Unlike αβ T cells, γδ T cells constitute only a small proportion (1–5%) of the lymphocytes that circulate in the blood and peripheral organs. γδ T cells share many cell-surface proteins and effector capabilities with both αβ T cells and natural killer (NK) cells, including cytokine production and cytotoxicity . In normal peripheral blood, Vδ2 T cells are the most prevalent type (> 80%), and they transiently expand in response to a variety of infectious diseases. They can recognize and kill certain tumour cells, abilities that underscore their integral role in host defence . This subset responds polyclonally to unprocessed, nonpeptidic phosphoantigens in a human leukocyte antigen (HLA)-unrestricted manner. A much smaller, but often sizable pool of γδ T cells, with the Vδ1 T cell receptor, also exists in the peripheral blood. These Vδ1 T cells apparently lack reactivity to specific antigens recognized by Vδ2 T cells .
The overall number of γδ T cells in the peripheral blood during HIV-1 infection is a topic of debate. Some studies have reported increased levels of this cell subset in HIV-infected patients [4,5], whereas others have suggested that their proportion in the peripheral blood is similar in uninfected and HIV-infected individuals [6,7]. All these studies, however, reported significant alterations in the distribution of the γδ subset in the peripheral blood of people with HIV infection, during which the proportion of Vδ2 T cells falls dramatically . In most HIV-infected subjects, loss of Vδ2 T cells also eliminates response to phosphoantigen stimulation . Remarkably, these changes occur before a significant decline in the CD4 T cells and are among the earliest changes in cellular immunity after infection . In contrast, Vδ1 T cells increase in number and frequency during HIV-1 infection, growing to account for 20–30% of CD3 T cells and thus dramatically shifting the Vδ2/Vδ1 ratio. Characterization of Vδ1–Jδ1 junctional diversity has demonstrated the stability of the Vδ1 repertoire over time. These T cells, nonetheless, exhibit a preactivated state suggestive of constant in-vivo stimulation of the Vδ1 T cells throughout HIV infection; it is characterized by CD38 expression and increased expandability in the presence of interleukin-2 (IL-2) . These cells also express high levels of perforin and display cytotoxic activity .
It has previously been shown that a subset of human T cells, characterized by either αβ or γδ T cell receptors, express natural killer receptors for HLA class I molecules, including not only members of the KIR family but also the CD94/NKG2A heterodimers. These receptors, expressed mainly by CD8 and γδ T cells, have been shown to inhibit T cell receptor-mediated T cell functions including cytolytic activity and cytokine production . Expressions of KIR and CD94/NKG2A decrease substantially within the first 6 months of HIV infection, correlated with a high plasma viral load and viral replication. KIR expression on CD8 T cells returns to normal levels after HAART .
To examine whether changes in the expression of activating and inhibitory NK cell receptors on γδ T cells may affect their ability to control HIV infection, the phenotype of Vδ1 and Vδ2 T cells expressing a member of the NKG2 receptor family were compared, and their ability to target cells was tested using HIV-infected and uninfected tumour cells expressing HLA-E as well as HIV-infected CD4 T cells.
Fresh blood samples from 25 HIV-1-infected patients were obtained at Hôpital Pitié-Salpêtrière. The hospital laboratory simultaneously determined the viral load and complete blood count. These subjects had no treatment for at least 6 months before blood collection; mean CD4 cell counts were 477 cells/μl (SD, 275) and plasma viral load ranged from 1000 to 100 000 copies/ml. For control purposes, leukocytes from 10 uninfected donors were obtained by leukapheresis from the hospital blood bank (Hôpital Pitié-Salpêtrière, Paris, France). All participants provided written informed consent.
