HIV infection is characterized by rapid turnover of HIV-specific T cells and natural killer (NK) cells [1–3]. NK cells are effector cells of the innate immunity arm, which rapidly kill their target cells without prior sensitization and are crucial in host defense against malignancies and viral infections . In humans, NK cells constitute 5–15% of the peripheral blood mononuclear cells (PBMCs). At least three distinct subpopulations can be distinguished based on their expression levels of CD56 and CD16:CD56bright/CD16− cells, which seem to have regulatory rather than cytolytic functions, as they are efficient producers of cytokines, CD56dim/CD16+ cells, which represent the largest NK cell subset in peripheral blood and which contain bulk amounts of granzyme and perforin [5,6], and CD56−/CD16+ NK cells, which have been described as a NK cell population with reduced functional properties emerging in HIV infection [7,8].
HIV possesses a number of mechanisms to efficiently evade NK cell surveillance . For instance, downregulation of activating receptors and upregulation of inhibitory receptors in HIV infection has been reported, thus leading to unresponsiveness of NK cells and impaired killing activity [10,11]. HIV infection has been strongly associated with loss of CD56+ NK cells and expansion of CD56−/CD16+ NK cells . This alteration of NK cell homeostasis seems to take place during acute infection . Alter et al.  demonstrated that loss of CD56+ NK cells is at least partly compensated by the expansion of CD56−/CD16+ cells; therefore, resulting in an overall stable NK cell pool. However, CD56−/CD16+ NK cells were shown to be highly dysfunctional in terms of cytotoxicity and cytokine release  and were furthermore characterized as ineffective interaction partners of monocyte-derived dendritic cells . A recent study  showed that Siglec-7 downmodulation precedes the rise of this subset. Thus, the replacement of CD56-expressing NK cells by functionally defective CD56−/CD16+ NK cells might be one of the major mechanisms of how HIV impairs overall NK cell response.
To gain further understanding of CD56−/CD16+ cells, we analyzed this population for early developmental marker CD122 , the lymph node-homing receptor C–C chemokine receptor type 7 (CCR7)  and the senescence marker CD57 . We demonstrate that the CD56−/CD16+ subpopulation in HIV patients is heterogeneous and that two subsets with distinct phenotypical and functional properties can be distinguished based on the expression of CCR7 and CD122. Furthermore, we found expression of the senescence marker CD57 in many CD122+ cells of HIV-seropositive individuals. Importantly, similar to CD56dim/CD16+ NK cells these CD57+ cells were less efficient in degranulation as measured by CD107a expression , thus indicating their functional impairment. These data suggest that a distinct CD122+ subset drives the expansion of CD56−/CD16+ NK cells.
Patients and methods
Thirty-four HIV-seropositive individuals and 15 healthy individuals were recruited in the HIV-outpatient clinic of the Medizinische Hochschule Hannover. HIV patients were either therapy-naive or untreated for more than 1 year (Table 1). All study participants gave written informed consent for their participation, and the study was approved by the local ethics committee (Votum der Ethikkommission der Medizinischen Hochschule Hannover no. 3150).
Isolation of mononuclear cells
PBMCs were isolated from fresh blood as described previously . Aliquots of 107 PBMCs each were cryopreserved in heat-inactivated fetal calf serum (FCS) supplemented with 10% dimethyl sulfoxide (Merck, Whitehouse Station, New Jersey, USA).
Phenotypical natural killer cell analysis and intracellular staining
Following mAbs were used in this study: anti-CD57 FITC (NK-1), anti-CD3 PerCP (SK7), anti-CD14 PerCP (MΦP9), anti-CD19 PerCP (J25C1), anti-CD16 APC-H7 (3G8), anti-human leukocyte antigen DR (HLA-DR) APC (G46-6)), antiperforin PE (δG9), anti-Ki-67 Alexa647 (B56), anti-Granzyme B Alexa700 (GB11) (BD Biosciences, Franklin Lakes, New Jersey USA), anti-CCR7 (150503) (R&D Systems, Minneapolis, Minnesota, USA), anti-CD158e1/e2 PE (Z27.3.7, recognizing KIR3DL1/3DS1), anti-CD158b1/b2j APC (GL183, recognizing KIR2DL2/2DL3/2DS2), anti-CD3 ECD (UCHT1), anti-CD56 PC7 (NKH-1), anti-CD38 PE (LS198-4-3), anti-CD45RO ECD (UCHL1), anti-CD122 PE (CF1) (Beckman Coulter, Brea, California, USA), anti-CD27 Alexa700 (O323) (BioLegend, San Diego, California, USA) and anti-CD8 Pacific Orange (3B5) (Invitrogen, Carlsbad, California, USA). Staining was performed as described before . Indirect staining of CCR7 using Pacific blue-conjugated goat antibodies against mouse immunoglobulins (Invitrogen) subsequently followed by washing and blocking of remaining goat-antimouse antibody using mouse serum (Dako, Glostrup, Denmark) preceded the staining with directly labeled antibodies. We used fixation and permeabilization kit from Invitrogen for intracellular detection of granzyme B, perforin and Ki-67 and followed the instructions given by the manufacturer.
