Natural killer (NK) cells are a subset of lymphoid cells that function as important mediators of the innate immune defense against viruses and tumor cells.1 They comprise approximately 15% of peripheral blood lymphocytes and are also found in liver, peritoneum, and placental tissue.1 They were originally identified functionally by their ability to lyse certain tumor cells without prior stimulation; however, they do not express clonally distributed antigen-specific receptors.1 The ability of NK cells to lyse virus or tumor targets does not depend on prior sensitization, nor is their activity restricted by the major histocompatibility complex (MHC).2,3 Furthermore, it has been established that human leukocyte antigen (HLA) class I molecules inhibit cytolytic activity of NK cells by their interaction with a series of cytotoxicity inhibitory receptors (iNKRs).1-6 The balance between the stimulation of iNKRs and activating receptors is fundamental to the regulation of NK-cell cytotoxic activity.1-6 On activation, NK cells release cytokines and chemokines that induce inflammatory responses; modulate hematopoiesis; control cell growth and function of monocytes, granulocytes, and dendritic cells; and influence the type of adaptive immune responses that follow.1-5
We have previously shown that HIV viremia, as a consequence of active viral replication, negatively influences the ability of NK cells to produce CC-chemokines, and thereby contains HIV replication ex vivo.2,3 Furthermore, NK-cell-mediated redirected cytotoxicity assays have demonstrated the inability of NK cells from patients with HIV viremia to lyse target cells in vitro as a consequence of increased expression of iNKRs and decreased expression of NK cytotoxicity receptors (NCRs).7-9 Recently, we have demonstrated that exposure of NK cells to HIV envelope proteins results in profound cellular abnormalities at the level of gene expression and generic cell functions, which are likely to be a consequence of a direct HIV gp120-mediated effect on NK cells.10 The underlying mechanisms whereby HIV viremia influences NK-cell receptor expression, cytokine secretion, and cytotoxic functions are not fully delineated at present. To provide a comprehensive picture of the complex cascades of gene expressions induced by HIV viremia on NK cells, we performed DNA microarray analyses using freshly isolated NK cells from HIV-infected viremic, HIV-infected aviremic, and HIV-seronegative donors. In addition, we performed functional assays in vitro to validate the findings from DNA microarray analyses, confirming the increased propensity of NK cells from HIV-infected viremic individuals to undergo CD95-mediated apoptosis (FMA) and cell turnover.
MATERIALS AND METHODS
Viremic and aviremic HIV-infected and HIV-seronegative individuals were recruited, and leukaphereses were performed to obtain peripheral blood mononuclear cells (PBMCs). All subjects signed an informed consent form approved by the National Institute of Allergy and Infectious Diseases (NIAID) Institutional Review Board (IRB) before the procedure. The demographics of patients who participated in the study are shown in Table 1.
Isolation of Natural Killer Cells
NK cells were isolated from PBMCs using column-based cell separation techniques (Stem Cell Technologies, Vancouver, British Columbia, Canada) as previously described.11 Cells obtained had a purity of >90%.
DNA Microarray Analyses
Total RNA was extracted from highly purified NK cells using Trizol (Invitrogen, Carlsbad, CA). DNA microarray analysis was performed using Affymetrix (Santa Clara, CA) Human Genome U133A oligonucleotide arrays according to the protocol specified by the manufacturer.12 A significant analysis of microarray (SAM) algorithm was used to determine the genes that were significantly differentially regulated after extensive prefiltering processes.12 Gene expression values (log2) were determined using Guanosine-Cytosine-Robust Multi-array (GC-RMA), followed by Loess normalization. Differentially regulated genes were selected using ANOVA.
To measure degrees of Fas-mediated apoptosis, purified NK cells were cultured with 1 μg/mL of sFasL (Kamiya Biomedical, Seattle, WA) and incubated in complete media containing 20 IU of interleukin (IL)-2 at 37°C overnight. For CD16- and NKG2D- receptor-mediated apoptosis, NK cells were treated with 2.5 μg of fMICA, or 2 μg/mL of D1D2, respectively, and incubated in complete media containing 20 IU of IL-2 at 37°C for 2 hours.13
NK cells were stained with Annexin-V fluorescein isothiocyanate (FITC; R&D Systems Inc., Minneapolis, MN), CD56-Allophycocyanin (APC), and CD16-phycoerythrin (PE; BD Biosciences, San Jose, CA) and then analyzed by flow cytometry according to manufacturer's instructions.