Flow cytometric analysis
A four-colour fluorescence-activated cell sorting (FACS) analysis was performed on freshly harvested blood cells from HIV-infected patients and healthy donors, as previously described . Isotype-matched immunoglobulin served as the negative control. NK cells were stained with the appropriate cocktail of monoclonal antibodies: anti-CD45 (HI30), anti-CD3 (SK7), anti-CD8 (SK1), anti-CD158a/KIR2DL1 (EB6B), anti-NKB1/KIR3DL1 (Z27.3.7) (all from BD Biosciences, Le Pont-de-Claix, France); anti-CD56 (N901), anti-CD158b/KIR2DL2 (CH-L), anti-CD94/NKG2A (Z199), anti-NKG2D (ON72), anti-NKp30 (Z25), anti-NKp44 (Z231) and anti-NKp46 (BAB281) (all from Beckman-Coulter, Villepinte, France); pan anti-γδ T cell receptor (IMMU510) and anti-Vδ2 (Innv389) (Immunotech, Marseille, France); anti-Vδ1 (TS8.2; Endogen, Montluçon, France); and anti-NKG2C (134591; R&D Systems, Lille, France). The FACS lysing solution kit (BD Bioscience) was used to lyse erythrocytes. After extensive washing, at least 100 000 events were analyzed on FACS Calibur (BD Bioscience). Results were analyzed with CellQuest Pro software (BD Bioscience) in the lymphocyte CD45 cell gate.
Isolation and culture of γδ T cells
Peripheral blood mononuclear cells (PBMC) from healthy and HIV-infected patients were labelled with biotin-coupled antipan γδ T cell receptor antibody (BD Bioscience) and then positively selected with streptavidin microbeads (Miltenyi Biotec, Paris, France). The purified γδ T cells were resuspended at 1 × 106 cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and recombinant human IL-2 (10 U/ml, Roche, Roches-Roussillon, France) for 7 days before use in the cytotoxicity assays. Isopentenyl pyrophosphate (30 μg/ml, Sigma, Lille, France) was used for phosphoantigen-specific stimulation of Vδ2 T cells . Vδ1 T cells were amplified after culture on plastic culture flasks coated with anti-CD3 monclonal antibody at 5 μg/ml (OKT3, Ortho Diagnostic Systems, Roissy, France) . Purity was evaluated by flow cytometry after staining with anti-CD3 (BD Biosciences), anti-Vδ1 (Endogen), and anti-Vδ2 (Immunotech).
Generation of HIV-infected primary CD4 T cells
PBMC were isolated from HIV-uninfected donors by Ficoll–hypaque gradient, and their CD4 T cells purified with anti-CD4 antibodies conjugated to magnetic beads, according to the manufacturer's instructions (Miltenyi). Positive isolated cells were shown to be more than 97% CD4 cells. Following stimulation with phytohaemagglutinin, the CD4 T cells were infected with HIVBRU at a median tissue culture infective dose of 1000, as previously described . Infected cells were cultured for 3 days, and then the HIV-infected cells were separated from uninfected cells according to the method of Ferrari et al. , which is based on the observation that CD4 receptors are downmodulated on HIV-infected cells. Uninfected cells were removed with anti-CD4-conjugated magnetic beads. Purification of HIV-infected CD4 T cells was determined with anti-HIVp24 capsid monclonal antibody (KC57; Beckman Coulter) and found to exceed 90%. After purification, the HIV-infected cells were extensively washed and then immediately used in functional assays.
Chromium release assay
Cytotoxicity was evaluated from purified Vδ1- and Vδ2-bearing T cells in a standard 4 h 51Cr-release assay, as previously described , against HLA class I-deficient human 721.221 cell line and transfected LCL-221-AEH cells, which express the E*0101 allele (generously provided by Dr D. Geraghty, Fred Hutchinson Cancer Research Center, Seattle, Washington State, USA) [17,18], or against purified uninfected and HIVBRU-infected CD4 T cells, as previously described [15,19].
The respective roles of NKG2A and NKG2C were determined by adding anti-NKG2A (Z199; Immunotech), anti-NKG2C (134522; R&D Systems) or IgG2b isotypic control (2735; BD Biosciences) at a final concentration of 20 μg/ml and incubating them with effector cells for 30 min at 37°C, as previously described .
