CD3ε Expression Defines Functionally Distinct Subsets of Vδ1 T Cells in Patients With Human Immunodeficiency Virus Infection

Human γδ T cells expressing the Vδ1 T cell receptor (TCR) recognize self and microbial antigens and stress-inducible molecules in a major histocompatibility complex-unrestricted manner and are an important source of innate interleukin (IL)-17. Vδ1 T cells are expanded in the circulation and intestines of patients with human immunodeficiency virus (HIV) infection. In this study, we show that patients with HIV have elevated frequencies, but not absolute numbers, of circulating Vδ1 T cells compared to control subjects. This increase was most striking in the patients with Candida albicans co-infection. Using flow cytometry and confocal microscopy, we identify two populations of Vδ1 T cells, based on low and high expression of the ε chain of the CD3 protein complex responsible for transducing TCR-mediated signals (denoted CD3εlo and CD3εhi Vδ1 T cells). Both Vδ1 T cell populations expressed the CD3 ζ-chain, also used for TCR signaling. Using lines of Vδ1 T cells generated from healthy donors, we show that CD3ε can be transiently downregulated by activation but that its expression is restored over time in culture in the presence of exogenous IL-2. Compared to CD3εhi Vδ1 T cells, CD3εlo Vδ1 T cells more frequently expressed terminally differentiated phenotypes and the negative regulator of T cell activation, programmed death-1 (PD-1), but not lymphocyte-activation gene 3, and upon stimulation in vitro, only the CD3εhi subset of Vδ1 T cells, produced IL-17. Thus, while HIV can infect and kill IL-17-producing CD4+ T cells, Vδ1 T cells are another source of IL-17, but many of them exist in a state of exhaustion, mediated either by the induction of PD-1 or by downregulation of CD3ε expression.

Vδ1 T cells are found at higher frequencies in the blood, intestinal mucosa, and bronchoalveolar fluid of patients with human immunodeficiency virus (HIV) compared with healthy subjects (28,29,30,31,32,33). We have examined the frequencies, phenotypes, and functions of circulating Vδ1 T cells in a cohort of untreated and antiretroviral therapy (ART)-treated patients with HIV and healthy control subjects. We find that percentage frequencies, but not absolute numbers of Vδ1 T cell are higher in the untreated patients compared to ART-treated patients and control subjects. We also have identified two subsets of Vδ1 T cells based on low and high levels of expression of the CD3ε polypeptide, denoted CD3ε lo and CD3ε hi Vδ1 T cells. Both were expanded in patients with HIV and, in particular, in the patients with Candida albicans co-infection. Phenotypic and functional analysis of these Vδ1 T cell subsets indicated that the CD3ε lo cells frequently express terminally differentiated (TD) and exhausted phenotypes and are unable to produce IL-17. These results suggest that HIV may induce a state of Vδ1 T cell inactivation.

MaTerials anD MeThODs study Population
Venous blood was obtained from 36 patients with HIV infection (21 males and 15 females) attending the Genitourinary Infectious Diseases Department at St. James's Hospital, Dublin. At the time of blood sample collection, 22 patients were receiving ART and 14 were not. The CD4 + T cell count ranged from 55 to 1,857 (median 529) cells/μl of blood in the treated patients and 261-1,115 (median 578) cell/μl in the untreated patients. The viral load ranged from <50 to 72,796 (median < 50) copies/ml in the treated patients and <50-51,000 (median 578) copies/ml in the untreated patients. Three patients were positive for hepatitis B virus and three were positive for hepatitis C. As controls, blood samples were obtained from 23 healthy age-and gender-matched control subjects. Ethical approval for this study was obtained from the Joint Research Ethics Committee of St. James's Hospital and Tallaght Hospitals, Dublin, and all participants gave written, informed consent. Buffy coat packs from healthy blood donors were kindly provided by the Irish Blood Transfusion Service. Whole blood was used for enumerating T cells, as described below. Peripheral blood mononuclear cells (PBMCs) were prepared by density gradient centrifugation over Lymphoprep (Nycomed Pharma, Oslo, Norway) and used immediately in all procedures.

enumeration of Vδ1 T cells
Absolute numbers of T cells per μl of blood were determined using Trucount tubes (BD Biosciences) according to the manufacturer's protocol. The percentages of CD3 + cells that expressed Vδ1 TCRs, were determined by flow cytometry, as described above, allowing us to calculate the absolute counts of Vδ1 T cells (per μl of blood).