Flow Cytometric Analysis of Natural Killer Cells
Freshly isolated NK cells were stained for intracellular Ki67 and CD95 surface expression using the following antibodies: CD56 APC and CD16 PE antibodies (BD Biosciences) were used to identify NK cells, and Ki67 PE or CD95 PE (BD Biosciences) was used to determine the expression of these receptors on NK cells intracellularly or on the cell surface.14,15 Approximate absolute numbers of NK-cell fractions were estimated by multiplying the percentage of cells by the total number of cells used for the assay.
Enzyme-Linked Immunoassay for Detection of Levels of Serum sFasL
Levels of sFasL in the serum of HIV-infected individuals were determined by the Quantikine enzyme-linked immunosorbent assay (ELISA) kit and reagents (R&D Systems) according to the manufacturer's instructions.
ANOVA with the Tukey multiple comparison test was used to compare means from 3 independent groups. Means from 2 independent groups were compared by the Student t test. The t test for paired data was used to determine the significance of the change in sFasL before and after 1 year's treatment with antiretroviral therapy (ART). Means with standard errors are reported. The Bonferroni method was used to adjust P values for multiple testing.
DNA Microarray Analyses of Freshly Isolated Natural Killer Cells From HIV-Infected Viremic, HIV-Infected Aviremic, and HIV-Seronegative Individuals
To delineate the mechanisms involved in impairment of NK-cell functions in HIV-infected viremic individuals, we performed DNA microarray analyses using RNA isolated from freshly isolated NK cells from 5 HIV-seronegative, 5 HIV-infected viremic, and 5 HIV-infected aviremic individuals. Using Affymetrix Human Genome U133 A oligonucleotide arrays consisting of probes encompassing more than 33,000 genes and a SAM algorithm,12 we identified differentially expressed genes (Fig. 1). The corresponding genes from the 3 groups of donors were grouped using K-means and hierarchic clustering, respectively. The hierarchic analyses classified the genes into 2 distinct categories or clusters (see Fig. 1). Of these, cluster I consisted of genes that were upregulated in NK cells of HIV-infected aviremic individuals but were downregulated in HIV-infected viremic and HIV-seronegative individuals. Cluster II was composed of genes that were upregulated in NK cells obtained from HIV-infected viremic individuals but were downregulated in NK cells obtained from HIV-infected aviremic and HIV-seronegative patients.
Hierarchic clustering analyses indicated that there was a striking similarity in the transcriptional profile of genes that were differentially expressed by NK cells from HIV-infected viremic, HIV-infected aviremic, and HIV-seronegative individuals (see Fig. 1). Statistical analyses demonstrated that relative to the frequency of all annotated genes on the DNA microarray, genes in cluster II were significantly associated with promoters of apoptosis, response to biotic stimulus, and cell communication (P < 0.01). These results strongly suggest that HIV viremia resulted in expression of genes involved in immune activation, along with those promoting programmed cell death.
Natural Killer Cells From HIV-Infected Viremic Individuals Undergo Fas-Mediated Apoptosis on Exposure to sFasL
To validate the data obtained from DNA microarray, we performed in vitro apoptosis assays on freshly isolated NK cells from HIV-infected viremic, HIV-infected aviremic, and HIV-seronegative individuals in the presence or absence of sFasL. On exposure to sFasL, a higher proportion of NK cells from HIV-infected viremic individuals underwent apoptosis, as detected by Annexin-V staining (Fig. 2). NK cells from HIV-seronegative and HIV-infected aviremic individuals did not undergo apoptosis. Spontaneous apoptosis was <5% in all experiments. The percentages and absolute numbers of NK cells undergoing FMA were higher for HIV-infected viremic individuals (22.1 ± 1.6% and 442,015 ± 31,182, respectively) when compared with HIV-infected aviremic individuals (3.1 ± 0.4% and 61,231 ± 7957, respectively; P < 0.001) and HIV-seronegative individuals (3.4 ± 0.4% and 68,846 ± 8662, respectively; P < 0.001; see Fig. 2).