The Vδ1 T cell subset degranulation assay was performed by CD107a detection, according to methods previously described [20,21]. Briefly, PBMC were stimulated on anti-CD3-coated plastic and then cultured in the presence of IL-2 for 10 days. Effector cells, 5 × 106 PBMC, were then added to target cells: 5 × 105 transfected LCL-221-AEH cells, primary uninfected CD4 T cells or HIVBRU-infected CD4 T cells. As negative and positive controls, PBMC were cultured in medium alone or were pretreated with phorbol 12-myristate 13-acetate/ionomycin. Cells were then incubated with anti-CD107a (H4A3; BD Pharmingen, Le Pont de Claix, France). Brefeldin A and monensin (Sigma) were added after 1 h to prevent CD107a recycling. After a further 3 h, cells were harvested and labelled with anti-CD3 (BD Biosciences), anti-Vδ1 (Endogen) and anti-NKG2C (R&D Systems).
The proportions of Vδ1+NKG2C+ T cells that degranulated in response to particular stimuli were estimated after extensive washing. At least 10 000 CD3+Vδ1+ T cells were analyzed by FACS.
Statistical analyses used the Mann–Whitney U test to compare the two groups. StatView 5.01 software (SAS Institute, Cary, North Carolina, USA) was used, and P values < 0.05 were considered significant.
Expression of natural killer receptors on natural killer cells, CD8 T cells and γδ T cells from HIV-infected patients
Peripheral blood NK cells, CD8 T cells and γδ T cells from HIV-infected individuals were analyzed for expression of NK receptors including NKp30, NKp44, and NKp46, KIRs (KIR2DL1/p58.1, KIR2DL2/p58.2 and KIR3DL1/p70) and the NKG2 receptor family (NKG2A, NKG2C, and NKG2D). As shown in Table 1, KIR expression on CD8 T cells and NK cells from HIV-infected patients was slightly higher than in the control cohort, as previously reported , but expression on γδ T cells was similar in both groups. Expression of NCR was observed only on NK cells, never on CD8 T cells or γδ T cells from uninfected or HIV-infected individuals. As previously reported, NKp44 was expressed only on NK cells from HIV-infected patients . The three cell subsets analyzed showed significant modulations in their proportions of inhibitory NKG2A and activating NKG2C receptors: the proportion of NKG2A cells was significantly lower in HIV-infected patients than in healthy donors (Table 1). Concomitantly, NKG2C expression on NK and γδ T cells increased sharply in HIV-infected patients, but remained close to the background on CD8 T cells from patients with and without HIV infection (Table 1).
Switch from inhibitory to activating NKG2 receptors on γδ T cells in HIV infection
Consistently with previous reports, the proportion of γδ T cells in HIV-infected individuals was similar to that in uninfected people (Fig. 1a) . Expression of the NKG2 family receptor was modulated, however, in HIV-infected patients. The percentage of γδ T cells expressing NKG2A was significantly correlated with the circulating CD4 cell count (P < 0.001; Fig. 1b). Concomitantly, NKG2C expression, which was close to the background level on γδ T cells from uninfected donors, was sharply higher on cells from HIV-infected subjects and was significantly and inversely correlated with their CD4 cell counts (P < 0.001; Fig. 1c). NKG2C expression was not, however, correlated with viral load (data not shown).
The Vδ1 and Vδ2 subsets of γδ T cells were distinct in both HIV-infected and uninfected individuals. A large increase in Vδ1 T cells was reported in HIV-infected patients: these cells accounted for an average of 10–15% of the CD3 T cells in patients with AIDS compared with < 1% in uninfected control donors . It was the main γδ subset in the HIV-infected patients, accounting for up to 94% of the total γδ T cells in patients with CD4 counts < 250 cells/μl but less than 30% in uninfected donors (Fig. 2).
