
Multiple cohorts of HESN individuals have exhibited HIV-specific T-cell responses and the production of soluble factors, including cytokines, B-chemokines, and interferon γ (IFN-γ). In addition, these subjects show low levels of CD4+ T-cell activation and cytotoxic and polyfunctional CD8+ T cells, as well as anti-HIV IgG or IgA antibodies capable of recognizing the envelope protein of HIV.7–16 However, it remains unknown whether these responses detected in some HESN individuals could be suggestive for persistence HIV-1 exposure rather than to provide protection.
Given the genotypic and functional association between increased natural killer (NK)-cell activity and resistance to HIV-1 infection in multiple cohorts of HESN subjects,17–20 the innate immune response may play a significant role in maintaining natural resistance to infection in high-risk subjects. NK cells play an important role in innate immunity by providing protection against viruses and tumor cells. The effector function of NK cells is the result of signals that are delivered by inhibitory and activating receptors.21 The majority of human NK cells are present in the blood, and approximately 90% of all NK cells present the CD56dim phenotype. In addition, there is a minor population (approximately 10%) that is CD56bright, and this type is the major NK-cell subtype found in tissues and secondary lymphoid organs. The CD56bright and CD56dim NK-cell subsets have been considered phenotypically, functionally, and developmentally different,22 with the immunoregulatory CD56bright NK cells producing abundant cytokines and the cytotoxic CD56dim NK cells functioning as efficient effectors of natural and antibody-dependent targeted cell lysis.22
Progressive HIV disease is associated with impaired NK responses and the selective depletion of CD56dim NK cells that occurs during chronic HIV-1 infection.23 The loss of CD56dim NK cells results in the enrichment of functionally anergic CD56null NK cells.24,25 These findings were also associated with the viral load (VL), suggesting that the virus itself may affect NK cells.
Heightened NK-cell activation, including CD69 expression and degranulation marker CD107a, has been associated with resistance to infection in several independent cohorts of HESN subjects.26,27 Moreover, an increased frequency of IFN-γ–producing NK cells after stimulation with phorbol 12-myristate acetate (PMA)/ionomycin28 and without stimulation has been documented in HESN subjects.26 Altogether, the findings suggest active NK-cell function in HESN subjects, which may be associated with their relative resistance to HIV infection.
Accumulating evidence indicates that at least some NK cells respond to specific antigens during the adaptive immune response.29,30 Human cytomegalovirus (HCMV) has been reported to induce the expansion of CD94+NKG2C+ NK cells in healthy adults and children as well as in HIV-infected and leukemia patients,31,32 and a subset of NKG2C+ NK cells was shown to be increased in CMV-seropositive healthy individuals and aviremic HIV-1–infected patients.29,32 Nevertheless, these long-lived NK cells have not yet been evaluated in HESN individuals. Given the interest in developing strategies to augment NK-cell function during chronic viral infections, such as HIV-1 and hepatitis C infections, NK cells have the potential to be exploited for long-term protection through vaccination.
Specific aspects of NK cells in HESN individuals must be evaluated to understand the differences between NK-cell subsets in terms of their maturation profiles and expression of memory-like markers. Information on such properties would be valuable to validate strategies for enhancing NK-cell effector functions in HIV infection.
MATERIALS AND METHODS
HIV-1 serodiscordant couples from an outpatient clinic at the Emílio Ribas Infectious Diseases Institute in São Paulo, the Ambulatory Service of the Department of Secondary Immunodeficiency Clinic of the Clinical Hospital, University of São Paulo Medical School (HC/FMUSP), and the Centro de Referência e Tratamento em DST-AIDS in São Paulo, Brazil, were enrolled. The HESN group (n = 19) comprised 7 men and 12 women, with a mean age of 44.3 (range, 25–66) years; the HIV-infected patients (n = 16) comprised 13 men and 3 women, with a mean age of 44.6 (range, 36–59) years; and healthy donors (n = 18) comprised 4 men and 14 women (age range, 22–58 years). Homosexual (n = 4) and heterosexual couples (n = 15) reported being with a single partner for more than 1 year. Couples reported participating in vaginal and oral sex, including unprotected sexual events, at a frequency of 3–4 times per month. The required entry criteria included at least 1 high-risk sexual exposure per month in the last 12 months. The HESN cohort was found to be seronegative at the time point that was studied. The majority (13/16) of HIV-1–infected individuals were receiving antiretroviral therapy (ART) treatment; of these subjects, 81.2% had an undetectable VL and 3 had detectable VLs (51,179, 179, and 50,930 copies RNA/mL). The CD4 counts and VL in the HIV-infected group are shown in Table 1. All subjects provided written informed consent under the approval of the São Paulo University Institutional Use Committee (CAPPesq n° 0683/09). Standard eligibility criteria were used for enrollment into the study. Exclusion criteria consisted of the use of immunosuppressants or immune-modifying drugs as well as pregnancy. The inclusion criteria included being over 18 years of age, reported participation in unprotected sex, and having a single partner for over 1 year.