For each sample, at least one million events were acquired using either FACSAria, LSR II (BD Biosciences) or FC500 flow cytometers (Beckman Coulter). Data were analyzed with FlowJo (TreeStar Inc., Ashland, Oregon, USA). Lymphocytes were defined by forward and side scatter. CD3+, CD14+, CD19+, dead cells and cell aggregates were removed from analysis based on PerCP and Viaprobe staining and pulse width analysis. NK cells and their distinctive subpopulations were defined based on their expression of CD56, CD16 or both. Fluorescence minus one staining was used to determine threshold values for the expression of specific markers.
CD107a degranulation assay
The assay was performed as described before . In brief, total NK cells, encompassing CD56bright/CD16− cells, CD56dim/CD16+ cells and CD56−/CD16+ cells, were highly purified by cell sorting on FACSAria II (BD Biosciences). One lakh NK cells/well were cultured in a 96-well plate in Roswell Park Memorial Institute-1640 medium, including 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mmol/l L-glutamine and 1 mmol/l sodium pyruvate. NK cells were exposed to 50 000 K562 cells and stimulated with 10 ng/ml interleukin (IL)-15 and 100 ng/ml IL-12. A medium control was run for every sample. CD107a antibody, Brefeldin A (Sigma, St Louis, Missouri, USA) and Golgi-Stop (BD Biosciences) were added after 1 h. After 5 h of incubation, cells were washed and surface staining was performed as described above.
GraphPad Prism (version 5.0) software (GraphPad Software, Inc., La Jolla, California, USA) was used for statistical evaluation of data. Unpaired t-test was performed when two groups were compared. All t-tests were two-tailed. Comparison of more than two groups was performed using one-way analysis of variance, followed by Tukey posttests. P values of less than 0.05 were considered significant.
Expansion of CD122+/CCR7− cells solely contribute to the expansion of CD56−/CD16+ natural killer cells
We defined NK cells as CD3− CD14− CD19− lymphocytes expressing either CD16 or CD56 or both as described previously . Utilizing CD56 and CD16, we defined three major NK cell subpopulations in the peripheral blood: CD56bright/CD16− cells, CD56dim/CD16+ cells and CD56−/CD16+ cells (Fig. 1a). In accordance with published reports [8,12,13], we found in our cohort of untreated HIV-seropositive patients a significant increase in CD56−/CD16+ NK cells as compared with the uninfected control individuals (mean, 14.2 versus 4.9%; P = 0.0013) (Fig. 1a).
We assessed the distribution of CD122 and CCR7 surface expression on CD56−/CD16+ cells. CD122 and CCR7 are not coexpressed in CD56−/CD16+ cells, neither in HIV-seropositive nor HIV-seronegative blood donors, and CD56−/CD16+ NK cells were not double negative for these surface receptors (Fig. 1b). Hence, the usage of CCR7 and CD122 allows the distinction of two clear subsets within the CD56−/CD16+ population.
To understand the relevance of these two CD56−/CD16+ subpopulations for NK cell homeostasis, we compared their relative distribution within the total NK cell pool (Fig. 1c). Almost all CD56bright/CD16− cells as well as CD56dim/CD16+ cells were positive for CD122 in healthy and HIV-infected individuals (Fig. 1c). There was a weak but significant loss of CD122 expression in these two NK cell subpopulations from HIV-seropositive patients as compared with uninfected individuals (mean, 93 versus 82.9%; P < 0.0001 and mean, 97.2 versus 83.8%; P = 0.0003, respectively).
