Although HAART induces immune reconstitution in most patients, up to 30% of HIV-infected patients receiving long-term HAART show no marked increase in their CD4+ T-cell count, despite complete suppression of their plasma HIV load. These patients are referred to as discordant or immunological nonresponders (InRs) [1–3]. Their impaired immunological recovery has been associated with variables including older age, therapeutic failure, poor adherence, genetic and virological characteristics, hepatitis co-infection, and low nadir CD4+ T-cell counts. This discordant response is often accompanied by a worse outcome and a faster clinical evolution than seen in full immunological responders [2–6]. In clinical terms, normalization of CD4+ T-cell count and function is a pivotal target in managing HIV infection, as the likelihood of developing non-AIDS comorbidities, including cardiovascular and renal diseases and cancers, is associated with the duration of CD4 cell counts below 500 cells/μl .
Despite intensive investigation, the mechanisms explaining this phenomenon remain largely unknown, although low production of new T cells, due to bone marrow or thymus impairment, and increased CD4+ T-cell destruction, revealed by high sensitivity to cell death ex vivo, contribute to InR status . The unbalanced T-cell homeostasis may also be influenced by undetectable, ongoing viral replication, especially in the tissues. Other suggested causes include microbial translocation from the gastrointestinal lumen and adverse effects of antiretroviral drugs at the mitochondrial level . Alternatively, failure of CD4+ reconstitution could be also explained by a CD4+ T-cell sequestration in lymph nodes, or by the potential dysregulation of inflammatory cytokines such IFN-α and IL-6, as well as IL-7 in T-cell homeostasis [10,11].
The CD4+ T-cell loss observed during the chronic phase of infection initially results from the death of productively infected CD4+ T cells, through the cytopathic effects of viral replication and CD8+ cytotoxic T lymphocyte (CTL)-mediated killing . Several studies have demonstrated, however, that most dying cells are uninfected and thus suggest that the death of uninfected bystander CD4+ T cells plays a major role in CD4+-cell depletion . Numerous studies have strengthened this hypothesis; they imply that several HIV-1 proteins, including gp120, Tat, and Nef, activate several disparate pathways to initiate apoptosis in uninfected cells . An alternative proposal is that the high level of immune activation in HIV-infected individuals causes the death of uninfected CD4+ T cells . We have suggested that NKp44L, the ligand of an activating NK receptor that is only expressed on uninfected bystander CD4+ T cells from HIV-1-infected patients, may play a major role in this phenomenon, rendering these cells sensitive to NK killing [16–19]. This mechanism has been thoroughly investigated at the cellular level and the pathway leading to NKp44L expression at the surface of human CD4+ T cells has been deciphered . Experiments in a model of simian-human immunodeficiency virus (SHIV)-infected macaques indicated that in-vivo inhibition of NKp44L expression correlates with prevention of CD4+-cell depletion and emphasized the ligand's critical role [21,22].
In view of the prognostic and functional role of NKp44L and in an attempt to shed light on the lack of CD4+ T-cell recovery during virologically successful HAART, we have characterized the phenotype and multifunctional features of CD4+ T cells in relation to their NKp44L expression in a well characterized cohort of InRs and compared them with those among full immunological responders and healthy donors. Our results lead us to propose a new phenomenon to explain the lack of CD4+ cells recovery in InRs linked to the persistence of NKp44L.
Uninfected healthy donors and HIV-infected immunological responders and immunological nonresponders
Table 1 summarizes the characteristics of the HIV-infected patients included in this study: 53 InRs and 82 immunological responders at the Pitié-Salpêtrière Hospital, Paris, France.
All recruited HIV-infected patients had been receiving HAART for at least 2 years and had undetectable viral loads (plasma HIV RNA copies <40 per ml). InRs were defined by a CD4+ cell count that remained below 350 cells/μl during the past 2 years, although the immunological responders had CD4+ cell count of 350 cells/μl or above throughout this period. These patients were negative for other infections. The hospital blood center (Etablissement Français du Sang) provided blood samples from 72 anonymous healthy donors as controls. All works were conducted in accordance with the Declaration of Helsinki. All patients provided written informed consent, and the relevant institutional review board (Pitié-Salpêtrière Hospital, Paris, France) approved the study.