To analyze the NK cells in the peripheral blood, venous blood was collected in EDTA-enriched tubes and staining was performed using the following antibodies: CD3-Qdot 605 (UCHT1; Invitrogen, Carlsbad, CA), CD19-Horizon V500 (HIB19), CD16-Alexa 647 (3G8), CD56-Alexa 700 (B159), CD127-PE-Cy7 (HIL-7R), CD69-FITC (FN50), NKG2D-APC (1D11), NKG2A-PE (131411; R&D Systems, Minneapolis, MN), NKG2C-FITC (134591; R&D Systems), CD94-APC (HP3D9), and CD57-FITC (NK-1). Antibodies were purchased from BD Pharmingen (San José, CA, USA). Approximately 70 μL of whole blood was stained for 20 minutes and then incubated for 15 minutes with FACS lysing solution (BD FACS Lysing; BD Biosciences, San Jose, CA) to lyse the erythrocytes. After 2 washes in isotonic solution (Hemoton SPEC; Brazil Hemoton SPEC; São Paulo, Brazil), 200,000 events were acquired using a flow cytometer (LSRFortessa; BD Biosciences) with the FACSDiva software (BD Biosciences). The data were analyzed using the FlowJo software version 9.4.11 (Tree Star, Inc., Ashland, OR). The forward scatter area versus height parameter was used to exclude cell doublets, and then T cells and B cells were excluded. Small lymphocytes were identified according to their forward and side scatter properties. NK cells were identified by gating cells that were positive for CD56 and excluding CD3+ and CD19+ cells. CD56bright and CD56dim NK cells were identified and analyzed for each receptor (NKG2A, NKG2C, NKG2D, CD127, CD69, and CD57). Boolean gate arrays were then created using the FlowJo software. This analysis determined the expression frequency of each receptor based on all possible combinations of the 4 receptors. To analyze the polychromatic flow cytometry data and to generate graphical representations of the NK cells, the SPICE Program was used (version 2.9; Vaccine Research Center, NIAID, Bethesda, MD).
CD107a Degranulation and Intracellular IFN-γ Detection
The frequency of degranulating NK cells (CD107a expression) and IFN-γ secretion was determined by flow cytometry. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient (GE Healthcare, Uppsala, Sweden) centrifugation. PBMCs (1 × 106 cells/mL) were incubated with 30-ng/mL PMA (Sigma-Aldrich, St. Louis, MO) and 0.3-μg/mL ionomycin (Sigma-Aldrich). CD107a PE-Cy5 was added to detect degranulating NK cells. The samples were plated and incubated at 37°C with 5% CO2 for 6 hours. After 2 hours of incubation, Brefeldin (10 μg/mL; Sigma) was added to the cultures and incubated for another 4 hours. After incubation, the cells were washed and incubated with human IgG for 15 minutes, followed by fixation with Cytofix/Cytoperm solution (BD Biosciences) for 20 minutes and permeabilization with Perm/Wash solution for 20 minutes at 4°C. The cells were then stained with CD3-Qdot 605 (Invitrogen), CD19-Horizon V500, CD16-APC-Cy7, CD56-Alexa 700, or IFN-γPE (BD Biosciences). Next, the samples were washed with Perm/Wash buffer and diluted in isotonic solution. A total of 250,000 events was collected and analyzed by flow cytometry (LSRFortessa; BD Biosciences) using the FACSDiva software (BD Biosciences). The expression of surface CD107a and intracellular IFN-γ in CD3−CD56dim and CD56bright NK cells was analyzed using the FlowJo software.
