Programmed death (PD)-1 molecule and its ligand PD-L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus-1-infected individuals

G Rosignoli; C H Lim; M Bower; F Gotch; N Imami

doi:10.1111/j.1365-2249.2009.03960.x

. 2009 Jul;157(1):90–97. doi: 10.1111/j.1365-2249.2009.03960.x

Programmed death (PD)-1 molecule and its ligand PD-L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus-1-infected individuals

G Rosignoli ^*, C H Lim ^*, M Bower ^†, F Gotch ^*, N Imami ^*

PMCID: PMC2710596 PMID: 19659774

Abstract

Human immunodeficiency virus (HIV)-1 causes T cell anergy and affects T cell maturation. Various mechanisms are responsible for impaired anti-HIV-1-specific responses: programmed death (PD)-1 molecule and its ligand PD-L1 are negative regulators of T cell activity and their expression is increased during HIV-1 infection. This study examines correlations between T cell maturation, expression of PD-1 and PD-L1, and the effects of their blockade. Peripheral blood mononuclear cells (PBMC) from 24 HIV-1⁺ and 17 uninfected individuals were phenotyped for PD-1 and PD-L1 expression on CD4⁺ and CD8⁺ T cell subsets. The effect of PD-1 and PD-L1 blockade on proliferation and interferon (IFN)-γ production was tested on eight HIV-1⁺ patients. Naive (CCR7⁺CD45RA⁺) CD8⁺ T cells were reduced in HIV-1 aviraemic (P = 0·0065) and viraemic patients (P = 0·0130); CD8 T effector memory subsets [CCR7⁻CD45RA^–(T_EM)] were increased in HIV-1⁺ aviraemic (P = 0·0122) and viraemic (P = 0·0023) individuals versus controls. PD-1 expression was increased in CD4 naive (P = 0·0496), central memory [CCR7⁺CD45RA^– (T_CM); P = 0·0116], T_EM (P = 0·0037) and CD8 naive T cells (P = 0·0133) of aviraemic HIV-1⁺versus controls. PD-L1 was increased in CD4 T_EMRA (CCR7⁻CD45RA⁺, P = 0·0119), CD8 T_EM (P = 0·0494) and CD8 T_EMRA (P = 0·0282) of aviraemic HIV-1⁺versus controls. PD-1 blockade increased HIV-1-specific proliferative responses in one of eight patients, whereas PD-L1 blockade restored responses in four of eight patients, but did not increase IFN-γ-production. Alteration of T cell subsets, accompanied by increased PD-1 and PD-L1 expression in HIV-1 infection contributes to anergy and impaired anti-HIV-1-specific responses which are not rescued when PD-1 is blocked, in contrast to when PD-L1 is blocked, due possibly to an ability to bind to receptors other than PD-1.

Keywords: anergy, HIV-1, PD-1, PD-L1, T cell subsets

Introduction

T cell anergy and clonal exhaustion are characteristic defects of chronic human immunodeficiency virus (HIV)-1 infection and account for the inability of the immune system to mount an effective response to the virus and ultimately clear it from the organism. Reduced interferon (IFN)-γ production and low proliferative capacity in response to stimulus are signs of this defect, even in patients receiving highly active anti-retroviral therapy (HAART) [1,2]. Various mechanisms are responsible for the defective anti-HIV-1 immune response. Ligation of the T cell receptor (TCR) complex is crucial for T cell activation, and is regulated by co-stimulatory molecules which can provide positive and negative signalling. Positive signals are mediated by molecules such as CD28, which is expressed constitutively on T cells and upon ligation is able to induce T cell proliferation and cytokine production [3,4].

The programmed death 1 (PD-1) molecule is an inducible negative regulator of T cell activity [5]. PD-1 is expressed on lymphocytes and in particular on T and B cells. PD-L1 is one of the ligands for PD-1 and is expressed both on antigen- presenting cells (APC) and T cells. Recent in vitro and in vivo studies, including a mouse model of choriomeningitis, have shown the importance of the PD-1/PD-L1 interaction in chronic viral infection [6–8]. In HIV-1⁺ patients, levels of PD-1 and PD-L1 are increased even when patients are receiving successful HAART and have undetectable viral loads, suggesting that the increase in the expression of these two molecules is linked to HIV-1 related anergy [7,9,10].