We found striking differences for the expression of CD122 within the CD56−/CD16+ population in which we detected considerable expansion of CD122+ cells in HIV-seropositive individuals (mean, 8.0 versus 1.2%; P = 0.0048) (Fig. 1c). However, there was no difference for frequency of the CD122− subpopulation of CD56−/CD16+ NK cells between HIV-seropositive patients and controls (Fig. 1c).
Expression of senescence marker CD57 on cells with high turnover, such as HIV-specific CD8+ T cells, has been reported, and CD57 was shown to be expressed on terminally differentiated effector cells [18,20]. We, therefore, questioned whether CD57 is also expressed on CD56−/CD16+ NK cells. Healthy individuals expressed CD57 on a substantial fraction of CD56dim/CD16+ NK cells but not on CD56bright/CD16− cells and scarcely on CD56−/CD16+ NK cells . Of note, a substantial fraction of CD56−/CD16+ NK cells from HIV-infected patients but not from healthy donors was positive for CD57 (mean, 7.1 versus 0.9%; P = 0.0045) (Fig. 1d).
We next addressed the question, which of the identified subsets within CD56−/CD16+ cells expressed CD57. There was no expression of CD57 on CCR7+ cells, neither in HIV-infected individuals nor in healthy controls, but there was a substantial CD57+ population on the CCR7− subpopulation in HIV-seropositive patients and, to a much lesser extent, on HIV-seronegative controls (Fig. 1e). Therefore, in terms of CCR7 and CD57 expression, altogether three subsets can be defined in HIV-seropositive individuals (Fig. 1e). On the basis of these results, we will refer to these subsets as CCR7+/CD57− cells, CCR7−/CD57− cells and CCR7−/CD57+ cells in the following. The latter two subsets lack CCR7 expression and are subfractions of the CD122+ subpopulation (Fig. 1b).
As we observed a clear expansion of the CD122+ subset within the CD56−/CD16+ population giving rise to CD57+ cells in HIV infection, we wanted to delineate how these three subsets contributed to the expansion of CD56− CD16+ cells during HIV infection. The frequencies of CCR7+/CD57− cells did not change in HIV-infected patients, whereas both the CCR7−/CD57− subset and CCR7−/CD57+ cells, strongly increased in our HIV-positive cohort (Fig. 1f). This demonstrates that CCR7 and CD57 double-negative cells and even more the CCR7−/CD57+ subset drive the expansion of CD56−/CD16+ cells.
In summary, we demonstrate two distinct subpopulations within the CD56−/CD16+ population, which can be defined by their expression of CD122 and CCR7, but only the CD122+ subpopulation expands in HIV infection. We show substantial expression of the senescence marker CD57 on the CD122+ NK cell population in HIV infection.
Differential phenotype of CCR7+/CD57−, CCR7−/CD57− and CCR7−/CD57+ cells
Having shown that HIV infection leads to expansion of CD122+ cells with a substantial proportion expressing CD57+, we next sought to answer the question, whether this alteration was also reflected in the expression of activation markers and surface receptors, which were shown to have relevance for human NK cells, such as CD8, CD27 and KIRs [21,22]. There was no expression of CD27 on CD56−/CD16+ NK cells irrespective of the HIV infection status (data not shown).
Subsets of CD56−/CD16+ NK cells in HIV patients according to their CD57 and CCR7 expression considerably varied in their expression patterns of HLA-DR and CD38. The majority of CCR7+/CD57− cells displayed expression of HLA-DR+ but low expression of CD38 (Fig. 2a). On the contrary, CCR7−/CD57+ NK cells were characterized by low expression of HLA-DR, whereas in most patients, these cells were mostly positive for CD38. CCR7−/CD57− cells resembled CCR7−/CD57+ cells rather than CCR7+ cells and we found no differences in terms of CD38 expression. The frequencies of HLA-DR-positive cells were significantly higher in the CCR7 and CD57 double-negative subset than in CCR7−/CD57+ cells (mean, 29.8 ± 16.1 versus 17.6 ± 14.9%; P < 0.01) (Fig. 2a).
The CD56−/CD16+ subsets also considerably varied in their CD8 expression patterns. CD8 is not expressed on CCR7+/CD57− cells but with increasing prevalence on CCR7−/CD57− cells (mean 13.9 ± 6.6%) and on CCR7−/CD57+ cells (mean, 20.0 ± 12.3%) (Fig. 2a).