Serological evidence of anti-IgG antibodies specific for HCMV was collected using an enzyme-linked immunosorbent assay (Virion/Serion, Würzburg, Germany) according to the manufacturer's suggestions. The cutoff point was established as 25 U/mL.
Kruskal–Wallis tests with Dunn posttest were used to compare the variables between HIV-infected patients, HESN subjects, and healthy controls (HCs). P ≤ 0.05 was considered statistically significant.
Phenotype of CD56bright and CD56dim NK Cells in HESN Subjects and HIV-1–Infected Individuals
The activating and maturating markers as well as the memory markers on NK cells were analyzed for the groups of HESN subjects, HIV-1–infected partners, and uninfected nonexposed individuals (Table 1).
We evaluated the expression of activating markers, including CD69, NKG2C, and NKG2D, or of an inhibitory/immature marker, that is, NKG2A, in the CD56bright and CD56dim NK-cell subsets. In addition, we evaluated the expression of CD127, which is the interleukin 7 (IL-7) receptor-α chain, in thymic emigrant cells, or immature NK cells, as well as CD57 expression in the highly mature and possibly terminally differentiated NK cells. The gating strategy is shown in Figure S1 (see Supplemental Digital Content, http://links.lww.com/QAI/A568).
Figure 1 shows an enrichment of CD56bright and CD56dim NK cells expressing NKG2A or NKG2C in the HESN and HIV groups compared with the HC. Of note, only HESN individuals showed an increased enrichment of CD56brightNKG2D+ and CD56dimNKG2D+ NK cells compared with the HIV and HC groups. Another marker that was differentially expressed in HESN was CD127, which was found at an increased frequency in CD56bright NK cells compared with the HIV and HC groups (Fig. 1A). No CD127+ expression was verified in CD56dim NK cells.
In particular, highly mature CD57+ NK cells were enriched in CD56dim NK cells of the HESN and HIV groups compared with the HC group (Fig. 1B). Absent CD57 expression in CD56bright NK cells was observed. Moreover, an increased frequency of CD56brightCD69+ expression was observed in the HIV group, whereas the percentage of CD56dimCD69+ NK cells was increased in the HESN group.
Taken together, the results revealed marked differences in NK surface marker expression in HESN individuals, including high expression of NKG2D+ in both NK subsets and enrichment of CD127+CD56bright NK cells. HESN and HIV groups have shown some similarities such as increased frequency of NKG2A and NKG2C in both NK subsets and enrichment of CD57+CD56dim NK cells.
Expansion of NKG2C+ Memory NK Cells and HCMV Seropositivity
Accumulating evidence indicates that at least some NK-cell subsets respond to specific antigens during an adaptive immune response, including the expansion of NKG2C+CD57+ NK cells during CMV infection.30,32 Therefore, we examined the relationship between CD56dimNKG2C+ or CD57+ NK cells and HCMV seropositivity.
The HESN, HIV-1, and HC groups showed a high correlation with CMV serology and the percentages of CD56dimNKG2C+ cells (Fig. 2A) or CD56dimCD57+ cells (Fig. 2B).
As shown in Figure 2C, HCMV serology revealed increased titers in the HIV group compared with the HC group. In particular, the HC group demonstrated a seronegative frequency of 35% (7/20), in contrast to the HESN and HIV-1 groups, which were CMV seropositive. Our data revealed a significant expansion of CD56dimNKG2C+ and CD56dimCD57+ cells with CMV seropositivity in HESN and HIV individuals.
NK Receptor Expression in NK-Cell Subsets
Next, we evaluated the frequency of NK cells expressing inhibitory (NKG2A) or activating (NKG2C, NKG2D) receptors as well as CD94 for both NK subsets. CD94 can form a complex with NKG2A or NKG2C, and the ligand for these receptors is the human leukocyte antigen E (HLA-E) molecule. Considering the numerous combinations that can be generated through Boolean analysis, the data for the expression of the 4 receptors, as well as the combinations of only 3 or 2 receptors, are shown in Figure 3. Gating strategy is shown in Figure 3A.
We observed a unique profile between the NK subsets. In particular, CD56bright NK cells simultaneously expressed all 4 receptors (CD94+NKG2A+NKG2C+NKG2D+), and there was an increased frequency of this combination in the HESN group (Fig. 3B). The frequency of NK CD56bright cells expressing 3 receptors, including CD94+NKG2A+NKG2C+ or CD94+NKG2A+NKG2D+ cells, was higher among HIV-infected individuals than HCs.