CD4⁺ T cells are the main target for HIV-1 infection [11]; their number is reduced progressively and their function impaired during the course of disease [12]. Differentiation/maturation of CD8⁺cytotoxic T lymphocytes (CTL), which are crucial for anti-viral action, is also affected by HIV-1 infection due in part to lack of help from CD4⁺ T cells [13]. The differentiation stages of CD4⁺ and CD8⁺ T cells can be categorized phenotypically according to the expression of CD45RA and CCR7. Four different subsets of T cells have been identified [13–15]: naive T cells (CCR7⁺CD45RA⁺); central memory T cells (T_CM) (CCR7⁺, CD45RA^–); effector memory T cells (T_EM) (CCR7⁻CD45RA^–); and terminally differentiated effector T cells (T_EMRA) (CCR7⁻CD45RA⁺). Naive T cells following priming can differentiate first into effector/effector memory cells and subsequently become T_CM. T_CM are cells able to proliferate and produce cytokines such as interleukin (IL)-2, and because of their ability to replicate and differentiate they are crucial for the maintenance of the memory pool. T_CM are affected particularly during HIV-1 infection [15], especially during the acute stage of disease during which they are rapidly depleted [12], but they can be restored partially by HAART [1,2]. T_EM are less able to divide and produce IL-2 but are characterized by the ability to produce IFN-γ and perforin during viral infection. T_EMRA are maturated further than the T_EM phenotype and are the T cells which are least prone to divide. T_EM and T_EMRA subsets' relative percentages are increased during HIV-1 infection, especially within the HIV-1-specific CD8⁺ T cell compartment [13].

In this study we investigated expression of PD-1 and PD-L1 on different CD4⁺ and CD8⁺ T cell subsets in HIV-1⁺ patients and healthy controls to determine whether PD-1 and PD-L1 expression may correlate with the abnormal distribution of subsets of CD4⁺ and CD8⁺ T cells, and whether expression may be related to HIV-1-induced anergy. We also attempted blockade of PD-1 and PD-L1 to assess whether HIV-1-specific T cell responses would be rescued in vitro.

Materials and methods

Study cohort

Blood samples taken from 17 healthy volunteers and 24 HIV-1⁺patients were used in the phenotypical study. Eighteen patients were on HAART and had plasma HIV-1 RNA below the detection limit (50 copies/ml), with median CD4 T cell counts of 412 cells/µl blood [interquartile range (IQR) 225–771]. The remaining six patients were treated with HAART but had detectable viral loads (median 16826 IQR 97–68831). The median CD4 count for this second group was 231 cells/µl blood (IQR 37–248). For the functional studies, consisting of lymphocyte proliferation assays (LPR) and enzyme-linked immunospot (ELISPOT) assays, peripheral blood mononuclear cells (PBMC) from eight HAART-treated HIV-1⁺ patients were used. All but two patients had undetectable viral load. Detailed patients' characteristics for each group are shown in Table 1. The patients' informed consent and Ethics Committee approval were obtained for the studies described.

Table 1.

Patients' characteristics.

Phenotyping	CD4 (cells/µl blood)	CD8 (cells/µl blood)	VL (copies/ml)
Aviraemic
1	1436	5060	< 50
2	97	416	< 50
3	403	832	< 50
4	811	452	< 50
5	1489	680	< 50
6	1117	990	< 50
7	120	997	< 50
8	565	391	< 50
9	420	741	< 50
10	157	986	< 50
11	643	1493	< 50
12	400	680	< 50
13	217	638	< 50
14	758	823	< 50
15	449	1011	< 50
16	339	745	< 50
17	328	668	< 50
18	228	792	< 50
Median (IQR)	412 (225–771)	768 (661–992)
Viraemic
1	255	2014	37019
2	246	676	108
3	224	524	31667
4	8	286	164267
5	238	691	1984
6	47	457	64
Median (IQR)	231 (37–248)	600 (414–1022)	16826 (97–68831)
Functional studies
1^†	291	1095	333
2^†	889	1221	< 50
3^†	272	953	1063
4^†	763	1377	< 50
5^†	97	416	< 50
6	1489	680	< 50
7	157	986	< 50
8	339	745	< 50
Median (IQR)	315 (186–858)	970 (696–1190)

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^†

Enzyme-linked immunospot assay and lymphocyte proliferative response. IQR: interquartile range; VL: viral load.

Collection and separation of blood

Twenty millilitres of whole blood were collected into lithium heparin tubes (Becton Dickinson, Oxford, UK). Fresh PBMC were separated by density gradient centrifugation using Histopaque-1077 (Sigma, Poole, UK).