Flow cytometry and monoclonal antibodies
CD4+ T cells were analyzed from the peripheral blood. Fresh cells were first incubated with 2 μg/ml of anti-NKp44L mAb (#7.1)  for 45 min at room temperature, washed in PBS/BSA 0.5%, and then stained with a 1/50 dilution of rat antimouse IgM-PE (Becton Dickinson) for 30 min . Cells were next labeled with an appropriate combination of antibodies: CD4-FITC or CD4-APC, CD127-FITC, HLA-DR-PeCy7, PD1-PeCy7, CCR7-PeCy7, CD38-APC, CD95-APC from Becton Dickinson), CD45RA-Texas Red (Invitrogen), and CD3-FITC or CD3-ECD (Beckman Coulter). After staining, FACS Lysing Solution (Becton Dickinson) was used to lyse erythrocytes. Acquisition was performed on a Gallios flow cytometer (Beckman Coulter) and analyzed with Kaluza Software.
Detection of early apoptotic NKp44L+CD4+ T cells
NKp44L-PE, CD3-ECD, and CD4-APC mAbs were used to stain 5 × 105 PBMCs that were then incubated with annexin V-FITC (Becton Dickinson) for 15 min, according to the manufacturer's recommendations. Cells were analyzed by flow cytometry in the CD3+CD4+ lymphocyte gate.
Ki67 evaluation in NKp44L+CD4+ T cells
NKp44L-PE, CD3-ECD, and CD4-APC mAbs were used to stain 5 × 105 PBMCs that were then fixed and permeabilized with the FoxP3 kit (e-biosciences). After washing, cells were stained for Ki67-PECy7 (Becton Dickinson) mAb and washed in PBS before flow cytometry on the CD3+CD4+ lymphocyte gate.
Degranulation of NK cells was performed according to a method previously described . Briefly, freshly isolated nonactivated PBMCs (5 × 105) were resuspended in the presence of anti-CD107a-FITC mAb (H4A3; Beckton Dickinson). After 1 h of incubation, monensin (Sigma–Aldrich) was added at 6 μg/ml for an additional 3 h of incubation before staining with CD3, CD56 and NKp44 mAbs, followed by flow cytometry analysis on the CD3−CD56+ NK-cells gate.
Intracellular cytokine staining and multifunctionality of NKp44L+CD4+ T cells
Freshly isolated PBMCs (5 × 105) were or were not stimulated with 4 μg/ml of phytohemagglutinin (PHA-P) (Oxoid) or 6 μg/ml of anti-CD3 (UCHT-1) and 6 μg/ml of CD28 (CD28.2) (Beckman Coulter) antibodies for 5 h at 37°C in the presence of Golgi Stop and Golgi Plug solutions (Becton Dickinson). Cells were then stained for cell-surface markers (NKp44L, CD3, and CD4), fixed (BD Cell Fix, Becton Dickinson), permeabilized with Cytofix/Cytoperm reagent (Becton Dickinson), and stained for intracellular IL-2 (PE-Cy7; MQ1–17H12; Becton Dickinson), IFN-γ (Alexa-Fluor-700; B27; Becton Dickinson), and TNF-α (APC; Mab11; E-Biosciences) expression. Data were analyzed with Flow Jo version 9 (TreeStar, Ashland, Oregon, USA), and the rate of CD4+ T cells positive for 0, 1, 2, or 3 functions was defined with the software's Boolean gate algorithm. Pestle software was used to remove the background. Pie charts generated by Spice software (NIAI freeware) present the frequency of CD4+ T cells positive for 0, 1, 2, or 3 responses (to IL-2, IFN-γ, and TNF-α), while arcs depict the frequency of cells specifically positive for each of these cytokines, as previously described .
All statistical analyses were performed with Prism 5 software (GraphPad, San Diego, California, USA). Nonparametric Wilcoxon and Mann–Whitney tests were performed to compare paired data and two independent groups, respectively. The Kruskal–Wallis test with the Dunn post-test for P-value calculation was used for multiple comparisons of independent groups. P-values < 0.05 were considered significant. *P < 0.05; **P < 0.01; ***P < 0.001.