Among other markers, mature NK cells are characterized by expression of KIRs . Presence of senescence marker CD57 on CD122+ cells prompted us to investigate the frequencies of either KIR2DL2/2DL3/2DS2+ or KIR3DL1/3DS1+ cells within CD57− versus CD57+ cells. Frequency of KIR+ cells among CD57− cells was significantly lower as compared with CD57+ cells for KIR2DL2/2DL3/2DS2+ cells (mean, 6.8 ± 8.8 versus 36.7 ± 21.8%; P < 0.0001) and KIR3DL1/3DS1+ cells (4.9 ± 8.7 versus 18.7 ± 15.7%; P < 0.0001) among CD57+ (Fig. 2b). Noteworthy, there was a high variance of the frequency of KIR-expressing cells within the CD57+ subset.
Taken together, we provide evidence for differential phenotype of the subsets within CD56−/CD16+ NK cells as shown by differential expression patterns of HLA-DR, CD38, CD8 and KIRs.
Differential functional properties of subsets within CD56−/CD16+ cells
We next evaluated the cytotoxic potential of CD56−/CD16+ cells by studying their intracellular content of granzyme B and perforin. CD57 is frequently coexpressed with granzymes and perforin [20,23] and we thus hypothesized that the CD57+ subset within the CD56−/CD16+ NK subpopulation expressed highest amounts of granzyme B and perforin. Indeed, a considerable fraction of CCR7−/CD57+ cells was positive for granzyme B and a majority of this subset also expressed perforin. In contrast to CD57-expressing CD56−/CD16+ NK cells, CCR7+/CD57− cells expressed very low amounts of granzyme B and were almost all negative for perforin (Fig. 3a). Analogous to the expression patterns of HLA-DR, CD38 and CD8, CCR7−/CD57− cells were similar to CD57+ cells, especially with regard to their perforin content. Furthermore, CCR7−/CD57− displayed considerably higher percentages of granzyme B expression than CCR7+/CD57− cells (P < 0.001) but significantly less than the CCR7−/CD57+ subset (P < 0.001) (Fig. 3a).
Having confirmed that CCR7−/CD57+ cells exhibited highest cytolytic potential as shown by granzyme B and perforin expression, we questioned whether this is also reflected in their actual ability to degranulate. We, therefore, performed a CD107a degranulation assay after stimulating sorted NK cells with IL-12, IL-15 and K562 cells. Despite high percentages of granzyme B and perforin-expressing cells, CD57+ cells were significantly less efficient in degranulation as compared with CD57− cells (mean, 27.3 versus 13.2%, P = 0.0076) (Fig. 3b). This indicates that senescence as shown by CD57 expression is accompanied by functional impairment as indicated by defective CD107a degranulation. Of note, there was a high variance in the efficiency of CD57− cells to degranulate.
We showed significant increase and accumulation of CD122+ cells within the CD56−/CD16+ population with concomitant CD57 expression. Presence of CD57 has been associated with shorter telomeres , which suggests that the CCR7−/CD57+ subset previously underwent vigorous proliferation. We, therefore, studied CD56−/CD16+ NK cells for the presence of nuclear Ki-67, which is strictly associated with cycling cells and is thus a useful marker to monitor growing cell populations . Ki-67 was hardly found in CCR7+/CD57− cells (Fig. 3c). However, a significant proportion of CCR7−/CD57− was positive for Ki-67, indicating that this population was the most dynamic in terms of proliferation (mean, 23.7 versus 2.5 and 7.8%, respectively; P < 0.001). Ki-67+ cells were significantly less frequent in the CD57+ subset yet higher in comparison with the CCR7+/CD57− subset (P < 0.01). This observation would be in accordance with proliferative exhaustion of CD57-expressing cells due to numerous prior cell cycles. In summary, our results indicate different functional capacities of different subsets within CD56−/CD16+ cells.
Expansion of the CD56− natural killer cell subsets does not correlate with clinical parameters
We showed that only CD122+ cells contribute to the overrepresentation of CD56−/CD16+ cells. CCR7+ cells neither expressed granzyme B nor perforin nor any other NK cell-related markers, such as CD161 or NKG2D (data not shown). To rule out dendritic cell contamination, we tested whether these cells express the blood dendritic cells antigens BDCA-1 or BDCA-4 and found that these cells were negative for these molecules (data not shown).