Vδ1 T cell sorting and expansion
Lines of Vδ1 T cells were generated from healthy blood donors as described previously (5). Briefly, PBMC were prepared from buffy coat packs and monocytes were isolated by positive selection using CD14 Microbeads (Miltenyi Biotec, Gladbach Bergische, Germany). Monocytes were allowed to differentiate into immature dendritic cells (DCs) by culturing them for 6 days in the presence of granulocyte-monocyte colony-stimulating factor and IL-4 as described (34). Immature DC were plated at densities of 100,000 cells/ml and stimulated overnight with medium only, with heator ethanol-killed C. albicans (5 × 10 6 cells/ml) (5). C. albicans strain 10231 was obtained from the American Type Culture Collection and cultured for 24 h on malt extract agar. Fungi were cultured for 24 h, isolated, counted, and then inactivated by heating at 96°C for 60 min. Samples were then centrifuged at 5,000 × g for 10 min, the supernatants discarded, and the pellets washed with phosphate buffered saline (PBS). Inactivation was confirmed by plating an aliquot onto malt extract agar and incubating for 7 days to check for growth. Total γδ T cells were enriched from PBMCs using human anti-TCR γ/δ Microbeads (Miltenyi Biotec). γδ-enriched cells (200,000 cells/ml) were cultured in the absence or presence of C. albicans-or curdlan-treated DCs at 2:1 ratios in complete serumfree AIM-V medium (AIM-V containing 0.05 mM l-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, 0.02 M HEPES, 55 µM β-mercaptoethanol, 1× essential amino acids, 1× nonessential amino acids, and 1 mM sodium pyruvate). Co-cultures were challenged with phytohemagglutinin (1 µg/ml; Sigma-Aldrich, Dublin, Ireland) and cultured with rIL-2 (40 U/ml; Miltenyi Biotec), which was added in fresh medium every 2-3 days. Cultures were restimulated every 2 weeks with activated DCs and phytohemagglutinin, which resulted in yields of >10 million Vδ1 T cells by day 28.

confocal Microscopy
Expanded Vδ1 T cells were sorted into cells with high and low surface expression of CD3ε using a MoFlo XDP Cell Sorter (Beckman Coulter). The cell populations were subsequently incubated on poly l-lysine-coated 8-well Lab-Tek glass chamber slides (Nunc; Thermo Fisher Scientific) for 30 min at 37°C. The cells were fixed with an equal volume of 8% paraformaldehyde for 15 min at 37°C, permeabilized with 0.3% triton X-100 in PBS for 5 min at room temperature and then blocked with 3% bovine serum albumin in PBS for 30 min at room temperature. The samples were incubated with a fluorescein isothiocyanate (FITC)-conjugated mouse antihuman CD3ε antibody (clone SK7, BioLegend, 1/50 dilution in 3% BSA/PBS) and incubated overnight at 4°C. After two washes in PBS, the slides were counter-stained with Hoechst 33258 (Molecular Probes) for 30 min at room temperature to visualize the nuclei. The slides were then imaged under 63× oil immersion with a Zeiss laser scanning confocal 510 microscope (Carl Zeiss, Hertfordshire, UK). The mean fluorescence intensity (MFI) of CD3 staining and Hoechst staining in individual cells was quantified using Zen 2009 imaging software (Carl Zeiss). The MFI of Hoechst served as an internal reference control between the different populations.

analysis of intracellular cytokine Production
Interleukin-17 expression by fresh, unexpanded Vδ1 T cells within γδ T cell-enriched PBMCs was examined by flow cytometry after stimulation of the cells for 6 h with medium alone or with 1 ng/ml phorbol myristate acetate (PMA) and 1 µg/ml ionomycin (PMA/I) in the presence of brefeldin A to prevent cytokine release from the cells (5,34).

statistical analysis
Prism GraphPad software (San Diego, CA, USA) was used for data analysis. Cell frequencies and numbers determined by flow cytometry in subject groups and cytokine levels in treatment groups were compared using the Mann-Whitney U test. P values <0.05 were considered significant. Correlations were defined using Pearson's correlation coefficient.