Interestingly, Vδ1 and Vδ2 T cells differed markedly in their expression of the NKG2 receptor family. In uninfected patients, NKG2A was principally expressed on Vδ2 T cells: 44.5% (SD, 2.6) of Vδ2 T cells but less than 17.1% (SD, 1.7) of Vδ1 T cells expressed NKG2A. NKG2C, in contrast, was found mainly on Vδ1 T cells, with 23.3% (SD, 2.6) of Vδ1 T cells expressing NKG2C compared with 9.5% (SD, 2.1) of Vδ2 T cells (Fig. 2). In HIV-infected patients, NKG2 receptor expression was modulated only on Vδ1 T cells (Fig. 2a). NKG2A cells decreased significantly in HIV-infected patients (P < 0.0001), whereas NKG2C cells increased progressively (Fig. 2a). CD4 cell counts in HIV-infected patients were strongly correlated with the proportion of Vδ1 T cells expressing NKG2C (P < 0.0001, r 2 = 0.6384; data not shown). By contrast, NKG2A and NKG2C levels remained similar on Vδ2 T cells from people with and without HIV infection (Fig. 2b).
NKG2C-triggered cytotoxicity by Vδ1 T cells from HIV-infected patients against HLA-E target cells
It has previous been reported that Vδ1 T cells induce cytotoxicity in disease situations, including HIV-1 infection [1,6,22]. It is possible, therefore, that the modulation of expression of the NKG2 receptor family on Vδ1 T cells during HIV infection is associated with modulation of their functional capacities. To address this hypothesis, purified Vδ1 T cells were activated by anti-CD3, whereas purified Vδ2 T cells were selectively stimulated with isopentyl pyrophosphate. Cytotoxicity experiments were then performed in the presence of target cells that did or did not express HLA-E, which is the ligand for CD94/NKG2A and NKG2C. As Fig. 3a shows, activated Vδ1 T cells from HIV-infected patients were more highly cytotoxic against HLA-E-expressing 721.221 cells than were the same cells from uninfected individuals. There was, however, no dramatic difference within the group of HIV-infected patients according to their CD4 cell count.
Cytotoxicity against 721.221 target cells that did not express HLA-E was similar in Vδ1 T cells from uninfected and HIV-infected patients. Cytotoxic responses in Vδ2 T cells from both uninfected and HIV-infected persons were also similar, regardless of the target cells (Fig. 3a). Furthermore, if anti-NKG2A was used block the inhibitory effect of NKG2A, Vδ2 T cells treated with anti-NKG2A were more highly cytotoxic against HLA-E-expressing 721.221 cells than were untreated Vδ2 T cells, although the anti-NKG2A treatment had little effect on the Vδ1 T cells (Fig. 3b). In contrast to the Vδ2 T cells, Vδ1 T cells from HIV-infected patients showed strong cytolytic activity, regardless of the presence of anti-NKG2A (Fig. 3b). This finding suggests that Vδ1 T cells, unlike Vδ2 T cells, are insensitive to the anti-NKG2A treatment. Yet, anti-NKG2C treatment resulted in a clear and specific decrease of the lytic potential of Vδ1 T cells toward 721.221 target cells expressing HLA-E, compared with untreated Vδ1 T cells, whereas lytic activities of Vδ2 T cells from either uninfected or HIV-infected persons remained closed to the background, regardless of whether anti-NKG2C was present or not (Fig. 3c). These results indicate that one of the major functions of NKG2C (killing of target cells expressing HLA-E) is mediated by Vδ1 T cells.
To confirm the involvement of NKG2C in Vδ1 T cell cytolysis of HLA-E-positive target cells, the degranulation capacity of Vδ1 T cells that express NKG2C was measured. These cells from HIV-infected patients showed much stronger degranulation in response to HLA-E-positive target cells than those from uninfected donors (Fig. 3d). In HIV-infected patients, 26.8–45.3% of Vδ1 T cells coexpressed NKG2C and CD107a markers, compared with < 0.4% of Vδ1 T cells from uninfected donors. Together, these data demonstrate that, during HIV-1 infection, a high frequency of Vδ1 T cells, which overexpressed NKG2C, provides strong cytolytic capacity against HLA-E-positive target cells.