Higher frequency of CD57 expression on CD8+ T cells was described to be associated with increased age of analyzed individuals . We thus questioned whether increased frequency of CD57+ cells in HIV-infected donors correlated with their age. We found no such correlation (r = 0.06, P = 0.72) (Fig. 4a), suggesting that HIV infection is the primary cause for accumulation of CD57+/CCR7− NK cells. Nevertheless, expansion of CD57+/CD56−/CD16+ NK cells did not correlate with viral load, CD4+ T-cell counts or with CD4+/CD8+ T-cell ratio (Fig. 4b–d). A recent study  had proposed that the expansion of CD56−/CD16+ cells is predominantly driven by HIV viral load. We compared the frequencies of CD56−/CD16+ cells in control individuals with HIV-infected donors, which we had subdivided according to their viral load in three groups: viral load below 2000 copies/ml, viral load between 2000 and 20 000 copies/ml and viral load exceeding 20 000 copies/ml. In contrast to the report mentioned above, we found no correlation between frequencies of CD56−/CD16+ cells and viral load (Fig. 4e). Remarkably, one of the HIV-infected patients (UV173) had no detectable viral load despite absence of treatment and still displayed relatively high numbers of CD56−/CD16+ cells of 23.2%.
To test whether there is a potential relationship between expansion of CD56−/CD16+ cells and immune activation, we performed correlation analysis of percentages of CD56−/CD16+ cells with percentages of CD38-expressing CD8+ T cells. There was no correlation between these two parameters (Fig. 4f). CD38 expression on CD8+ T cells from patient UV173 mentioned above was similar to values obtained from uninfected controls further arguing against a direct relationship between immune activation observed on CD8+ T cells and expansion of CD56−/CD16+ cells.
Our cohort comprised five patients who had interrupted their antiviral treatment for longer than 1 year. We tested whether former antiviral treatment affected the frequencies of CD56−/CD16+ cells in comparison with therapy-naive patients and found no substantial difference (mean, 14.7 versus 16.9%, P = 0.7). We thus show that the increase of CD56−/CD16+ cells is not directly correlated with clinical parameters.
HIV infection has been associated with severe disturbances in the NK cell compartment . HIV affects homeostasis of NK cell subsets by inducing loss of CD56-expressing NK cells and accumulation of CD56−/CD16+ NK cells, which were shown to be dysfunctional [12,13]. The overall biological role of CD56−/CD16+ NK cells is poorly understood. Interestingly, expansion of this subset has not only been described in HIV infection but also in other disease settings, such as HIV/hepatitis C virus (HCV) coinfection [26,27], HCV infection , tuberculosis , myasthenia gravis  and in cord blood transplant recipients . These reports altogether suggest that expansion of CD56−/CD16+ NK cells in the peripheral NK cell compartment can be found in several diseases, in which NK cells might play a substantial role.
Here, we provided evidence for differential CD122 expression within CD56−/CD16+ NK cells. CD122 is also known as IL-2 receptor β-chain, which is as well shared by the IL-15 receptor . This molecule is one of the early surface markers expressed on committed NK cell precursors and its expression is crucial for NK cell development . Absence of CD122 on CCR7+/CD57− cells, as well as the fact that these cells were devoid for virtually every NK cell-related marker, we have tested, including NKG2D and CD161 (data not shown), raises the question whether these cells are classical NK cells. Our gating strategy excluded all monocytes from the analysis (Fig. 1a). As mentioned earlier, we sought to rule out dendritic cell contamination by analyzing whether these cells express BDCA-1 or BDCA-4. We found that these cells were negative for these molecules (data not shown). We clearly showed that this particular subset is not involved in disturbed homeostasis of the NK cell pool in HIV infection.
CCR7 and CD57 double-negative cells resemble CD57+ cells but express Ki-67 indicating active proliferation. This population was positive for granzyme B and perforin but exhibited less numbers of KIR-expressing cells.