Vδ1 T cell Frequencies but not numbers are higher in Patients With Untreated hiV infection
Peripheral blood mononuclear cells were prepared from blood samples of 36 patients with HIV infection and 23 healthy donors, stained with mAbs specific for CD3ε and the Vδ1 TCR and analyzed by flow cytometry (Figure 1A). Figure 1B shows that the frequencies, as percentages of lymphocytes, of Vδ1 T cells were significantly higher in the HIV patient samples. Absolute counts of Vδ1 T cells were not significantly different between patients and controls ( Figure 1C), suggesting that the percentage increases in Vδ1 T cells are a result of the depletions of CD4 + T cells by HIV. When the patients were divided into untreated (n = 14) and ART-experienced (n = 22) groups, the frequencies of Vδ1 T cells were found to be higher only in the untreated patients ( Figure 1D). Vδ1 T cell numbers did not correlate significantly with total CD4 + T cell counts ( Figure 1E), suggesting that the increases in Vδ1 T cells in patients with HIV do not simply compensate for the depletions of CD4 + T cells. These data confirm and extend previous observations of altered Vδ1 T cell frequencies in patients with HIV.

significant numbers of Vδ1 T cells Do not appear to express cD3ε
A surprising observation made, while determining the frequencies of Vδ1 T cells in patients and control subjects, was that significant numbers of Vδ1 T cells do not appear to express CD3ε. CD3ε-negative Vδ1 T cells were detected in PBMC and in γδ T cell-enriched PBMC from both patients and control subjects using three different anti-CD3ε mAbs (clones MEM-1, SP4, and HIT-3a) after gating out dead cells, doublets and using FMO controls (Figure 2A). This allowed us to subdivide Vδ1 T cells into two groups on the basis of low and high expression of the TCR co-receptor, denoted CD3ε lo and CD3ε hi Vδ1 T cells, respectively. The levels of Vδ1 TCR expression were slightly higher in CD3ε hi compared to CD3ε lo Vδ1 T cells in both HIV patients and control subjects, although these differences did not   reach statistical significance ( Figure 2B). Further flow cytometric analysis revealed that both CD3ε lo and CD3ε hi Vδ1 T cells express the CD3ζ polypeptide ( Figure 2C).

cD3ε lo Vδ1 T cells express Very low levels or no intracellular cD3ε
The low levels of CD3ε expression by some Vδ1 T cells may be due to internalization of the CD3ε chain. To investigate if CD3ε lo Vδ1 T cells express intracellular CD3ε, Vδ1 T cells were purified from two healthy donors and sorted by flow cytometry into cells with high and low surface expression of CD3ε ( Figure 3A).  Figure 4A shows that both subsets of Vδ1 T cells are expanded in the untreated patients, whereas CD3ε hi Vδ1 T cells, only, are expanded in treated patients. There were no significant differences in the frequencies of CD3ε lo and CD3ε hi Vδ1 T cells in patients with HIV. We previously reported that Vδ1 T cells expand and release IL-17 in response to C. albicans, a common co-infection in patients with HIV (5). Figure 4B shows that the frequencies of both subsets of Vδ1 T cells were significantly higher in patients with Candida co-infection (n = 13) compared to patients with no evidence of fungal infection (n = 19), indicating that fungal infection makes a significant contribution to the increased frequencies of Vδ1 T cells reported in patients with HIV infection (28)(29)(30)(31)(32)(33).

