Dysfunctional natural killer cells, in vivo, are governed by HIV viremia regardless of whether the infected individual is on antiretroviral therapy

Barker, Edwarda; Martinson, Jeffa; Brooks, Cicelya; Landay, Alana; Deeks, Stevenb

doi: 10.1097/QAD.0b013e3282f1d658
Author Information

aDepartment of Immunology and Microbiology, Rush University Medical Center, Chicago, IL, USA

bDepartment of Medicine, University of California, San Francisco, California, USA.

Received 1 February, 2007

Revised 1 May, 2007

Accepted 11 May, 2007

Article Outline

Progression to AIDS during HIV-infection is associated with a decrease in the number and function of natural killer (NK) cells [1–4]. The reduction in NK cells expressing CD56 and CD16 correlates with low CD4+ T-cell count [5] and increasing viral load [6,7]. Moreover, higher viral loads are associated with an increased presence of a highly dysfunctional NK cell subpopulation lacking CD56 but retaining CD16 [7,8], whereas suppression of viral replication with highly-active antiretroviral therapy (HAART) results in improvements in both NK cell function and NK cell phenotype [7]. These observations have been used to propose that high level viral replication is a consequence rather than a cause of alterations in the NK cell phenotypic patterns [7], although this remains controversial [9]. Of note, most of these studies involved individuals who were either not controlling HIV replication or whose virus was fully controlled with HAART. No study has systematically included individuals whose virus was low in the absence of therapy, and presumably being controlled immunologically.

The present study determines the characteristics of NK cell subsets across a broad range of chronically-infected patients. A total of 76 individuals from four well characterized groups of individuals were enrolled: (i) ‘elite’ controllers: plasma HIV RNA levels < 75 copies/ml in the absence of any therapy (n = 19) (Fig. 1a); (ii) ‘partial’ controllers on antiretroviral therapy (PCAT): plasma HIV RNA levels between 75 and 104 copies/ml (n = 19) (Fig. 1a); (iii) ‘HAART suppressed’: plasma HIV RNA levels < 75 copies on long-term HAART (n = 19) (Fig. 1a); and (iv) ‘noncontrollers’: plasma HIV RNA levels > 104 copies RNA/ml (n = 19) (Fig. 1a). All subjects were recruited from the UCSF Study of the Consequences of the Protease Inhibitor Era (SCOPE) cohort and all provided their written informed consent. The first two groups were selected because previous work from these individuals have the strongest evidence for immune-mediated virus control [10].

Peripheral blood lymphocytes from the study subjects were stained with flurochrome-conjugated CD56, CD16, CD3 and CD69 antibodies (BDIS, San Jose, California, USA). After staining, cells were washed twice with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin and 0.1% sodium azide. Cells were fixed with 2% paraformaldehyde in PBS, pH 7.2, acquired on a FACsCalibur (BDIS) flow cytometer and analyzed using Cell Quest Software, version 3.2.1 (BDIS). Plasma HIV RNA levels were determined by the bDNA amplification technique (Quantiplex HIV RNA; Chiron, Emeryville, California, USA), as described previously [11].

The virologic noncontrollers had the lowest total percentage of classically defined NK cells (CD56+/CD3−) (mean ± SD = 3.4 ± 2.3%). This percentage was significantly lower than that observed in the HAART-suppressed group (mean ± SD = 5.7 ± 3.9%; P = 0.03) and is consistent with prior observations [1–4]. The percentage of CD56+ NK cells was intermediate in the elite controllers and PCAT subjects, and did not differ significantly from the noncontroller group (Fig. 1b).

Of the three different NK cell subsets, based on CD56 and CD16 expression, the CD56dim/CD16+ NK cell subset possesses the greatest capacity to mediate a cytotoxic response [6,7]. The two viremic groups (noncontrollers and PCAT) had the lowest percentage of these cells (Fig. 1c) and were not statistically different from one another. The differences in the percentage of CD56dim/CD16+ NK cells between the noncontrollers and either aviremic group were statistically significant (P = 0.004 in noncontrollers versus elite controllers, and P = 0.007 in noncontrollers versus HAART-suppressed).

CD56neg/CD16+ subset of NK cells are thought to be largely dysfunctional [8]. In contrast to the trends observed above, we saw a striking association between high viral load and a high percentage of these cells. Subjects whose virus was controlled therapeutically (HAART) or presumably immunologically (elite controllers) had the lowest percentage of these cells overall compared with the viremic individuals. Since little if any of these cells exist in uninfected individuals [12], our findings suggest that the presence of this subpopulation of cells occurred as a consequence of virus infection and that virus control appears to maintain this population of NK cells to significantly lower levels than when virus is not controlled (Fig. 1d; P < 0.0001 for either elite or HAART suppressed compared to either viremic population). The percentage of CD56bright/CD16neg NK cells, which tend to be effector NK cells with the greatest capacity to produce cytokines [13], was similar across all groups tested (Fig. 1e).