The study included 135 HIV-infected patients with undetectable plasma viral levels (<40 copies/ml): 53 InRs and 82 immunological responders (Table 1). These groups were also compared with the group of 72 healthy donors. The two groups of HIV-infected individuals did not differ significantly for sex, age, or history of AIDS-defining conditions (Table 1). In contrast, at analysis, they differed markedly for their CD4/CD8 ratios and for both the proportion of CD4+ cells and the absolute CD4+ T-cell count. Their median CD4+ T-cell counts differed significantly (P < 0.0001): 253 ± 55 cells/μl for the InRs and 547 ± 122 cells/μl for the immunological responders (Fig. 1a). A significant difference was also observed in their frequency of CD4+ T cells (P < 0.0001) (Fig. 1a).
NKp44L expression disrupted CD4+ T-cell maturation in immunological nonresponders
We have previously shown that NKp44L is expressed on CD4+ T cells during HIV-1 infection and is correlated with their progressive depletion . In line with these data, Fig. 1b shows that NKp44L was expressed in InRs significantly more often (33 ± 23%), than in immunological responders (P < 0.0001) or healthy control individuals (P < 0.0001), both with levels close to background (Fig. 1b and c). Consistently, NKp44L is mainly expressed in CD4+ T cells with CD4 cell count below 350 cells/μl (Fig. 1d). We also assessed the frequency of CD3−CD56+ NK cells and observed similar levels in each subset of individuals (data not shown). However, NK cells from some InRs harbored NKp44, the specific NK cytotoxic receptor of NKp44L, which is only observed on activated cells . Thus, NKp44 was present at the surface of a significant fraction of NK cells from InRs, but not on NK cells from immunological responders or healthy donors (Fig. 1e and f). More importantly, we next assessed the degranulation capacities of NK cells in the whole PBMC, and shown that the frequency of CD107a is significantly higher in InRs, compared with healthy control and immunological responders, in accordance to the frequency of NKp44 expression on NK cells (Fig. 1g and h). Altogether, these data are in line with the sensitivity of NKp44L+CD4+ T cells for activated NKp44+ NK killing .
To characterize CD4+ T cells expressing NKp44L more fully in InRs, we first assessed peripheral CD4+ T-cell maturation positive or negative for NKp44L. Figure 2a shows that the frequency of CD4+ naive CD45RA+CCR7+ T cells (CD4+ TN) was significantly lower, and the frequency of CD4+ effector memory CD45RA−CCR7− T cells (CD4+ TEM) higher in NKp44L+, compared with NKp44L negative cells, Thus, CD4+ TN accounted for 12 ± 9% of NKp44L+ cells and 26 ± 13% of the negative subset (P < 0.001) in the InRs, whereas CD4+ TEM accounted for 45 ± 15% and 34 ± 16%, respectively (P < 0.05) (Fig. 2a). Note that in the InR group, these two subsets (NKp44L positive and negative) did not differ for the frequency of CD45RA−CCR7+ central memory CD4+ T cells (CD4+ TCM).
In line with these data, CD38, which is constitutively expressed on phenotypically naive CD4+ T-cells, offsetting its role as an activation marker in the CD4 subset [26,27], was significantly lower in NKp44L+CD4+ T cells (28 ± 14%), than in their NKp44L− counterparts (46 ± 17%; P = 0.0025) (Fig. 2b). Importantly, we found no associations between NKp44L expression and any of our measures of CD4+ T-cell activation, defined by co-expression of CD38 and HLA-DR, or HLA-DR alone (Fig. 2c and data not shown). In addition, the frequency of CD127+ cells is similar related to NKp44L expression (data not shown). Overall, these data indicate that the disruption of T-cell subset maturation that is observed in InRs appears to be associated with NKp44L expression, but independent of the IL-7 pathway, despite the control of viral replication.
NKp44L expression is associated with CD4-cell proliferation and apoptosis in immunological nonresponders
We next assessed the proliferative capacities of CD4+ T cells by analyzing expression of the intracellular proliferation-associated antigen Ki67. As expected, the proportion of Ki67+CD4+ T cells was significantly higher in CD4+ T cells expressing NKp44L (39 ± 20%) compared with the negative counterpart (5 ± 4%; P < 0.0001) (Fig. 3a). In contrast, the level of senescent CD4+ T cells expressing programmed-death 1 (PD-1) did not differ substantially in CD4+ T cells according to their expression of NKp44L (data not shown).