cD3ε expression by Vδ1 T cells can Be Modulated by activation
We next investigated if CD3ε expression by Vδ1 T cells is stable or if it can be modulated by activation. CD3ε hi and CD3ε lo Vδ1 T cells were sorted from lines of Vδ1 T cells that were expanded from three donors. Cells were restimulated with PMA/I (Figure 5A) or DC pulsed with heat-killed C. albicans and PHA ( Figure 5B) and   (34,35). Figure 6A shows that significant proportions of total lymphocytes and gated CD3ε hi Vδ1 T cells within γδ T cell-enriched PBMC from patients and controls expressed naïve (CD45RA + CD27 + ), central memory (CD45RA − CD27 + ), effector memory (CD45RA − CD27 − ), and TD (CD45RA + CD27 − ) phenotypes. By contrast, CD3ε lo Vδ1 T cells from control subjects exhibited significantly higher frequencies of TD cells compared to CD3ε hi Vδ1 T cells (Figures 6A,B). A similar increase in TD cells among CD3ε lo Vδ1 T cells was found in the patients with HIV, with 90-100% of these cells being CD45RA + CD27 + in some patients, but this did not reach statistical significance. Interestingly, the proportions of CD3ε hi Vδ1 T cells that expressed TD phenotypes were higher in the HIV patients compared to control subjects. When the HIV-infected patients were divided into untreated (n = 13) and ART-treated (n = 14) subjects, the proportions of CD3ε lo Vδ1 T cells expressing TD phenotypes was only marginally higher than those of CD3ε hi Vδ1 T cells (Figure 6B). These results show that significant proportions of CD3ε lo Vδ1 T cells express TD phenotypes, suggesting that they are exhausted as a result of HIV infection.
cD3ε lo Vδ1 T cells More Frequently express PD-1, but not lag-3 or cD31, Than cD3ε hi Vδ1 T cells

Human immunodeficiency virus can induce the expression of the inhibitory receptors PD-1 and LAG-3 on HIV-specific T cells leading to their inactivation (36-42). Since Vδ1 T cells with TD phenotypes are preserved in patients with HIV infection, we investigated if CD3ε lo and CD3ε hi Vδ1 T cells from five untreated patients with HIV infection and eight control
subjects express PD-1 or LAG-3. We also investigated if these cells express the naïve T cell marker CD31 (43). Figure 7 shows that PD-1 is expressed at higher levels on CD3ε lo Vδ1 T cells compared to CD3ε hi Vδ1 T cells from eight healthy donors. A similar trend, although not statistically significant was found in five untreated HIV patients ( Figure 7B). PD-1 expression by CD3ε lo and CD3ε hi Vδ1 T cells was similar in patients and control subjects. By contrast, neither CD3ε lo nor CD3ε hi Vδ1 T cells from patients or controls expressed LAG-3. CD31 was expressed by variable proportions of CD3ε lo and CD3ε hi Vδ1 T cells and its expression was not altered in patients with HIV (Figure 7).

cD3ε lo Vδ1 T cells exhibit impaired il-17 Production
The increased expression of PD-1 and TD phenotypes of CD3ε lo Vδ1 T cells suggest that these cells are in a state of exhaustion. We and others have shown that Vδ1 T cells are rapid and potent producers of IL-17 (4,5). We investigated if CD3ε lo and CD3ε hi Vδ1 T cells from patients with HIV infection and control subjects differ in their ability to produce IL-17. γδ T cell-enriched PBMC from 13 healthy donors and 11 patients with HIV were stimulated for 6 h with PMA/I or incubated in medium alone and IL-17A expression by gated CD3ε lo and CD3ε hi Vδ1 T cells was examined by flow cytometry (Figure 8A). Figures 8B,C show that PMA/I treatment induced the production of IL-17 by significant numbers of CD3ε hi Vδ1 T cells from both control subjects and HIV patients. However, stimulation of CD3ε lo Vδ1 T cells with PMA/I did not lead to IL-17 production, suggesting that these cells are at least partially inactivated (Figure 8).