Vδ1 T cells expressing NKG2C effectively kill HIV-infected CD4 T cells
Finally, the cytolytic activities of activated Vδ1 T cells against HIV-infected CD4 T cells were assessed. As shown in Fig. 4A, autologous activated CD4 T cells in uninfected individuals were not particularly sensitive to either Vδ1 or Vδ2 T cells. Similarly, Vδ2 T cells from HIV-infected patients were essentially unable to kill autologous CD4 T cells. By contrast, Vδ1 T cells from HIV-infected patients were highly lytic, regardless of CD4 cell counts. This phenomenon was effectively blocked in the presence of anti-NKG2C (Fig. 4a).
To confirm the cytotoxic capacities of Vδ1+NKG2C+ T cells against CD4 T cells from HIV-infected persons, uninfected and HIVBRU-infected CD4 T cells were used as target cells. Since CD4 molecules are downmodulated on HIV-infected cells (data not shown) , phytohaemagglutinin-stimulated primary CD4 T cells infected with HIV-1BRU were separated from the uninfected cells. HLA-E expression on uninfected and HIVBRU-infected CD4 T cells, determined by Western blot, revealed that HLA-E expression doubled on HIV-infected compared with uninfected cells (data not shown), as previously described . As shown in Fig. 4b, the lytic activity of Vδ1 T cells from HIV-infected patients toward uninfected CD4 T cells was more effective than that of the same cell population from uninfected patients. In contrast, the cytotoxicity of Vδ1 T cells increased sharply against HIVBRU-infected CD4 T cells, although the lysis of HIVBRU-infected CD4 T cells in the presence of anti-NKG2C fell significantly, to a level similar to that of uninfected CD4 T cells (Fig. 4b). The results obtained with five healthy donors and 10 HIV-positive patients are summarized in Fig. 4c and confirm that Vδ1 T cells from HIV-infected patients and treated with anti-NKG2C were significantly less cytotoxic against autologous CD4 T cells (Fig. 4c upper panel; P = 0.0025) and against CD4 T cells infected by HIVBRU (Fig. 4c lower panel; P = 0.0018). This phenomenon was observed only with Vδ1 T cells from HIV-infected individuals, regardless of the CD4 cell count. However, these data also suggested that Vδ1 T cells are relative efficient at killing autologous uninfected CD4 T cells, regardless of whether NKG2C is present. This suggests that Vδ1 T cells also could eliminate uninfected CD4 T cells by an NKG2C-independent mechanism. Concomitantly, as shown in Fig. 4d, a high percentage of Vδ1+NKG2C+ T cells from HIV-infected patients were highly capable of degranulation in the presence of HIVBRU-infected CD4 T cells. Specifically, 20.9–24.8% of these Vδ1 T cells coexpressed NKG2C and CD107a, compared with < 9.0 and 7.4%, respectively, in the presence of uninfected CD4 T cells or in the absence of target cells. Taken together, these data clearly show that Vδ1 T cells expressing NKG2C are highly cytotoxic against HIV-infected CD4 T cells.
This study clearly demonstrated the polarization of γδ T cells during untreated HIV-1 infection into two major populations defined by their NKG2 receptor family expression and showed the predominance of the Vδ1 T cell subset expressing NKG2C in HIV-infected patients compared with healthy control subjects. It also showed that the NKG2C receptor is functional in Vδ1 T cells from HIV-infected individuals and mediates target cell lysis. This phenomenon raises the possibility that emerging NKG2C expression on Vδ1 T cells may contribute to a decline in the number of CD4 T cells observed in HIV-infected patients with chronic untreated viraemia. The data presented here support the existence of an association between CD4 cell counts and the proportion of Vδ1+NKG2C+ T cells but also show the strong cytolytic capacities of these cells against HIV-infected CD4 T cells, but not against uninfected CD4 T cells. However, cytolytic activity, unlike NKG2C expression, was not strictly correlated with CD4 cell counts. This suggests that the presence of NKG2C on Vδ1 T cells is sufficient to induce a cytolytic response against target cells expressing HLA-E, such as HIV-infected CD4 T cells.