Viral load

Plasma viral load was measured using Versant HIV-1 RNA 3·0 branched assay (Siemens Healthcare, Camberley, UK) with a lower detection limit of 50 HIV-1 RNA copies/ml.

Lymphocyte subsets

Whole blood lymphocyte measurement was performed by staining with murine anti-human monoclonal antibodies (mAb) against CD3, CD4 and CD8 (Tetra One, Beckman Coulter, High Wycombe, UK). Analysis was carried out using an FC-500 Beckman Coulter flow cytometer.

PBMC phenotypic analysis

Freshly isolated PBMC (10⁶) were stained for 30 min with murine anti-human mAb: fluorescein isothiocyanate (FITC) PD-1, phycoerythrin (PE)-Cy7 PD-L1, APC CD45RA, peridinin chlorophyll (PerCP) CD3, APC-Cy7 CD8 (Becton Dickinson) and PE CCR7 (R&D Systems, Abingdon, UK). Matched isotype controls were used for each fluorochrome analysed. Multi-colour flow cytometric analysis was performed on an LSRII six-colour flow cytometer (Becton Dickinson) using Diva software (Becton Dickinson) for acquisition and post-acquisition analysis.

Antigens and peptide pools

Peptide pools used for the proliferation assays and IFN-γ ELISPOT assays were a pool of HIV-1 Gag p24 20mer overlapping peptides (NIBSC, Potters Bar, UK), and a pool of 33 HIV-1 major histocompatibility complex (MHC) class I restricted Gag p17 and p24 9mer peptides (Sigma-Genosys Ltd, Cambridge, UK). The peptide pools were used at a final concentration of 5 µg/ml. Phytohaemoagglutinin (PHA) (Sigma) was used as a positive control at a concentration of 5 µg/ml [16,17].

Blocking antibodies

Murine anti-human PD-1 (clone J116) and PD-L1 (clone MIH1) and matched isotype control antibodies (eBiosciences, San Diego, CA, USA) were used for the blocking experiments at a final concentration of 10 µg/ml.

Proliferation assay

Freshly isolated PBMC (10⁵) were cultured for 5 days in 10% AB plasma/RPMI-1640 (Sigma), with either 5 µg/ml Gag 20mer pool, 5 µg/ml Gag 9mer pool, 5 µg/ml PHA (positive control) or 10% AB plasma/RPMI-1640 only (negative control), in round-bottomed microtitre plates (Greiner, Stonehouse, UK). Cultures were performed in the presence of 10 µg/ml of PD-1 or PD-L1 blocking antibody, isotype control or phosphate-buffered saline (PBS), the latter used as negative control (no difference was found between isotype control antibody or PBS; data not shown). After 5 days each well was pulsed with 1 µCi [³H]-methylthymidine [³H]-TdR (Amersham International, Amersham, UK) and after a further 6 h incubation cells were harvested onto glass fibre filtermats (Wallac Oy, Turku, Finland). Proliferation was measured by liquid scintillation spectroscopy using a 1205 Betaplate counter (Wallac Oy). A positive response is defined as a stimulation index score (obtained by dividing the experimental value by the value of the tissue culture media negative control) of ≥ 3 coupled with a Δ count per minute of > 600.

IFN-γ ELISPOT assays

Detection of single cell release of IFN-γ in ELISPOT assays was carried out as recommended by the manufacturer (Mabtech, Nacka Strand, Sweden) and as described previously [16]. All assays were performed in duplicate with freshly isolated PBMC. Polyvinylidene difluoride (PVDF) 96-well plates (Millipore, Livingston, UK) were coated with 100 µl anti-IFN-γ mAb at 10 µg/ml (Mabtech) overnight at 4°C. Cells were stimulated with Gag 20mer, Gag 9mer peptide pools or 10% AB plasma/RPMI-1640 in the presence of 10 µg/ml of PD-1 or PD-L1 blocking antibody, isotype control antibody or PBS (negative controls). Plates were incubated for 20 h at 37°C in 5% CO₂. After the incubation plates were washed, 100 µl biotinylated anti-IFN-γ (Mabtech) was added at 1 µg/ml in PBS to each well, and plates incubated for 2 h at room temperature. After washing, 100 µl streptavidin–alkaline phosphatase (ALP) antibody (Mabtech) was added at 1/1000 dilution in PBS to each well for a final incubation at room temperature for 1 h. Plates were washed, developed and counted. Background negative control values were < 30 spot-forming cells (SFC) per million PBMC. PHA was used as a positive control and values of > 250 SFC/million PBMC were obtained in all assays.