DiscUssiOn
Numerous studies have shown that Vδ1 T cells are proportionally expanded in patients with HIV (28)(29)(30)(31)(32)(33). Vδ1 T cells may contribute to immunity against HIV by killing infected CD4 + T cells (21, 25), releasing antiviral cytokines (4,25,27) and chemokines (23). They may also contribute to the immunodeficiency associated with HIV infection, by depleting CD4 + T cells (26). In this study, we have shown that Vδ1 T cells are not expanded in our patients with HIV infection, but their overall percentages are increased, suggesting that these cells are merely preserved in patients with HIV, while other cells are depleted. Since Vδ1 T cells are an important source of innate IL-17 (4,5), it is also possible that their main role in patients with HIV is to stimulate immunity against co-infecting bacteria and fungi (1)(2)(3)(4)(5). Consistent with this hypothesis, we and others have found that Vδ1 T cells expand and produce IL-17 in response to C. albicans and that their frequencies are highest in HIV-positive patients with Candida co-infection (4,5). Vδ1 T cells are also thought to be major producers of IL-17 in patients with colorectal cancer, in whom they have reduced IFN-γ production (44,45). However, very few of these cells from healthy donors and patients with primary immunodeficiencies were reported to produce IL-17 (46), suggesting that IL-17 production by Vδ1 T cells is dependent on environmental factors, such as infection.
The TCR consists of a clonotypic αβ or a γδ glycoprotein heterodimer, generated by somatic recombination of germline gene segments, that recognizes antigens associated with antigenpresenting molecules, such as MHC, MR1, or CD1 (10). The TCR polypeptides associate with the CD3 complex, formed by the CD3 γ, δ (not to be confused with the TCR γ and δ polypeptides), ε and ζ subunits, which are invariable and mediate signal transduction. CD3ε can form heterodimers with CD3γ and CD3δ, while CD3ζ frequently exists as a homodimer, and CD3δε, CD3γε, and CD3ζζ are all capable of transducing activating signals in response to TCR ligation (9, 10). The CD3 γ, δ, ε, and ζ polypeptides all contain ITAMs in their cytoplasmic domains, which are required for intracellular assembly and surface expression of the TCR and signal transduction events that mediate thymocyte maturation and mature αβ T cell activation (47)(48)(49)(50). Humans and mice lacking CD3ε have no αβ or γδ T cells (49,51), indicating an absolute requirement for CD3ε in early T cell development. However, unlike in αβ T cells, γδ TCR rearrangement can occur in the absence of CD3ε (50) and some mature γδ T cells do not express CD3ε (52).
In this study, we have identified two populations of Vδ1 T cells, one of which expresses normal levels of CD3ε and the other which appears to express no or low levels of CD3ε, but normal levels of CD3ζ. CD3ε lo and CD3ε hi Vδ1 T cells were present in PBMC from patients with HIV and in control subjects and in expanded lines of Vδ1 T cells. Using confocal microscopy of sorted CD3ε lo and CD3ε hi Vδ1 T cells, we show that the absence of CD3ε is unlikely to be due to internalization of the polypeptide, since intracellular CD3ε was not detected. To investigate the stability of CD3ε expression, CD3ε lo and CD3ε hi Vδ1 T cells were sorted from lines of Vδ1 T cells and restimulated with C. albicans and cultured in the presence of IL-2. We found that CD3ε hi Vδ1 T cells could downregulate CD3ε and CD3ε lo Vδ1 T cells could upregulate CD3ε expression, suggesting that the expression of this component of CD3 can be modulated by activation and that its downregulation is reversible. CD3ε expression is required for progression of thymocyte maturation from the double positive CD4 + CD8 + stage to the single positive CD4 + or CD8 + stage and for assembly of the pre-TCR (49,50,53,54), but appears to be dispensible in mature T cells, where it may act to amplify weak signals from the TCR (55,56). Thus, it is possible that CD3ε lo Vδ1 T cells have a lower responsiveness to antigenic stimulation than CD3ε hi Vδ1 T cells. Interestingly, Vδ1 TCR expression was slightly lower in CD3ε lo compared to CD3ε hi Vδ1 T cells in HIV patients and control subjects, adding further support to this idea. CD3ε contains endocytosis determinants that may contribute to the up-and downregulation of CD3ε on T cells (57) and recent studies have provided evidence that CD3ε expression can be downregulated by tumor-educated tolerogenic DC (58) and possibly by HIV (59,60). We found that both CD3ε lo and CD3ε hi Vδ1 T cells are expanded in patients with untreated HIV infection compared to control subjects, but especially in patients with C. albicans co-infection. Thus, CD3ε lo Vδ1 T cells accounted for 0.1% of lymphocytes in controls, compared to 0.5% in untreated HIV patients (P = 0.03) and >1% in patients with HIV and Candida infection (P = 0.0001). Likewise, CD3ε hi Vδ1 T cells accounted for 0.2% of controls, compared to 2.3% of untreated HIV patients (P = 0.0001) and >3% of patients with HIV and Candida infection (P = 0.015). Future studies are required to identify the antigenic specificities of the Vδ1 TCR and to ascertain if Vδ1 T cell numbers or the ratios of CD3ε hi to CD3ε lo Vδ1 T cells can be used as a prognostic marker of Candida co-infection.
