HLA-G⁺ HIV-1-specific CD8 T cells are associated with HIV-1 immune control

Abstract

Objective(s)

To assess the frequency and function of HIV-1-specific HLA-G⁺ CD8 T cells in HIV-1 controllers and progressors.

Design

We performed an observational cross-sectional cohort analysis in untreated (n=47) and treated (n=17) HIV-1 patients with different rates of disease progression and n=14 healthy individuals.

Methods

We evaluated the frequency, the proportion and the function of total and virus-specific HLA-G⁺ CD8 T cells by tetramer or intracellular cytokine staining, followed by flow cytometric analysis. Cytokine secretion of sorted CD8 T cell subsets was evaluated by Luminex assays.

Results

The proportion and the absolute frequency of HLA-G⁺ HIV-1-specific CD8 T cells were directly associated with CD4 T cell counts and inversely correlated with viral loads, while total or HLA-G-negative HIV-1-specific CD8 T cells were not. In functional assays, HLA-G⁺ CD8 T cells from HIV-1-negative individuals had higher abilities to produce the antiviral CCR5 ligands MIP-1β, MIP-1α and Rantes.

Conclusions

HLA-G⁺ HIV-1-specific CD8 T cells may represent a previously-unrecognized correlate of HIV-1 immune control.

Introduction

A large number of studies suggest that natural HIV-1 disease progression in untreated patients can be modulated by T cell-mediated immune responses [1–3]. In individuals with progressive disease, HIV-1-specific CD8 T cells mostly consist of IFN-γ secreting effector-memory cells, and although these immune responses can exert antiviral immune pressure and influence viral sequence evolution, they are not very effective in suppressing HIV-1 replication [4]. In persons with natural control of HIV-1 infection, HIV-1-specific CD8 T cells have a more polyfunctional profile that includes a higher proportion of IFN-γ/IL-2 co-secreting central-memory CD8 T cells [5]. Cellular immune responses in persons receiving suppressive antiretroviral therapy seem to be enriched for HIV-1-specific CD8 T cells with a stem cell memory phenotype [6].

Surface expression of HLA-G, a non-classical HLA class Ib molecule typically expressed on placental trophoblasts, denotes a subset of CD4 and CD8 T cells with immunoregulatory properties that do not express the Forkhead Box P3 transcription factor [7]. HLA-G-expressing T cells have the ability to suppress T cell proliferation and reduce bystander immune activation, most likely through direct interactions between HLA-G and the inhibitory HLA-G ligand LILRB1 [8, 9]. HLA-G⁺ CD4 T cells are reduced during untreated progressive HIV-1 infection, and are inversely associated with levels of cellular immune activation, suggesting that these cells may have beneficial effects on HIV-1 disease outcome [9]. In the present study, we analyzed the expression of HLA-G⁺ HIV-1-specific CD8 T cells in untreated patients with different stages of HIV-1 disease progression. Our results indicate an increase of HLA-G⁺ HIV-1-specific CD8 T cells in patients with controlled HIV-1 disease, an inverse association between proportions of HLA-G⁺ HIV-1-specific CD8 and viral loads, and an increased ability of HLA-G⁺ CD8 T cells from HIV-1-negative individuals to secrete CCR5-binding chemokines, such as Rantes, MIP-1β and MIP-1α. Together, these results suggest that HLA-G-expressing antigen-specific cytotoxic T cells can represent a previously-unrecognized component of antiviral immune defense.

Material and Methods

Patients

Samples from 27 patients with chronic progressive HIV-1 infection, (median viral loads 39,200 HIV-1 RNA copies/ml and CD4 T cell counts 505 cells/ul), 20 controllers (median viral loads 62.5 RNA copies/ml and CD4 T cell counts 829.5 cells/ul) and 17 ART-treated patients (median viral loads 50 RNA copies/ml and CD4 T cell counts 784.5 cells/ul) were used for this study. 14 HIV-1-negative individuals were also recruited. All subjects gave written informed consent and the study was approved by the Institutional Review Board of Massachusetts General Hospital/Partners Healthcare.