Statistical analysis

Lymphocyte subsets, viral loads and immune response data were not distributed normally. GraphPad5 (GraphPad Software, La Jolla, CA, USA) was used for all statistical calculations. Initial analysis of data was performed using the t-test and further analysis was carried out using the Mann–Whitney U-test. P-values below 0·05 were considered significant.

Results

Distribution of T cell memory subsets

First we investigated the distribution of CD4⁺ and CD8⁺ T cells among the different memory subsets according to the expression of CCR7 and CD45RA (an example of the gating strategy is shown in Fig. 1a).

Fig. 1 — The plots show gating strategy for T cell memory subsets on CD4 and CD8 T cells of a representative human immunodeficiency virus (HIV)-1⁺ aviraemic patient (a). The graphs show combined data for flow cytometric analysis of distribution of naive, central memory T cells (T_CM), effector memory T cells (T_EM) and terminally differentiated effector T cells (T_EMRA) subsets in CD4⁺ (b) and CD8⁺ (c) T cells in healthy donors (▭) compared to HIV-1⁺ aviraemic patients () and HIV-1⁺ viraemic patients (). Box plots on graphs show median (central bar), interquartile range (IQR) (box), 10th and 90th percentile (whiskers) and outliers (black dots).

Inline graphic — The plots show gating strategy for T cell memory subsets on CD4 and CD8 T cells of a representative human immunodeficiency virus (HIV)-1⁺ aviraemic patient (a). The graphs show combined data for flow cytometric analysis of distribution of naive, central memory T cells (T_CM), effector memory T cells (T_EM) and terminally differentiated effector T cells (T_EMRA) subsets in CD4⁺ (b) and CD8⁺ (c) T cells in healthy donors (▭) compared to HIV-1⁺ aviraemic patients () and HIV-1⁺ viraemic patients (). Box plots on graphs show median (central bar), interquartile range (IQR) (box), 10th and 90th percentile (whiskers) and outliers (black dots).

We observed no significant difference in the distribution in any of the CD4⁺ T cell subsets. However, we found a trend to increased percentages of T_EM in both the viraemic and aviraemic HIV-1⁺ groups compared to healthy controls. We also observed a slight reduction in the naive CD4⁺ T cell subsets in HIV-1⁺ patients when compared with data from healthy donors (Fig. 1b). When the CD8⁺ T cell compartment was analysed we found a reduction in the frequency of cells in the naive T cell compartment in aviraemic HIV-1⁺ (P = 0·0065) and viraemic HIV-1⁺ patients (P = 0·0130) compared with healthy controls. We also found an increased percentage of T_EM in the viraemic HIV-1⁺ (P = 0·0023) and in the aviraemic HIV-1⁺ (P = 0·0122) compared to controls (Fig. 1c).

Levels of PD-1 expression on CD4 and CD8 T cell subsets

We measured the expression of PD-1 in the different CD4⁺ and CD8⁺ T cell subsets (examples of PD-1 expression profiles are shown in Fig. 2f). We found an increase in the percentage of CD4⁺ cells expressing PD-1 in the T_CM subset in aviraemic HIV-1⁺ patients compared to healthy controls (P = 0·0116) (Fig. 2a). PD-1-expressing T_CM cells were also increased in the viraemic group, but the difference did not reach statistical significance. Expression of PD-1 was also increased in the CD4⁺ subset of T_EM in aviraemic patients compared to healthy controls (P = 0·0037), and in the CD4⁺naive subset in aviraemic patients (P = 0·0496) compared to controls (Fig. 2a). No differences were observed between the groups when CD4⁺ T_EMRA were analysed. Mean fluorescence intensity (MFI) for PD-1 did not differ between the three different groups, although there was a trend towards increased PD-1 expression in both aviraemic and viraemic patients in different CD4 subsets (Fig. 2b). In the CD8⁺T cell subsets we found a statistically significant increase in the percentages of naive T cells expressing PD-1 in the aviraemic patient groups (P = 0·0133) (Fig. 2c). Again, MFI for PD-1 did not differ between the three different groups of CD8 T cell subsets (Fig. 2d).