Several other studies provide a comparative phenotypic analysis of NK cell receptors that are shared by NK and T cell subsets in normal and HIV-1 infected individuals. These results show that the pathogenesis of HIV disease involves alterations in different cell subsets [1,6,9,22,24]. In particular, NK cells in HIV-1 infection switch their NKG2 receptor expression from inhibitory to activating .
Vδ1 T cells have an overall surface phenotype that differs from most peripheral blood T cells but is similar to that of T cells found in the intestinal epithelium. Most human intestinal epithelium cells use αβ T cell receptors; however, among the γδ T cells, almost all use Vδ1 receptors . This suggests that the source of expanded Vδ1 T cells during HIV disease might be the intestinal epithelium cells that migrate into peripheral blood as a consequence of HIV-associated intestinal inflammation.
The potent effector functions of Vδ1 T cells during HIV-1 infection present the question of their specificity. It has been clearly demonstrated that Vδ2 T cells recognize phosphated nonpeptidic ligands, but no similar antigens have been identified for the Vδ1 subset. Groh and colleagues  showed, however, that Vδ1 T cells, like NK cells, express mainly NKG2D and can thus lyse tumour cells expressing MICA and MICB, two stress-induced molecules. Tumours positive for MICA/B had significantly higher frequencies of Vδ1 T cells than tumours negative for them. Accumulation of Vδ1 T cells has been also reported in the peripheral blood and gut of patients with coeliac disease, the lungs of patients with sarcoidosis, in cutaneous leprotic lesions, in synovial fluid of patients with rheumatoid arthritis and in cerebrospinal fluid of patients with acute multiple sclerosis [28,29]. Similarly, we confirmed the accumulation of Vδ1 T cells during HIV-1 infection and observed that these cells expressed significant levels of NKG2C, which specifically recognizes HLA-E, an HLA class Ib molecule. HIV-1 has been observed to selectively downregulate class I complex proteins during infection, thereby avoiding detection by cytotoxic T cells ; however, we observed here, consistent with a previous report , that the virus induced significant HLA-E expression. In this context, our data clearly showed that the HIV-infected CD4 T cells were significantly more sensitive than uninfected cells to Vδ1 T cells expressing NKG2C, the ligand of HLA-E. However, our data showed also that the level of cytoxicity was associated more with the quality of the cell response, including expression of the receptor/ligand complex, and with the balance between activating and inhibitory receptors than with the quantitative responses mediated only by the frequency of the activating receptor.
Chronic infections with opportunistic pathogens may also play a role in the polarization of the NK receptor repertoire on γδ T cells during HIV infection. The frequency of NKG2C cells increases in individuals seropositive for cytomegalovirus . Furthermore, other viruses, including hepatitis C virus, and heat shock protein HSP60 contain epitopes that, even though they inhibit NKG2A cells, may push NKG2C cells to expand in a similar HLA-E-dependent manner .
Although blood Vδ1 T cells are normally a rare subset, their expansion in the blood of people with HIV infection and their functional differentiation strongly suggest that this subset could plays a key role in HIV disease. We hypothesis that Vδ1 T cells could play different functions during the evolution of the disease; first in the destruction of HIV-infected CD4 T cells by an NKG2C-dependent mechanism and, second, in the depletion of uninfected CD4 T cells by an NKG2C-independent mechanism. The in-vivo influence of Vδ1 T cells on the immune system in HIV infection remains to be determined.
We thank Yasmine Dubois for skilled technical help.
Sponsorship: This work was supported by grant from the Agence Nationale de Recherche sur le SIDA (ANRS). H. Fauster-Bovendo was supported by doctoral fellowship from the ANRS.
Note: Hugues Fausther-Bovendo and Nadia Wauquier contributed equally to this work.
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