In contrast, NK CD56dim cells expressing all 4 NK receptors were increased in the HIV-1 group when compared with the HC group (Fig. 3C). Other combinations of 2 or 3 receptors were also found at an increased frequency in HIV-infected individuals.
These results showed that HESN individuals presented a unique CD56bright NK-cell subset expressing the activating and inhibitory receptors CD94, NKG2A, NKG2C, and NKG2D, whereas the CD56dim NK-cell subset was more common in HIV-infected patients.
Effector Molecules in NK Subsets
Next, we evaluated the frequency of degranulation (CD107a expression) and IFN-γ secretion among CD56bright and CD56dim NK cells after 6 hours of PMA plus ionomycin stimulation. Gating strategy is shown in Figure 4A.
CD56bright NK cells exhibited an increased percentage of single-positive CD107a+, IFN-γ+CD107−, and double-positive IFN-γ+CD107+ cells (Fig. 4B) in the HESN group when compared with the HIV and HC groups. CD56brightCD107a+ cells were predominantly observed in the HESN group (76%) and were detected at lower frequencies in HIV-infected (53.5%) and HC (43%) subjects (Fig. 4B). Moreover, an enrichment of IFN-γ−CD107a+CD56dim cells was found in the HESN group, reaching a frequency of 46% in comparison with the 26% and 21% frequencies observed in the HIV and HC groups, respectively (Fig. 4C). In addition, the single expression of IFN-γ− in cells was increased in HESN individuals for both NK-cell subsets. These findings show that HESN subjects contain an unusual enrichment of CD107a+IFN-γ+CD56bright NK cells.
Our findings uncovered a distinct NK-cell profile in HESN individuals; this cell subset was enriched for activating NKG2D+ expression, immature CD127+ expression as well as CD107a+ and/or IFN-γ–secreting cells, and mature NK “memory” CD57+CD56dim cells. Moreover, HESN individuals showed CD56bright cells that expressed CD94+NKG2A+NKG2C+NKG2D+, whereas this combined receptor expression was found in CD56dim NK cells in HIV individuals. These findings showed that HESN individuals possess a peculiar activated NK-cell profile most likely due to frequent exposure to HIV.
The unique changes in NK-cell surface marker expression included differential CD127 (IL-7 receptor-α chain) and NKG2D expression in the HESN group when compared with the HC group. The evidence suggesting that CD127+ NK cells may represent emigrant cells from the thymus indicates that NK cells may undergo dynamic replacement. In mice, a subset of NK cells in the lymph nodes expresses CD127, and these cells are thought to be derived from the thymus.33 Moreover, the accumulation of immature CD127+ NK cells was reported in mice bearing progressing tumors or suffering from chronic viral infection.34 However, further investigation is required to assess whether peripheral blood CD127+CD56bright NK cells could represent cells emigrating from the thymus and homing to other sites in humans.
A unique increase in NKG2D expression was observed for both NK-cell subsets in HESN individuals, and this finding may be related to HIV resistance. Elite controller and long-term nonprogressor patients have been shown to have conserved peripheral NK cells with increased NKG2D expression, reduced NKp46 surface expression, HLA-DR upregulation, and a mature NKG2A−KIR+CD57+CD56dim effector NK-cell phenotype.35 Some cytokines such as IL-2, IL-15, and IL-7 may upregulate NKG2D surface expression, whereas tumor growth factor β1 may downmodulate NKG2D.36 Understanding the mechanisms controlling the expression of NKG2D, which is an activating receptor that is important for the antiviral and antitumor activities of NK cells, has implications for immunotherapy.