Peptide-MHC class I multimer complexes

MHC class I multimers refolded with epitopic HIV-1 (n=6) or CMV/EBV (n=2) peptides were purchased from ProImmune (Oxford, UK). A list of all class I multimers included in this study is included in Table S1.

Flow cytometry

Cryo-preserved blood mononuclear cells were stained with blue viability dye (Life Technologies, 4°C for 20’), followed by incubation with appropriately titrated peptide-MHC class I multimer complexes at room temperature for 20 min in Ca²⁺-free media as described [10]. Cells were then washed and stained with antibodies against CD3, CD8, CD4, HLA-G at 4°C for 20 min. For intracellular cytokine staining, cells were stimulated overnight with optimal CD8 T cell peptides in presence of brefeldin A. Cells were then stained with blue viability dye (Life Technologies, 4°C for 20’), followed by incubation with appropriately titrated antibodies against CD3, CD4, CD8, HLA-G. After fixation and permeabilization for 20 min at 4°C using a commercial kit (Caltag), cells were stained intracellularly for IFN-γ, TNF-α, MIP-1β and IL-2. Cells were acquired on a Fortessa flow cytometer (Becton Dickinson) and analyzed using FlowJo X (Tree star). Analysis and presentation of cell distributions were performed using Graph Pad prism (version 6) and SPICE version 5.32, downloaded from <http://exon.niaid.nih.gov/spice/>.

Cell sorting

Cryo-preserved blood mononuclear cells were thawed and CD3⁺ T cell isolation was performed using immunomagnetic enrichment (Miltenyi Biotech). T cells were stained with blue viability dye (Life Technologies) at 4°C for 20min. Afterwards, cells were stained with antibodies directed against CD3, CD8, CD4, HLA-G, CD25, CD127. The following T-cell populations were live-sorted, after exclusion of CD25⁺ CD127⁻ CD4⁺ Tregs, using an Aria cell sorting device (Becton Dickinson): CD4⁺CD25^−/lowHLA-G⁻, CD4⁺CD25^−/lowHLA-G⁺, CD8⁺CD25^−/lowHLA-G⁻, CD8⁺CD25^−/lowHLA-G⁺.

Luminex assays

The sorted cell populations were stimulated in 96-well plates with anti-CD3 (plate-bound) and anti-CD28 (soluble) antibodies or were left unstimulated for 4 days. Afterwards, cell supernatants were analyzed by Luminex assays (Millipore) for the following cytokines: Eotaxin, G-CSF, GM-CSF, IFN-α2, IL-10, IL-12p70, IFN-γ, IL-13, IL-17α, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IP10, MCP1, TNF-α, TNF-β, VEGF.

Statistics

Data are plotted as means or medians, as indicated. Statistical significance was tested using Mann-Whitney U tests or paired Wilcoxon tests, as appropriate. Correlations have been tested using Spearman test.

Results

HLA-G is expressed on a small number of bulk CD8 T cells [7], however, the expression of HLA-G on HIV-1-specific CD8 T cells is unknown. To investigate this, we analyzed proportions of HLA-G-positive cells within 36 HIV-1-specific CD8 T cell populations targeting different CTL epitopes (Table S1) and 23 CMV/EBV-specific CD8 T cell populations in cohorts of untreated HIV-1 patients. The frequency of HLA-G⁺ HIV-1-specific cells within total CD8 T cells was higher in persons with spontaneous control of HIV-1 infection (n=19) than in patients with progressive disease (n=17) (Figure 1A). This was also true when the proportion of HLA-G⁺ CD8 T cells within a given population of multimer⁺ HIV-1-specific CD8 T cells was evaluated (Figure S1A). No difference was found between the frequencies or the proportions of CMV/EBV-specific HLA-G-positive CD8 T cells in these two study groups (Figure 1A and figure S1A). Moreover, the frequencies of total or HLA-G-negative HIV-1-specific CD8 T cells were also not significantly different (Figure 1A and S1B). Of interest, the frequencies, the relative proportions and the absolute numbers of HIV-1-specific HLA-G-expressing CD8 T cells were positively associated with total CD4 T cell counts and inversely with viral loads (Figure 1B and S1C). There was no association between total or HLA-G-negative HIV-1-specific CD8 T cells (Figure 1B and S1D) or HLA-G-positive CMV/EBV-specific CD8 T cells and CD4 T cell counts or HIV-1 viral loads (Figure S1E). Interestingly, quantitative differences of HLA-G⁺ HIV-1-specific CD8 T cells between progressors and controllers were most obvious for immune responses restricted by protective HLA class I alleles (HLA-B57/B27), but not detectable for responses restricted by HLA-A2 or –B8 (Figure 1C and S2A); however, this was not true for total or HLA-G-negative HIV-1-specific CD8 T cells (Figure S2B and 1C).