To determine if CD3ε lo Vδ1 T cells display phenotypic or functional differences from CD3ε hi Vδ1 T cells, PBMC freshly isolated from healthy donors were enriched for γδ T cells and further analyzed by flow cytometry. We found that CD3ε lo Vδ1 T cells more frequently have TD phenotypes and express PD-1, but not LAG-3, compared to CD3ε hi Vδ1 T cells, suggesting that they have previously been activated and exist in a state of inactivation. PD-1 and LAG-3 expression by HIV-specific CD4 + and CD8 + T cells is a feature of HIV infection, is associated with T-cell exhaustion and disease progression, and is thought to promote viral persistence (36)(37)(38)(39)(40)(41)(42). Our finding that Vδ1 T cells, and especially the CD3ε lo subset of Vδ1 T cells, frequently express PD-1 indicates that this induction of exhaustion in HIV infection extends to γδ T cells and suggests that mAb blocking of PD-1 may benefit patients with HIV (61). Previous workers have reported a skewing of Vγ9Vδ2 T cells toward TD in patients with HIV (32,62), which is associated with impaired IFN-γ production (63). We tested if CD3ε lo Vδ1 T cells display properties of exhaustion by testing their ability to produce IL-17, a cardinal function of Vδ1 T cells (4,5). We found that significant proportions of CD3ε hi Vδ1 T cells, but not CD3ε lo Vδ1 T cells, produced IL-17 in response to PMA/I stimulation ex vivo. Therefore, CD3ε lo Vδ1 T cells may represent a population of inactive, TD T cells.
Since IL-17 production is only one of multiple effector activities of Vδ1 T cells, future studies are required to determine if other activities, such as IFN-γ production, are deficient in CD3ε lo Vδ1 T cells. Vδ1 and Vγ9Vδ2 T cells expressing low levels of CD3 and exhibiting impaired responses to stimulation have been reported to accumulate in sites of active Mycobacterium tuberculosis infection (64,65) and Paget et al. (66) reported that murine Vγ6Vδ1 + T cells with low levels of CD3 predominantly produce IFN-γ whereas the same cells with high levels of CD3 produce IL-17. Thus, modulation of CD3ε expression may be a general mechanism for the regulation of γδ T cell activity.
The results of this study indicate that Vδ1 T cells persist in the blood of patients with untreated HIV infection, and especially in patients with Candida co-infection, while other T cells are depleted. Although it is not known if Vδ1 T cells can directly recognize HIV or HIV-infected cells, a recent study has shown that γδ TCR exposure to viruses can promote the expansion of virus-reactive T cells, providing strong evidence that γδ T cells mediate adaptive immune responses to viruses (67). Previous studies have demonstrated that Vδ1 T cells proliferate and release IL-17 in response to C. albicans, by a mechanism that requires IL-23 release from DC (4,5). The preservation of Vδ1 T cells in patients whose IL-17-producing CD4 + T cells may be depleted by HIV, identifies Vδ1 T cells as an alternative potential source of IL-17. However, it appears that significant proportions of Vδ1 T cells in patients with HIV have been driven to a state of inactivation, expressing TD phenotypes and the inhibitory receptor PD-1 and failing to produce IL-17 upon stimulation. Downregulation of CD3ε, a signaling molecule known to augment TCR-mediated responses (55,56), may represent another mechanism by which the effector functions of Vδ1 T cells can be inhibited.

eThics sTaTeMenT
Ethical approval for this study was obtained from the Joint Research Ethics Committee of St. James's Hospital and Tallaght Hospitals, Dublin, and all participants gave written, informed consent. acKnOWleDgMenTs This study was supported by a grant from the Health Research Board of Ireland. JO was supported by a training grant from Irish Aid (Combat Diseases of Poverty Consortium). We thank Dr. Balbino Alcarón for providing the anti-CD3ε mAb (clone SP4). We thank Conleth Feighery, Mark Little, Yasmeen Ghnewa, Vincent O'Reilly, Serena Arduini, Tanya Coulter, and Eilis Dockry for helpful discussions.