Some studies report that HIV-1-induced CD4+ T-cell depletion is mediated by Fas-FasL interactions. Thus, high levels of Fas (CD95) expression found in CD4+ T cells before HAART have been profoundly reduced after treatment . Importantly, we observed significantly higher expression of Fas in NKp44L+ CD4+ T cells (92 ± 6%), than in negative cells (69 ± 16%; P = 0.0004) (Fig. 3b). Consistent with these data, annexin-V staining revealed a significant increase in apoptosis of NKp44L+ T cells (90 ± 8%), but not of NKp44L− cells (5 ± 3%; P < 0.0001) (Fig. 3c).
NKp44L expression was associated with uncontrolled cytokine release
To gain deeper insight into the functional capacities of CD4+ T cells, intracellular cytokine profiles were assessed both before and after PHA or anti-CD3/CD28 stimulation. Specifically, we investigated the expression of IL-2, IFN-γ, and TNF-α in CD4+ T cells that did and did not express NKp44L. Figure 4a shows that intracellular cytokine expression was significantly higher in NKp44L+ cells, compared with NKp44L− cells. Interestingly enough, in the absence of stimulation, cytokine production remained close to the background level in NKp44L− cells but was sharply higher in NKp44L+ cells (Fig. 4a). After PHA stimulation, a high proportion of NKp44L+ cells, compared with NKp44L− subset, expressed IL-2 (69 ± 15% versus 9 ± 12%, respectively; P < 0.0001), IFN-γ (63 ± 14% versus 10 ± 11%; P < 0.0001), and TNF-α (64 ± 16% versus 10 ± 11%; P < 0.0001). Similar results were obtained after anti-CD3/CD28 antibodies stimulation (Fig. 4a).
We next determined the presence of CD4+ T cells expressing IL-2, IFN-γ, TNF-α or various combinations thereof by a multifunctional assay. Figure 4b shows that cytokine production was observed mainly in NKp44L+CD4+ T cells, with a greater tendency towards multifunctionality; most positive cells expressed some combinations of these cytokines. After stimulation by anti-CD3/CD28 antibodies or even later by PHA, a higher frequency of NKp44L+CD4+ cells expressed all three cytokines, compared with NKp44L− cells that were multifunctional only after PHA stimulation and then only slightly (Fig. 4b).
Up to 30% of HIV-infected patients show an incomplete response to HAART, and become InRs. In addition to the standard characterization based on clinical parameters reported in the Table 1, assessment of a comprehensive panel of immune markers on whole CD4+ T cells (Supplemental Fig. 1, http://links.lww.com/QAD/A347), very consistent with previous reports [2,6,29], confirmed the categorization of the patients in immunological responders and InR groups
This article suggested that InR status is associated with an expansion of highly differentiated, multifunctional CD4+ T cells expressing NKp44L, a cellular ligand for the activating NKp44 NK receptor . Expression of this ligand is specifically induced by a highly conserved motif of the gp41 envelope protein of HIV-1 on CD4+ T cells from HIV-infected patients . It might be mediated by low-level ongoing HIV replication and the subsequent presence of HIV antigens, including gp41, despite HAART, especially in tissues and lymphoid organs, as indicated both by theoretical considerations, and molecular analyses: detection of HIV mRNA in lymphoid tissue of patients on HAART with plasma viral loads less than 50 copies/ml [30–32], and of episomal HIV-1 infection intermediates in patients with undetectable plasma HIV RNA levels during HAART . Notably, longitudinal measures of NKp44L expression before and after HAART clearly showed an inverse correlation between the CD4 cell count elevation and the progressive decrease of NKp44L expression after HAART . Alternatively, an increase of NKp44L expression was associated with the CD4 cell count decrease in macaques infected with the SHIV162P3 .