Figure 1. Association between HIV-1-specific HLA-G⁺ CD8 T cells and HIV-1 disease progression.

A. Frequency of HIV-1- or CMV/EBV-specific (multimer-positive) HLA-G⁺ or HLA-G⁻ CD8 T cells within the total CD8 T cell pool in HIV-1-infected patients with untreated progressive disease or in controllers. B. Association between the frequency of HIV-1-specific HLA-G⁺ or HIV-1-specific HLA-G⁻ CD8 T cells within the total CD8 T cell pool, and CD4 T cell counts or HIV-1 viral load. C. Frequency of HIV-1-specific HLA-G⁺ or HLA-G⁻ CD8 T cells restricted by non-protective and protective HLA in indicated HIV-1-infected patients. D. (left panel): Frequency of HLA-G⁺ CD8 T cells in untreated progressors (UP), untreated controllers (UC) and ART-treated HIV-1-infected patients. (middle and right panel): Association between the frequency of HLA-G⁺ CD8 T cells and CD4 T cell counts or HIV-1 viral load.

For comparative purposes, we analyzed the proportions of total HLA-G-positive CD8 T cells in the two study cohorts. HIV-1-infected controller patients had significantly higher frequencies of HLA-G-expressing CD8 T cells than progressors, while frequencies of total and HLA-G-negative CD8 T cells were highest in progressors (Figure 1D and S3A–C). Notably, the frequencies and the absolute counts of HLA-G-expressing CD8 T cells were positively associated with total CD4 T cell counts and inversely with viral loads (Figure 1D and S3D), as opposed to total or HLA-G-negative CD8 T cells that were unrelated to CD4 T cell counts, and positively associated with HIV-1 viral loads (Figure S3E–F). Initiation of antiretroviral therapy did not restore the frequency and number of HLA-G⁺ CD8 T cells in chronic progressive patients (Figure 1D and S3A).

Production of chemokines and cytokines by HLA-G⁺ CD8 T cells was analyzed in 14 HIV-1-negative individuals, using Luminex assays. For these studies, CD8⁺CD25^−/lowHLA-G⁻ (Tc1) and CD8⁺CD25^−/lowHLA-G⁺ (HLA-G⁺ CD8 T cells) T cell populations were sorted after exclusion of classical Tregs (CD25^high/CD127⁻ CD4 T cells). CD4⁺CD25^−/lowHLA-G⁻ (Th1) and CD4⁺CD25^−/lowHLA-G⁺ (HLA-G⁺ CD4 T cells) were sorted as controls. Cells were then stimulated for 4 days with anti-CD3/anti-CD28 antibodies. HLA-G⁺ CD8 T cells produced higher levels of IL-10 and IL-2 than Tc1 cells (Figure 2A). Moreover, HLA-G-positive CD8 T cells produced higher levels of the CC chemokines MIP-1α, MIP-1β and RANTES (Figure 2A), which can act as competitive entry inhibitors for R5-tropic HIV-1 isolates [11]. No difference was observed between the cytokine profiles of Th1 and HLA-G⁺ CD4 T cells (data not shown). To extend these investigations, we analyzed secretion of IFN-γ and IL-2 in HLA-G⁺ and HLA-G⁻ CEF (CMV-, EBV-, Flu-)-specific CD8 T cells from HIV-1-negative donors as well as in HIV-1-specific CD8 T cells from controller patients using intracellular cytokine staining after overnight stimulation with peptides spanning HIV-1 gag (Figure 2 B and C). These data confirmed that virus-specific HLA-G⁺ CD8 T cells produced more IL-2, consistent with a relative phenotypic enrichment for more immature CCR7⁺ T cell subsets (Figure S4), while IFN-γ secretion was higher in virus-specific HLA-G⁻ cells compared to HLA-G⁺ CD8 T cells (Figure 2 B and C).

