Natural killer cells in HIV controller patients express an activated effector phenotype and do not up-regulate NKp44 on IL-2 stimulation

Abstract

Control of HIV replication in elite controller (EC) and long-term nonprogressor (LTNP) patients has been associated with efficient CD8(+)cytotoxic T-lymphocyte function. However, innate immunity may play a role in HIV control. We studied the expression of natural cytotoxicity receptors (NKp46, NKp30, and NKp44) and their induction over a short time frame (2-4 d) on activation of natural killer (NK) cells in 31 HIV controller patients (15 ECs, 16 LTNPs). In EC/LTNP, induction of NKp46 expression was normal but short (2 d), and NKp30 was induced to lower levels vs. healthy donors. Notably, in antiretroviral-treated aviremic progressor patients (TAPPs), no induction of NKp46 or NKp30 expression occurred. More importantly, EC/LTNP failed to induce expression of NKp44, a receptor efficiently induced in activated NK cells in TAPPs. The specific lack of NKp44 expression resulted in sharply decreased capability of killing target cells by NKp44, whereas TAPPs had conserved NKp44-mediated lysis. Importantly, conserved NK cell responses, accompanied by a selective defect in the NKp44-activating pathway, may result in lack of killing of uninfected CD4(+)NKp44Ligand(+) cells when induced by HIVgp41 peptide-S3, representing a relevant mechanism of CD4(+) depletion. In addition, peripheral NK cells from EC/LTNP had increased NKG2D expression, significant HLA-DR up-regulation, and a mature (NKG2A-CD57(+)killer cell Ig-like receptor(+)CD85j(+)) phenotype, with cytolytic function also against immature dendritic cells. Thus, NK cells in EC/LTNP can maintain substantially unchanged functional capabilities, whereas the lack of NKp44 induction may be related to CD4 maintenance, representing a hallmark of these patients.

Fig. 1.

Flow cytometric analysis of peripheral NK cells in HIV-1–positive patients, ECs/LTNPs, and HDs. In the box plots, bars represent range, lines represent median values, and boxes represent 75% and 25% percentiles. Data are representative of all patients and HDs. *P < 0.05, **P < 0.001, Mann–Whitney u test. (A) Activating receptor expression. Proportion of NK cell expressing each receptor. (B) NK cell expression of HLA-DR, NKG2A, CD85j, and CD57.

Fig. 2.

Correlation analysis of NKG2A with HLA-DR and CD85j expression (%) on CD56 ^dimCD16⁺–positive cells (Spearman test).

Fig. 3.

Cytokine-induced in vitro modulation of NK cell receptors. Expression of (Upper) NKp46 and (Lower) NKp30. (Left) Line plots represent the mean expression of given receptors (± SD) at baseline (T0) and after 2 (T2) and 4 (T4) d (Kruskall–Wallis test). Box plots indicate fold change compared with respective baseline values in the proportion of (Center) NK cells expressing given receptors or (Right) NK cell molecule density (MFI; 30 experiments). *P < 0.05, **P < 0.001, Mann–Whitney u test.

Fig. 4.

IL-2–induced expression of NKp44 by purified NK cells in vitro. (Left) Line plot represents the mean percentage of NK cells (± SD) expressing NKp44 before and after 4 d (T4) of in vitro culture with rhIL-2 (Kruskall–Wallis test). (Right) Box plot indicates fold change in the proportion of activated NK cells expressing NKp44 (*P < 0.05, Mann-Whitney u test; 20 experiments).

Fig. 5.

Functional activity of purified NK cells after in vitro activation in the presence of rhIL-2 (200 U/mL). PKH-26/TO-PRO3 flow cytometric cytotoxicity assay. Bars represent variation of percentage of lysis (percentage of TO-PRO3⁺/PKH⁺ cells). Data are representative of 21 experiments. Mann–Whitney u test was used for comparison. Bars indicate percentage of purified NK cell-mediated FcγR⁺P815 cell lysis in the presence of (Left) NKp44-specific mAbs and (Right) NKp46-specific mAbs after (Left) 4 or (Right) 2 d of culture in HD, nonprogressor (EC and LTNP), and progressor patients (mean + SD). Data are representative of 21 experiments. *P < 0.05, Mann-Whitney u test.