We, furthermore, demonstrated that CD56−/CD16+ NK cells from HIV patients rather than healthy individuals exhibit a higher portion of CD57+ cells. CD57 is a marker for replicative senescence and indicates history of numerous cell divisions as shown by shorter telomere lengths . In addition, CD57 expression is associated with high cytolytic potential in CD8+ T cells as well as NK cells  and this marker, previously termed HNK-1 or Leu-7, thus had been formerly utilized to define human NK cells. High expression of CD57 is found on CD56dim/CD16+ NK cells and has been associated with a terminally differentiated phenotype . To our knowledge, we are the first ones to demonstrate that this marker is also found on CD56−/CD16+ NK cells in HIV-infected patients and that CD57 expression on CD56− NK cells is associated with the expression of KIRs, CD8 and cytolytic molecules. In our experiments, we were not able to differentiate between activating and inhibiting KIRs. Nevertheless, CD57+/CCR7− cells also displayed higher frequencies of KIR2DS4+ cells (data not shown), another activating KIR. This suggests that CD57 expression is in general associated with higher numbers of KIR-expressing cells, irrespective of whether these are activating or inhibiting receptors. We also compared numbers of cells expressing NKG2D or ILT2 on CCR7−/CD57− cells versus CCR7−/CD57+ cells and found no significant differences in their frequencies (data not shown), which further suggests that CD57 expression is not necessarily associated with differential expression patterns of activating and inhibiting NK cell receptors.
Replicative senescence was accompanied by functional impairment in terms of degranulation. Several studies reported that CD56−/CD16+ NK cells are poor effector cells in terms of cell cytotoxicity in comparison with CD56dim/CD16+ cells [8,13,33]. Our data support this observation and we identified a novel subset within CD56−/CD16+ NK cells, which might be responsible for the overall measured functional impairment of CD56−/CD16+ cells. In addition, our data support the hypothesis that CD57 is a marker for terminal differentiation on NK cells [18,20,34].
Terminal differentiation and exhaustion of CD8+ T cells have been identified as prominent hallmarks in the immunopathology of HIV-1 infection . In HIV-1 infection, substantial alterations of markers of CD8+ T-cell memory differentiation, such as CD45RO/RA, CD27, CD28, CCR7 among others, as well as markers that relate to exhaustion or terminal senescence, such as PD-1 or CD57, have been described. How these alterations of CD8+ memory T cells relate to the differentiation of NK cells in HIV infection will be crucial for the understanding of the immunopathology of this disease.
Our data raise the question why significant numbers of CD57+/CCR7− cells arise in HIV-infected people but not in healthy individuals. Our untreated study cohort comprised HIV patients with persistently low or undetectable viral load (less than 50 copies/ml) as well as nonviral controllers with high viremia (more than 20 000 copies/ml) (Table 1). Occurrence of the CD57+/CCR7− subpopulation did neither correlate with CD4+ T-cell counts nor with their viral load nor any other clinical parameters we tested. We were also able to identify this population in treated HIV patients with ideally suppressed viral replication (data not shown). A recent cross-sectional and longitudinal study  suggested that HIV viral load is the major determining factor for the rise of CD56−/CD16+ cells. In our study, we did not observe significant differences between untreated individuals with low (<2000 copies/ml) and high (>2000 copies/ml) viral load. Our cohort included so-called ‘elite’ controller who suppress viral load beyond the limit of detection but with substantial frequencies of CD56−/CD16+ NK cells. In addition, there was no correlation between viral load and the frequency of CD56−/CD16+ NK cells. The reasons for the different findings are not clear and might be explained by yet unidentified host factors such as genetic background.
Our study provides a thorough phenotypical and functional description of novel expanding subsets within CD56−/CD16+ cells, which is characterized by terminal differentiation. Our data indicate that this terminal differentiation is accompanied by impaired cytotoxicity similar to that observed for CD56dim NK cells. This study thus helps to unravel the dysfunctional nature of this enigmatic NK cell subpopulation.
D.M.O. is supported by grants from the Bundesministerium für Bildung und Forschung, and the Helmholtz-Zentrum für Infektionsforschung (IG-SCID-TwinPro02). H.S.H. is supported by a fellowship of the MD/PhD program of the Hanover Biomedical Research School at Hanover Medical School. R.E.S. is supported by grant IND 06/20 from Bundesministerium für Bildung und Forschung. The authors want to thank Christina Reimer for her excellent assistance in flow cytometry and Mathias Rhein for his support in cell sorting.
D.M.O., H.S.H. and R.E.S. planned the project, designed the experiments or both; H.S.H., J.M.E., P.K., B.A.B., F.A. and N.B. performed the experiments. M.Z.S. provided technical assistance, M.B. helped with data analysis. H.S.H., M.B., R.E.S. and D.M.O. wrote the manuscript.