Figure 2. HLA-G⁺ CD8 T cells produce cytokines involved in HIV-1-infection control.

A. Amount (pg/ml for 10⁵ cells) of IL-10, IL-2, MIP-1α, MIP-1β and RANTES produced by Tc1 CD8 T cells and HLA-G⁺ CD8 T cells quantified by Luminex in 13 HIV-1 negative donors. B. Proportion of IL-2 (left) or IFN-γ (right) producing CEF-specific HLAG⁺ or HLA-G⁻ CD8 T cells in n=6 HIV-1 negative donors (blue dots) and in n=2 HIV-1 infected patients (black dots). C. Proportion of IL-2 (left), or IFN-γ (right) producing HIV-1-specific HLA-G⁺ or HLA-G⁻ CD8 T cells in n=8 HIV-1 infected patients.

Discussion

A large number of studies [1] show that T cell-mediated immune responses can influence HIV-1 disease progression. However, a perplexing aspect in HIV-1 immunobiology is that large numbers of HIV-1-specific CD8 T cells are encountered in almost all untreated HIV-1 infected patients, but do not represent a correlate of HIV-1 immune control [12]. This suggests that not all antigen-specific CD8 T cells contribute to antiviral immune defense, and that specific subpopulations of antigen-specific CD8 T cells may have improved antiviral effector functions. In previous studies, “polyfunctional” central-memory HIV-1-specific CD8 T cells with increased antigen-specific secretion of IL-2, stronger proliferative activities and enhanced expression of the Th1 master transcription factor T-bet were found to be associated with HIV-1 immune control [13, 14], but it is likely that alternative, as of yet unidentified subsets of antigen-specific T cells are also involved in HIV-1 immune defense. Here, we described a subset of HIV-1-specific CD8 T cells expressing HLA-G that is associated with immune control in untreated HIV-1 patients, and at least in HIV-1-negative individuals, these cells are able to secrete higher levels of the CC chemokines MIP-1α, MIP-β and Rantes, the physiological ligands for CCR5 [11]. These cells may therefore be able to actively participate in antiviral immune defense by competitively blocking CCR5, the viral co-receptor used by the majority of circulating viruses. Future studies will be necessary to determine if enhanced secretion of these chemokines can also be detected in virus-specific HLA-G⁺ CD8 T cells from HIV-1-infected patients, and whether these cells indeed have increased functional abilities to restrict HIV-1 replication steps or kill HIV-1-infected cells through enhanced cytotoxic properties. Given the extreme rarity of HIV-1-specific HLA-G⁺ CD8 T cells, which in many cases include less than 0.01% of all CD8 T cells, such a functional analysis will be technically challenging, and will require large quantities of patient cells. Notably, prior studies suggested that HLA-G positive T cells have elevated expression levels of lymphoid tissue homing markers [15], and for this reason may have higher abilities to penetrate lymph nodes and increase immune surveillance in this anatomical compartment. A closer examination of HLA-G⁺ CD8 T cells in lymphoid tissues, specifically in germinal centers that may serve as hotspots for viral replication [16], therefore also represents a highly informative future research perspective. Finally, it is noteworthy that HLA-G⁺ expressing T cells also have immunosuppressive properties [8], consistent with our observation of higher IL-10 secretion in this cell subset. Although IL-10 has been associated with immune dysfunction and accelerated immune exhaustion in prior studies [17], it is also possible that elevated regulatory properties of HLA-G⁺ CD8 T cells may improve HIV-1 disease progression by inhibition of immune over-activation [9]. An improved understanding of the complexity and heterogeneity of the CD8 T cell-mediated immune responses against HIV-1 may ultimately allow the development of improved immune interventions for HIV-1 treatment and prevention.