We next evaluated the activation status of NK cells in the different infected individuals studied, focusing on the expression of CD69 on CD56dim/CD3−/CD16+ and CD56neg/CD3−/CD16+ cells (Fig. 1f). In contrast to what has been observed from this cohort with T cell activation [11,14], patients who were viremic either in the presence or absence of therapy had lower levels of NK cell activation (P < 0.0001 for comparison between either aviremic group with either viremic group).

In summary, we did not find any consistent NK cell phenotypic pattern in our subsets of individuals who are likely to be controlling their virus immunologically (i.e. elite controllers, PCAT subjects) as compared to either HAART-suppressed or the noncontroller populations. These data suggest that NK cells, at least as measured in the peripheral blood using classical cell surface markers, are not causally associated with control of HIV replication. We did, however, observe consistent trends that tracked more closely with the level of viremia. For example, undetectable viremia in either elite controllers or HAART-suppressed subjects was associated with a higher percentage of activated effector NK cells (CD56dim/CD16+) and lower numbers of presumably dysfunctional NK cells (CD56neg/CD16+). As these observations were independent of whether the virus was controlled immunologically or therapeutically, they suggest that the level of viral replication was a cause rather than a consequence of the observed NK cell phenotypic patterns. This conclusion is supported by our previous study performed in vitro indicating that NK cells do not kill HIV infected cells effectively [15]. Further work is needed to correlate these phenotypic changes with NK cell function in both peripheral blood and in tissues of infected individuals. Given that NK cells are important in control of tumors and virus infected cells, it would be important to understand how HIV replication leads to an alteration in NK cells.

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1. Landay A, Poon MC, Abo T, Stagno S, Lurie A, Cooper MD. Immunologic studies in asymptomatic hemophilia patients. Relationship to acquired immune deficiency syndrome (AIDS). J Clin Invest 1983; 71:1500–1504.
2. Bonavida B, Katz J, Gottlieb M. Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. I. Defective trigger on NK cells for NKCF production by target cells, and partial restoration by IL 2. J Immunol 1986; 137:1157–1163.
3. Plaeger-Marshall S, Spina CA, Giorgi JV, Mitsuyasu R, Wolfe P, Gottlieb M, Beall G. Alterations in cytotoxic and phenotypic subsets of natural killer cells in acquired immune deficiency syndrome (AIDS). J Clin Immunol 1987; 7:16–23.
4. Mitchell WM, Forti RL, Vogler LB, Lawton AR, Gregg CR. Spontaneous and interferon resistant natural killer cell anergy in AIDS. AIDS Res 1983; 1:221–229.
5. 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+CD56+ cells and expansion of a population of CD16dimCD56− cells with low lytic activity. J Acquir Immune Defic Syndr Hum Retrovirol 1995; 10:331–340.
6. 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 USA 2003; 100:15011–15016.
7. 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.
8. Mavilio D, Lombardo G, Benjamin J, Kim D, Follman D, Marcenaro E, et al. Characterization of CD56−/CD16+ natural killer (NK) cells: a highly dysfunctional NK subset expanded in HIV-infected viremic individuals. Proc Natl Acad Sci USA 2005; 102:2886–2891.
9. Alter G, Malenfant JM, Altfeld M. CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods 2004; 294:15–22.
10. Emu B, Sinclair E, Favre D, Moretto WJ, Hsue P, Hoh R, et al. Phenotypic, functional, and kinetic parameters associated with apparent T-cell control of human immunodeficiency virus replication in individuals with and without antiretroviral treatment. J Virol 2005; 79:14169–14178.
11. Hunt PW, Martin JN, Sinclair E, Bredt B, Hagos E, Lampiris H, Deeks SG. T cell activation is associated with lower CD4+ T cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy. J Infect Dis 2003; 187:1534–1543.
12. Gaddy J, Broxmeyer HE. Cord blood CD16+56− cells with low lytic activity are possible precursors of mature natural killer cells. Cell Immunol 1997; 180:132–142.
13. Fehniger TA, Cooper MA, Nuovo GJ, Cella M, Facchetti F, Colonna M, Caligiuri MA. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity. Blood 2003; 101:3052–3057.
14. Hunt PW, Deeks SG, Bangsberg DR, Moss A, Sinclair E, Liegler T, et al. The independent effect of drug resistance on T cell activation in HIV infection. AIDS 2006; 20:691–699.
15. Bonaparte MI, Barker E. Inability of natural killer cells to destroy autologous HIV-infected T lymphocytes. Aids 2003; 17:487–494.
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