Phenotypically and functionally distinct subsets contribute to the expansion of CD56S/CD16R natural killer cells in HIV infection Henoch S. Honga,b, Johanna M. Eberharda, Phillip Keudela, Benjamin A. Bollmanna, Fareed Ahmada,c, Matthias Ballmaierd, Nupur Bhatnagara, Margot Zielinska-Skowroneka, Reinhold E. Schmidta and Dirk Meyer-Olsona Downloaded from https://journals.lww.com/aidsonline by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3mH5nK33R3QiiXeuLTYgosU/aM0m3agF4s+KUlsIDbf0= on 05/14/2018
Objective: Chronic HIV infection has been associated with activation and increased turnover of natural killer (NK) cells as well as with disturbed homeostasis of the NK cell compartment, including loss of CD56þ NK cells and accumulation of dysfunctional CD56/CD16þ NK cells. We performed a comprehensive phenotypical and functional characterization of this population. Design: A cross-sectional study was performed to analyze CD56/CD16þ NK cells from 34 untreated HIV-infected and 15 seronegative individuals. Methods: NK cells were analyzed by flow cytometry. Degranulation was assessed by measuring their expression of CD107a after stimulation with K562 cells, interleukin-12 and interleukin-15. Results: CD56/CD16þ NK cells are heterogeneous and composed of two populations, namely CD122/CCR7þ cells and CD122þ/CCR7 cells. We show that expanded CD122þ but not CCR7þ cells in HIV-seropositive individuals are characterized by expression of senescence marker CD57 similarly to CD56dim/CD16þ NK cells along with expression of KIRs, CD8, perforin and granzyme B. Despite expression of perforin and granzyme B, CD57 expressing cells exhibited less numbers of degranulating cells as measured by CD107a, indicating their functional impairment. However, there was no correlation between expansion of total CD56/CD16þ NK cells or the distinct subpopulations and viral load or CD4 cell count. Conclusion: These data indicate that expansion of CD56/CD16þ cells in HIV infection is driven by a distinct subset within this population with high expression of terminal differentiation marker with a phenotype resembling CD56dim/CD16þ NK cells. ß 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins
AIDS 2010, 24:1823–1834 Keywords: C–C chemokine receptor type 7, CD57, cytotoxicity, innate immunity, natural killer cells
a
Klinik fu¨r Immunologie und Rheumatologie, Medizinische Hochschule Hannover, Hannover, Germany, bDivision of Immunology, New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts, USA, cInternational Centre for Genetic Engineering and Biotechnology, New Delhi, India, and dPa¨diatrische Ha¨matologie und Onkologie, Medizinische Hochschule Hannover, Hannover, Germany. Correspondence to Dirk Meyer-Olson, MD, Klinik fu¨r Immunologie und Rheumatologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel: +49 511 532 3641; e-mail:
[email protected] Received: 9 December 2009; revised: 14 April 2010; accepted: 23 April 2010. DOI:10.1097/QAD.0b013e32833b556f
ISSN 0269-9370 Q 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins
1823
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
1824
AIDS
2010, Vol 24 No 12
Introduction HIV infection is characterized by rapid turnover of HIVspecific 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 [4]. 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/CD16þ cells, which represent the largest NK cell subset in peripheral blood and which contain bulk amounts of granzyme and perforin [5,6], and CD56/CD16þ 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 [9]. 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 CD56þ NK cells and expansion of CD56/CD16þ NK cells [12]. This alteration of NK cell homeostasis seems to take place during acute infection [8]. Alter et al. [8] demonstrated that loss of CD56þ NK cells is at least partly compensated by the expansion of CD56/CD16þ cells; therefore, resulting in an overall stable NK cell pool. However, CD56/CD16þ NK cells were shown to be highly dysfunctional in terms of cytotoxicity and cytokine release [13] and were furthermore characterized as ineffective interaction partners of monocyte-derived dendritic cells [14]. A recent study [15] showed that Siglec-7 downmodulation precedes the rise of this subset. Thus, the replacement of CD56expressing NK cells by functionally defective CD56/ CD16þ NK cells might be one of the major mechanisms of how HIV impairs overall NK cell response. To gain further understanding of CD56/CD16þ cells, we analyzed this population for early developmental marker CD122 [16], the lymph node-homing receptor C–C chemokine receptor type 7 (CCR7) [17] and the senescence marker CD57 [18]. We demonstrate that the CD56/CD16þ 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 CD122þ cells of HIV-seropositive individuals. Importantly, similar to CD56dim/CD16þ NK cells these CD57þ cells were less efficient in degranulation as measured by CD107a expression [12], thus indicating their functional impairment. These data
suggest that a distinct CD122þ subset drives the expansion of CD56/CD16þ NK cells.
Patients and methods Study participants 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 [3]. 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 (MFP9), anti-CD19 PerCP (J25C1), antiCD16 APC-H7 (3G8), anti-human leukocyte antigen DR (HLA-DR) APC (G46-6)), antiperforin PE (dG9), 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), antiCD3 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 [19]. 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,
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
CD56S/CD16R NK cell subsets in HIV infection Hong et al.
1825
Table 1. Demographic data on HIV-seropositive subjects. Patient ID
Disease status
UV147 UV173 UV162 UV146 UV169 UV096 UV180 UV163 UV171 UV178 UV090 UV189 UV191 UV187 UV129 UV002 UV046 UV190 UV181 UV085 UV167 UV182 UV114 UV151 UV160 UV161 UV174 UV179 UV175 UV168 UV036 UV188 UV185 UV105
VC VC VC VC VC VC VC VC VC VIC VIC VIC VIC VIC VIC VIC VIC VIC VIC VIC VIC VNC VNC VNC VNC VNC VNC VNC VNC VNC VNC VNC VNC VNC
Age (years) 43 34 57 43 32 40 73 22 42 48 32 49 24 24 47 26 31 26 61 69 28 42 48 45 41 20 44 28 30 64 31 48 31 37
Sex Female Male Male Male Female Male Male Female Male Male Female Female Male Female Male Female Female Male Male Female Male Male Male Male Female Male Male Male Male Male Female Male Female Male
% CD4 T cells
CD4 T-cell count (cells/ml)
Viral load (copies/ml)
% NK cells
Total NK cell number
33 39 48 42 33 19 24 30 38 15 27 28 27 17 21 24 32 30 19 12 18 22 11 15 12 27 11 29 23 12 38 25 14 16
845 1355 1281 736 754 363 393 514 748 382 630 510 400 351 353 286 338 357 217 180 337 332 264 467 283 442 219 480 356 200 770 291 304 199