1. Douek DC, Roederer M, Koup RA. Emerging concepts in the immunopathogenesis of AIDS. Annu Rev Med 2009; 60:471–484.
2. Kottilil S, Jackson JO, Reitano KN, O'Shea MA, Roby G, Lloyd M, et al
. Innate immunity in HIV infection: enhanced susceptibility to CD95-mediated natural killer cell death and turnover induced by HIV viremia. J Acquir Immune Defic Syndr 2007; 46:151–159.
3. Meyer-Olson D, Brady KW, Bartman MT, O'Sullivan KM, Simons BC, Conrad JA, et al
. Fluctuations of functionally distinct CD8+
T-cell clonotypes demonstrate flexibility of the HIV-specific TCR repertoire. Blood 2006; 107:2373–2383.
4. Yokoyama WM, Kim S, French AR. The dynamic life of natural killer cells. Annu Rev Immunol 2004; 22:405–429.
5. Jacobs R, Hintzen G, Kemper A, Beul K, Kempf S, Behrens G, et al
. CD56bright cells differ in their KIR repertoire and cytotoxic features from CD56dim NK cells. Eur J Immunol 2001; 31:3121–3127.
6. Cooper MA, Fehniger TA, Caligiuri MA. The biology of human natural killer-cell subsets. Trends Immunol 2001; 22:633–640.
7. Sondergaard SR, Ullum H, Pedersen BK. Proliferative and cytotoxic capabilities of CD16+
natural killer cells. Apmis 2000; 108:831–837.
8. Alter G, Teigen N, Davis BT, Addo MM, Suscovich TJ, Waring MT, et al
. Sequential deregulation of NK cell subset distribution and function starting in acute HIV-1 infection. Blood 2005; 106:3366–3369.
9. Fauci AS, Mavilio D, Kottilil S. NK cells in HIV infection: paradigm for protection or targets for ambush. Nat Rev Immunol 2005; 5:835–843.
10. De Maria A, Fogli M, Costa P, Murdaca G, Puppo F, Mavilio D, et al
. The impaired NK cell cytolytic function in viremic HIV-1 infection is associated with a reduced surface expression of natural cytotoxicity receptors (NKp46, NKp30 and NKp44). Eur J Immunol 2003; 33:2410–2418.
11. Mavilio D, Benjamin J, Daucher M, Lombardo G, Kottilil S, Planta MA, et al
. Natural killer cells in HIV-1 infection: dichotomous effects of viremia on inhibitory and activating receptors and their functional correlates. Proc Natl Acad Sci U S A 2003; 100:15011–15016.
12. Hu PF, Hultin LE, Hultin P, Hausner MA, Hirji K, Jewett A, et al
. Natural killer cell immunodeficiency in HIV disease is manifest by profoundly decreased numbers of CD16+
cells and expansion of a population of CD16dimCD56−
cells with low lytic activity. J Acquir Immune Defic Syndr Hum Retrovirol 1995; 10:331–340.
13. Mavilio D, Lombardo G, Benjamin J, Kim D, Follman D, Marcenaro E, et al
. Characterization of CD56−
natural killer (NK) cells: a highly dysfunctional NK subset expanded in HIV-infected viremic individuals. Proc Natl Acad Sci U S A 2005; 102:2886–2891.
14. Mavilio D, Lombardo G, Kinter A, Fogli M, La Sala A, Ortolano S, et al
. Characterization of the defective interaction between a subset of natural killer cells and dendritic cells in HIV-1 infection. J Exp Med 2006; 203:2339–2350.
15. Brunetta E, Fogli M, Varchetta S, Bozzo L, Hudspeth KL, Marcenaro E, et al
. The decreased expression of Siglec-7 represents an early marker of dysfunctional natural killer-cell subsets associated with high levels of HIV-1 viremia. Blood 2009; 114:3822–3830.
16. Minami Y, Kono T, Miyazaki T, Taniguchi T. The IL-2 receptor complex: its structure, function, and target genes. Annu Rev Immunol 1993; 11:245–268.
17. Worbs T, Forster R. A key role for CCR7 in establishing central and peripheral tolerance. Trends Immunol 2007; 28:274–280.
18. Brenchley JM, Karandikar NJ, Betts MR, Ambrozak DR, Hill BJ, Crotty LE, et al
. Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+
T cells. Blood 2003; 101:2711–2720.