Interestingly, HESN subjects showed increased IFN-γ production and/or CD107a degranulation in both CD56bright and CD56dim NK cells when compared with the HIV and HC groups. The NK cells expressing CD107a+IFN-γ+ after PMA and ionomycin stimulation were predominantly detected (76%) not only within the CD56dim subset but also among CD56bright (46%) cells. Moreover, IFN-γ+ or CD107a+ cells were mainly detected in HESN individuals. In general, the CD56bright subset is known to be more secretory than cytotoxic; however, the percentage of CD107a-expressing cells was markedly increased when combined or not combined with the percentage of IFN-γ–producing cells. These observations in HESN individuals suggest that cells within each NK-cell subset may be differentially activated in the peripheral blood. Moreover, an increased frequency of IFN-γ–producing NK cells from HESN subjects compared with controls has been reported after PMA and ionomycin treatment, although the specific NK subset was not discriminated.28 Thus, determining the factors that trigger the activation of CD56bright NK cells and promote their cytotoxic role in peripheral tissues is a relevant issue that should be further explored.
Our HESN cohort was entirely composed of heterosexual people who were in stable relationships with HIV-infected partners with an undetectable VL, the majority of whom had received ART treatment. The chronically HIV-infected group exhibited similar frequencies of CD107a+IFN-γ+CD56dim and CD56bright NK cells as HC individuals as well as some similarities with HESN subjects such as NKG2A and NKG2C expression in both NK subsets. These data revealed that NK cells were not fully impaired in HIV-infected subjects, possibly because of their undetectable VLs. The impairment in NK-cell frequency in the absence of ART,25,37 with the selective depletion of CD56dim NK cells and their diminished ability to perform cytolysis and secrete cytokines, is well established.23,37,38
One important question that emerged was whether alterations in surface marker expression on NK-cell subsets in HESN individuals were due to frequent exposure to HIV or other viral infections. Interestingly, HESN subjects demonstrated 100% seropositivity for HCMV, similar to their HIV-infected partners, whereas the healthy group demonstrated 35% seronegativity. Humans can be infected with CMV (50%–100%, depending on geographical location) and remain infected for life.39 Although it has been shown in a mouse model that the generation of long-lived memory NK cells is more protective during a secondary encounter with this pathogen,40 it is not clear whether humans possess virus-specific memory NK cells. Moreover, during acute CMV infection, preferential expansion of a unique subset of NK cells that coexpress the activating CD94–NKG2C receptor undergoes transition into NKG2Chi cells and finally acquire CD57 expression, which suggests that CD57 may serve as a marker of memory NK cells.41
Interestingly, an intense expression of CD57 and NKG2C on CD56dim NK cells was observed in HESN and HIV subjects, with strong correlations between NKG2C and CD57 expression and IgG titers for HCMV. To the best of our knowledge, the expansion of memory-like NK cells in HESN subjects has not been described previously and is most likely the result of CMV transmission from an HIV-infected partner.
The frequency of NK cells expressing the receptors CD94, NKG2A, NKG2C, and NKG2D, in combined form, which was verified by Boolean evaluation, allowed us to identify the unique phenotypic profiles of NK cells between the HESN and HIV groups. NK CD56bright cells simultaneously expressing CD94+NKG2A+NKG2C+NKG2D+ were more abundant in HESN subjects, whereas CD56dim NK cells that expressed these 4 receptors were more prominent in HIV-infected individuals compared with HCs. The role of NK cells simultaneously expressing several receptors is unknown, but it most likely contributes to the activation or inhibition of NK-cell subsets. Alterations in CD56dim NK cells expressing these receptors were found mainly in the HIV group, and, in fact, this NK subset was more susceptible to chronic HIV-1 infection. Inhibitory NKG2A and activating NKG2C both specifically recognize the nonclassical major histocompatibility complex I molecule HLA‐E,42 and it has been described that HIV-1 mediates the upregulation of HLA-E expression, resulting in the impaired function of NK cells as a viral evasion strategy.43
Unique differences, such as in the activating profile due to an increased NKG2D+ expression and a high percentage of CD107a+ and IFN-γ–producing cells within the CD56bright and CD56dim NK-cell subsets, were detected in HESN individuals. Other characteristics of HESN subjects included an increased CD127 expression in NK CD56bright cells, revealing a high frequency of immature and thymic emigrant cells, and in parallel, the presence of memory-like CD56dimCD57+NKG2C+ cells, an activating phenotype and CD56bright NK cells that simultaneously express activating and inhibitory NK receptors. However, additional studies are needed to address whether the expression of activating/inhibitory receptors on regulatory CD56bright NK cells and cytotoxic CD56bright and CD56dim NK cells plays a role in controlling early HIV infection.
The authors are grateful to all individuals who participated in this study.
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