Natural Killer Cells From HIV-infected Viremic Individuals Express Higher Levels of CD95 on Their Surface and Have Increased Levels of Circulating sFasL in Serum
To address the role of CD95 expression on the surface of NK cells with regard to their susceptibility to undergo apoptosis on exposure to sFasL, we performed flow cytometry to detect the CD95 receptor on the surface of NK cells from the study subjects. HIV-infected viremic individuals expressed a higher level of CD95 receptor on their surface (79.5 ± 2.5%) when compared with HIV-infected aviremic individuals (15.1 ± 1.2%; P < 0.001) and HIV-seronegative individuals (8.7 ± 1.1%; P < 0.001; Fig. 3A). To address whether a specific subset of NK cells exhibited higher levels of Fas, we performed CD95 staining on CD56bright CD16low and CD56dim CD16bright NK cells (see Fig. 3B). Among HIV-infected viremic patients, CD56dim CD16bright NK-cell subsets showed higher levels of CD95 (74.6 ± 4.5% and 1079,485 ± 46,789, respectively) when compared with CD56bright CD16low NK cells (34.2 ± 3.4% and 48,954 ± 998, respectively; P < 0.001).
We also investigated whether HIV-infected viremic patients have increased serum levels of sFasL that could suggest a possible mechanism for increased in vivo apoptosis of NK cells. Serum sFasL was measured from patients before initiation of ART and 1 year after therapy. All except 2 of the 16 patients we examined had a decrease in the pretreatment level of their serum sFasL at the end of 1 year of therapy (see Fig. 3C). The mean level of sFasL before initiation of ART (70.3 ± 7.9 pg) was significantly higher than that at the end of 1 year of therapy (43.1 ± 4.9 pg; P = 0.001).
Natural Killer Cells From HIV-Infected Viremic Individuals Do Not Have Increased Propensity to Undergo Apoptosis Mediated by CD16 or NKG2D
To address whether NK cells from HIV-infected viremic individuals have a selective propensity for apoptosis mediated by CD95, we tested the ability of NK cells to undergo apoptosis mediated by 2 other cell surface receptors, CD16 and NKG2D. Recently, a highly polymerized chimeric IgG1-IgA fusion protein that binds extensively to CD16 and activates NK cells was described.13 At higher concentrations, this protein (D1D2) is capable of triggering apoptosis of NK cells (Fig. 4A). The percentages and absolute numbers of NK cells from HIV-infected viremic, HIV-infected aviremic, and HIV-seronegative individuals undergoing apoptosis on exposure to the D1D2 polymer were similar (25.1 ± 1.5% and 502,333 ± 29,003, respectively, vs. 25.7 ± 1.4% and 513,600 ± 28,812, respectively, vs. 25.6 ± 0.9% and 512,333 ± 18,268, respectively; P > 0.5). Also, when another polymerized chimeric IgG1-IgA fusion protein that binds to NKG2D (fMICA) was used to induce apoptosis of NK cells from HIV-infected viremic, HIV-infected aviremic, and HIV-seronegative individuals, the rates of apoptosis were similar (19.3 ± 0.9% and 386,900 ± 17,252, respectively, vs. 21.4 ± 1.8% and 428,480 ± 36,229, respectively, vs. 21.2 ± 1.3% and 424,333 ± 26,033; P > 0.5; see Fig. 4B).
Natural Killer Cells From HIV-Infected Viremic Individuals Have Increased Intracellular Ki67 Suggesting Increased Cell Turnover In Vivo
Given that NK cells from HIV-infected viremic individuals have increased levels of CD95 expression on their surface and increased circulating sFasL in serum resulting in enhanced FMA, we investigated whether there is any evidence of increased NK-cell turnover in vivo. Because intracellular Ki67 is a good correlate of in vivo cell turnover,14 we performed intracellular Ki67 staining of NK cells from the study subjects ex vivo. A higher percentage of NK cells obtained from HIV-infected viremic individuals expressed intracellular Ki67 staining (14.0 ± 0.9% and 280,800 ± 18,121, respectively) when compared with those from HIV-infected aviremic individuals (3.5 ± 0.5% and 70,540 ± 9882, respectively; P < 0.001) and HIV-seronegative individuals (3.1 ± 0.3% and 62,360 ± 9647, respectively; P < 0.001; Fig. 5A). To address whether a specific subset of NK cells exhibited higher levels of cell turnover, we performed Ki67 staining on CD56bright CD16low and CD56dim CD16bright NK cells (see Fig. 5B). Among HIV-infected viremic patients, a higher percentage of CD56dim CD16bright NK-cell subsets showed intracellular Ki67 (9.6 ± 0.5% and 245,071 ± 15,343, respectively) when compared with CD56bright CD16low NK cells (4.2 ± 0.4% and 10,614 ± 815, respectively; P < 0.001).