19. Meyer-Olson D, Shoukry NH, Brady KW, Kim H, Olson DP, Hartman K, et al
. Limited T cell receptor diversity of HCV-specific T cell responses is associated with CTL escape. J Exp Med 2004; 200:307–319.
20. Hong HS, Eberhard JM, Keudel P, Bollmann BA, Ballmaier M, Bhatnagar N, et al
. HIV infection is associated with a preferential decline in less-differentiated CD56dim CD16+NK cells
. J Virol84
21. Vossen MT, Matmati M, Hertoghs KM, Baars PA, Gent MR, Leclercq G, et al
. CD27 defines phenotypically and functionally different human NK cell subsets. J Immunol 2008; 180:3739–3745.
22. Addison EG, North J, Bakhsh I, Marden C, Haq S, Al-Sarraj S, et al
. Ligation of CD8alpha on human natural killer cells prevents activation-induced apoptosis and enhances cytolytic activity. Immunology 2005; 116:354–361.
23. Chattopadhyay PK, Betts MR, Price DA, Gostick E, Horton H, Roederer M, De Rosa SC. The cytolytic enzymes granzyme A, granzyme B, and perforin: expression patterns, cell distribution, and their relationship to cell maturity and bright CD57 expression. J Leukoc Biol 2009; 85:88–97.
24. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol 2000; 182:311–322.
25. McNerlan SE, Rea IM, Alexander HD, Morris TC. Changes in natural killer cells, the CD57CD8 subset, and related cytokines in healthy aging. J Clin Immunol 1998; 18:31–38.
26. Gonzalez VD, Falconer K, Bjorkstrom NK, Blom KG, Weiland O, Ljunggren HG, et al
. Expansion of functionally skewed CD56-negative NK cells in chronic hepatitis C virus infection: correlation with outcome of pegylated IFN-alpha and ribavirin treatment. J Immunol 2009; 183:6612–6618.
27. Gonzalez VD, Falconer K, Michaelsson J, Moll M, Reichard O, Alaeus A, Sandberg JK. Expansion of CD56−
NK cells in chronic HCV/HIV-1 co-infection: reversion by antiviral treatment with pegylated IFNalpha and ribavirin. Clin Immunol 2008; 128:46–56.
28. Zarife MA, Reis EA, Carmo TM, Lopes GB, Brandao EC, Silva HR, et al
. Increased frequency of CD56Bright NK-cells, CD3−
NK-cells and activated CD4+
T-cells or B-cells in parallel with CD4+
CDC25High T-cells control potentially viremia in blood donors with HCV. J Med Virol 2009; 81:49–59.
29. Barcelos W, Sathler-Avelar R, Martins-Filho OA, Carvalho BN, Guimaraes TM, Miranda SS, et al
. Natural killer cell subpopulations in putative resistant individuals and patients with active Mycobacterium tuberculosis
infection. Scand J Immunol 2008; 68:92–102.
30. Nguyen S, Morel V, Le Garff-Tavernier M, Bolgert F, Leblond V, Debre P, Vieillard V. Persistence of CD16+
natural killer cells: a highly dysfunctional NK subset expanded in ocular myasthenia gravis. J Neuroimmunol 2006; 179:117–125.
31. Lu X, Kondo Y, Takamatsu H, Ohata K, Yamazaki H, Takami A, et al
NK cells in the peripheral blood of cord blood transplant recipients: a unique subset of NK cells possibly associated with graft-versus-leukemia effect. Eur J Haematol 2008; 81:18–25.
32. Goldman JP, Blundell MP, Lopes L, Kinnon C, Di Santo JP, Thrasher AJ. Enhanced human cell engraftment in mice deficient in RAG2 and the common cytokine receptor gamma chain. Br J Haematol 1998; 103:335–342.
33. Alter G, Suscovich TJ, Kleyman M, Teigen N, Streeck H, Zaman MT, et al
. Low perforin and elevated SHIP-1 expression is associated with functional anergy of natural killer cells in chronic HIV-1 infection. AIDS 2006; 20:1549–1551.
34. Hoji A, Connolly NC, Buchanan WG, Rinaldo CR Jr. CD27 and CD57 expression reveals atypical differentiation of human immunodeficiency virus type 1-specific memory CD8+
T cells. Clin Vaccine Immunol 2007; 14:74–80.