The phenotype of NKp44L+ cells, combined with naive T-cell loss, is likely to reflect accelerated transitional proliferation of naive T cells into more differentiated effector memory cells. This bias toward more mature dysfunctional lymphocytes is in agreement with earlier studies reporting skewed CD4+ T-cell maturation in InRs, with an accretion of effector memory cells and a reduction of unprimed-naive T cells . Thus, we set out to determine whether the differentiated maturation profile of NKp44L+ cells is associated with other major characteristics of CD4+ T cells observed in InRs. In fact, we observed that NKp44L+ cells display a heightened proliferative capacity. However, such effect might not result in higher CD4 cell count in InRs, as more than 92% of the NKp44L+CD4+ T cells were Fas+ and at least 90% stained positive for annexin-V, consistently with higher caspase-3 levels among the discordant patients . In addition, the capacity of NKp44L+CD4+ T cells to secrete multiple cytokines (IL-2, IFN-γ, and TNF-α), compared with their NKp44− counterparts is consistent with the onset of a cytokine storm in InR NKp44L+ cells The abnormal cytokines production of NKp44L+ cells may be related to their important proliferating capacity, consistently with Combadière et al. . Interestingly, patients with an immune reconstitution inflammatory syndrome (IRIS), have also a significantly higher fraction of multifunctional cells linked to increased IL-2, IFN-γ, and TNF-α T-cell responses, associated with a massive increase in the frequency of effector memory cells among CD4+ T cells reactive at the time of IRIS . Thus, our functional and phenotypic results support the conclusion that highly differentiated, multifunctional NKp44L+CD4+ T cells are present in large numbers in some situations of uncontrolled falling T cell counts.
HIV infection was also characterized by high levels of immune activation, which could impair immunological recovery in course of HAART [8,36]. Interestingly, however, when evaluating activation pattern, HLA-DR+ and HLA-DR+CD38+ levels did not vary according to the level of NKp44L, despite trend to higher Ki67 expression. Notably, the proportion of CD4+CD38+ T cells is significantly lower in the NKp44L+ subset. Consistently, Scalza-Inguanti et al.  showed that CD4+CD38+ cells exhibit limited proliferation ability and a reduced capacity to produce Th1 cytokines, including IFN-γ, and TNF-α.
In line with our previous in-vitro and in-vivo investigations [12,18], our data on NK degranulation, associating NKp44 expression on NK cells, together with a high percentage of NKp44L on CD4+ T cells, could explain the T-cell depletion observed in such InRs patients. Knowing the sensitivity of NKp44L+CD4+ T cells to NK killing , the persistence of NKp44L expression could indicate why CD4+ T cells in those HIV-infected patients fail to revert to the phenotype found in immunological responders, despite 2 years of HAART. This discordant immune response linked to NKp44L, accompanied by the worse outcome and the faster clinical evolution observed in InRs, requires extensive further investigation. The establishment of a link between abnormalities in CD4+ T-cell differentiation, cell death, and NKp44L expression must encourage us to develop new therapeutic strategies to prevent the appearance of NKp44L and then to stimulate immune recovery in InRs.
We thank all of the clinicians and patients who participated in the study. The authors also thank Thibault Roguet, Anaïs Loubiere (Service de Médecine Interne. Hôpital La Pitié-Salpêtrière, Paris, France), Erell Guillerm, and Sophie Rondeau (Département d’Immunologie, Hôpital La Pitié-Salpêtrière, Paris, France) for providing blood samples to develop the assays. We are grateful to Christophe Parizot (Département d’Immunologie. Hôpital La Pitié-Salpêtrière, Paris, France), Dr Assia Samri, and Dr Laura Papagno (INSERM UMR-S 945, Paris, France) for excellent technical support and helpful discussion.
Author contributions: A.S., C.K., A.S., J.C., P.D. and V.V. conceived and designed the experiments; A.S. and F.B. performed the experiments; A.S., F.B., R.H.T.F., C.K., A.S., J.C., P.D. and V.V. analyzed data; A.G., I.N., R.C., C.K. and A.S. enrolled patients; and A.S. and V.V. wrote the article.
Conflicts of interest
V.V. is a researcher from the Centre National de la Recherche Scientifique (CNRS). J.C. is chief executive officer of InnaVirVax, and R.H.F. is an employee of InnaVirVax.
The authors have no other conflicts of interest to disclose.
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