In the present study, we have demonstrated that active HIV replication in vivo resulting in viremia alters the profiles of gene expression in NK cells, leading to the upregulation of genes involved in apoptosis. These findings were validated by a number of functional assays that confirmed increased FMA of NK cells. In this regard, CD95 was found to be upregulated on the surface of NK cells and sFasL was increased in the serum of HIV-infected viremic individuals. Moreover, NK cells from HIV-infected viremic individuals, particularly the CD56dim CD16bright subset, expressed increased levels of intracellular Ki67, suggesting a higher rate of cell turnover in vivo.
HIV infection is associated with several phenotypic and functional defects of NK cells, including increased expression of iNKRs,7,16 decreased expression of NCRs,7,9 reduced ability to secrete CC-chemokines and block HIV replication,11,17,18 and decreased capability of receptor-meditated lysis of target cells.7-9 Moreover, several studies have suggested that there is a progressive loss of NK cells in HIV-infected individuals,19-25 although the exact mechanism(s) of this phenomenon is unclear. Certain studies have reported an increased susceptibility of NK cells obtained from HIV-infected individuals to undergo apoptosis.26,27 Although a study has suggested that a subset of NK cells can be productively infected with HIV,28 there was no associated loss of NK cells attributable to infection by HIV. To understand the defects seen in NK cells in HIV infection better, we performed a series of experiments using freshly isolated NK cells from HIV-infected viremic, HIV-infected aviremic, and HIV-seronegative individuals.
DNA microarray analysis has been used previously in studies of the pathogenesis of HIV infection and associated immune dysfunction.10,29-32 In the present study, DNA microarrays were performed using RNA isolated from freshly isolated NK cells from HIV-infected viremic, HIV-infected aviremic, and HIV-seronegative individuals. Our results demonstrated that HIV viremia resulted in expression of genes promoting programmed cell death. These results suggest an important effect of HIV viremia on NK-cell function, most probably resulting in activation and activation-induced cell death. These activation-induced defects could lead to progressive NK-cell loss and increased NK-cell turnover in vivo.
To validate DNA microarray findings that NK cells from HIV-infected viremic individuals have an increased propensity to undergo activation-induced apoptosis, we performed in vitro apoptosis assays. When NK cells from HIV-infected viremic individuals were exposed to sFasL, they underwent apoptosis at a higher rate than NK cells from HIV-infected aviremic or HIV-seronegative individuals. In addition, a higher proportion of NK cells from HIV-infected viremic individuals expressed CD95 on their surface when compared with NK cells from HIV-infected aviremic and HIV-seronegative individuals. There was no increase in spontaneous apoptosis of NK cells from HIV-infected viremic individuals when compared with those from HIV-infected aviremic and HIV-seronegative individuals, however. Considering the fact that the difference in FMA was only seen in the presence of sFasL, we investigated whether HIV-infected viremic patients have increased sFasL in their serum, thereby providing a potential mechanism for increased triggering of FMA of NK cells in vivo. When serum levels of sFasL were measured longitudinally from HIV-infected viremic individuals immediately before initiation of ART and after 1 year of ART, serum sFasL levels were significantly elevated during the viremic state. Moreover, 14 of the 16 patients had a decrease in the serum levels of sFasL at the end of 1 year of ART. Thus, increased levels of CD95 expression on NK cells, along with increased serum levels of sFasL, provide a plausible explanation for the increased propensity of NK cells from HIV-infected viremic individuals to undergo FMA in vitro. Several studies have shown that immune activation induced by HIV viremia results in increased expression of CD95 on various lymphocyte subsets and facilitates apoptosis of lymphocytes.31,33-35 Lymphocytes from HIV-infected individuals express higher levels of CD95 on their surface during the HIV viremic state.33 Our findings further extend these observations and show that the HIV-infected viremic state results in global immune activation leading to increased CD95 expression on NK cells, rendering them susceptible to FMA.
We further investigated whether NK cells from HIV-infected viremic individuals were susceptible to activation-induced cell death by mechanisms other than that mediated by the CD95-FasL. We have previously demonstrated that a highly polymerized chimeric IgG1-IgA fusion protein that binds extensively to CD16 activates NK cells and, at higher concentrations, is capable of triggering apoptosis of NK cells.14 When the ability of D1D2 protein to induce apoptosis mediated by maximal stimulation of CD16 was tested on NK cells from HIV-infected viremic, HIV-infected aviremic, and HIV-seronegative individuals, no difference in the degree of apoptosis was observed. Furthermore, when another polymerized chimeric IgG1-IgA fusion protein that binds to NKG2D and, at higher concentrations, induces apoptosis of NK cells was used, NK cells from HIV-infected viremic, HIV-infected aviremic, and HIV-seronegative individuals underwent apoptosis to similar degrees. These results suggest that NK cells from HIV-infected viremic individuals have a unique susceptibility to undergo apoptosis mediated by the CD95-sFasL interaction.
If NK cells from HIV-infected viremic individuals expressed higher levels of CD95, had increased serum levels of sFasL, and had an increased susceptibility to undergo FMA in vivo, there should be increased NK-cell turnover in vivo. To evaluate the degree of NK-cell turnover, we measured the level of intracellular Ki67 staining in NK cells ex vivo. It has been shown previously that intracellular Ki67 staining is an excellent correlate of cell turnover, as detected by in vivo labeling of cells.14 NK cells from HIV-infected viremic individuals expressed a significantly higher degree of intracellular Ki67 staining when compared with that from HIV-infected aviremic and HIV-seronegative individuals, suggesting that NK cells from HIV-infected viremic individuals do have an increased turnover rate in vivo. Moreover, previous studies have demonstrated that the CD56dim CD16bright subset of NK cells accounts for most of the dysfunction of total NK cells in HIV-infected patients.19,20,23 When we investigated the intracellular expression of Ki67 on NK cells from both subsets, the CD56dim CD16bright subset expressed significantly higher levels of intracellular Ki67 staining when compared with the CD56bright CD16dim subset. These findings suggest that CD56dim CD16bright NK cells are more susceptible to apoptosis and cell turnover in vivo.
Collectively, our data signify an increased propensity of NK cells, particularly the CD56dim CD16bright subset, from HIV-infected viremic individuals to undergo FMA and cell turnover in vivo. Most likely, these changes are attributable to the effect of immune activation induced by HIV viremia. Such changes over time could lead to loss and dysfunction of NK cells. Two recent studies examining the lymphocyte kinetics in simian immunodeficiency virus (SIV)-infected macaques using in vivo labeling showed that NK cells from SIV-infected macaques undergo cell turnover at a higher rate during the viremic state.36,37 Furthermore, another study using in vivo labeling of lymphocytes with 5′-bromo-2′-deoxyuridine (BRDU) in HIV-infected individuals suggested a higher rate of cell turnover among the various lymphocyte subsets in the viremic state.14 Our data are consistent with these observations but further suggest that it is the CD56dim CD16bright subset of NK cells that undergoes cell death and turnover in HIV-infected viremic individuals. This subset of NK cells comprises up to 90% of all peripheral NK cells and is predominantly composed of cytotoxic effector cells.38 Several studies on NK-cell dysfunction in HIV-infected individuals have described the CD56dim CD16bright subsets of NK cells as being the most susceptible to HIV viremia-induced changes in phenotype and function.7,8,19,23,26 Conceivably, the increased turnover of the effector CD56dim CD16bright subset of NK cells could result in some of the described dysfunction of NK cells in HIV-infected viremic individuals.7,8,19,23,26 Studies of lymphocyte dynamics specifically focusing on in vivo labeling of NK cells may validate the results of this study in HIV-infected viremic patients.
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