Levels of PD-L1 expression on CD4 and CD8 T cell subsets

Next, we measured percentages of T cells positive for the expression of PD-L1 (as shown in the histograms in Fig. 3f). We observed an increase in the percentage of PD-L1-expressing cells in all CD4⁺ T cell subsets, but only the difference on CD4⁺ T_EMRA in aviraemic patients compared to controls reached statistical significance (P = 0·0119) (Fig. 3a). Percentages of PD-L1-expressing cells were not reduced substantially in the viraemic group in all CD4⁺ T cells subsets when compared to controls. MFI values for PD-L1 were not statistically different in the CD4⁺ T cells subsets, although some reduction was observed in the naive subsets in both viraemic and aviraemic patients (Fig. 3b).

Numbers of cells expressing PD-L1 were increased in the CD8⁺ T cell subsets in the aviraemic HIV-1⁺group compared to healthy controls, especially in the T_EM (P = 0·0494) and the T_EMRA subsets (P = 0·0282) (Fig. 3c). A trend towards increased PD-L1-expressing cells was also observed in CD8 T_EMRA of viraemic HIV-1⁺patients. MFI levels of PD-L1 were not statistically different between the three groups in all CD8⁺ T cell subsets; they appeared to be slightly increased in the healthy control group (Fig. 3d).

PD-1 and PD-L1 blocking

We tested the effect of PD-1 and PD-L1 blockade on HIV-1-specific responses in proliferative and IFN-γ production assays using Gag peptides as stimulus (patients' characteristics are illustrated in Table 1). We found that only one of eight patients responded to Gag in proliferative assays while four of five patients produced IFN-γ (Fig. 4a,b). PD-1 blockade increased Gag-induced proliferation in the responsive patient but failed to rescue responses in any of the other eight patients (Fig. 4a). When we used anti-PD-L1 blocking antibody we measured increased Gag-induced proliferation in the responsive patient and also rescued responses in four more patients who had no response to Gag before PD-L1 blockade (Fig. 4b). In five of the patients, IFN-γ ELISPOT was also performed, but there was no change in IFN-γ production after Gag stimulation either in the absence or presence of PD-1 or PD-L1 blocking antibodies (Fig. 4a,b).

Fig. 4 — Graphs showing stimulation index results and interferon-γ production of human immunodeficiency virus-1⁺ patients' peripheral blood mononuclear cell stimulation with peptide pools of Gag 20mers and Gag 9mers. The first set of graphs show results using anti-programmed death (PD-1) antibody in lymphocyte proliferative response (LPR) and enzyme-linked immunospot assays (ELISPOT) (a); the last four graphs show LPR and ELISPOT assays using anti-PD-ligand-1 (b).

Discussion

In this study we dissected the phenotypical changes in differentiation and maturation markers affecting CD4⁺ and CD8⁺ T cells during HIV-1 infection. It has been reported previously that changes in the distribution of T cells in different memory subsets occur during HIV-1 infection, and may affect effective responses negatively against HIV-1. Our results display a phenotypical picture showing increased numbers of CD4⁺ and CD8⁺T_EM cells, which are preterminally differentiated producing cytokines but are less capable of dividing in HIV-1⁺ patients. The increase of T_EM seems to be influenced in particular by the presence of virus for the CD8⁺ T cells. Another important difference observed was the decrease of naive CD8⁺ T cells in HIV-1 patients, due possibly to impaired haematopoiesis and lack of signalling from the CD4⁺ T cells [18–20]. We could not find any substantial difference in the T_CM subset in CD8⁺ and CD4⁺ T cells, which may be explained by the positive effect of HAART in regenerating T cell populations [21,22].

Abnormal distribution between the subsets is accompanied by an increased number of cells expressing PD-1; this phenomenon was particularly evident in the CD4⁺ T cell compartment, especially in the T_EM, T_CM and naive T cell subsets in the aviraemic patient group. This observation may explain the lack of help and stimulation by CD4⁺ T cell to CTL during HIV-1 infection, resulting in an abnormal composition of the CD8⁺ T cell memory subsets, despite successful HAART [13]. Numbers of cells expressing PD-L1 are increased in the HIV-1⁺ aviraemic group's CD4⁺ subsets reinforcing a picture of negative regulation and anergy [8,10]. We found lower numbers of cells expressing PD-L1 in the aviraemic patient group CD4⁺ T cell subset due probably to the fact that, in this group of patients, cells expressing PD-L1 go into apoptosis more rapidly by inducing production of IFN-α, as reported by Boasso et al.[23].

Interestingly, more naive CD8⁺ T cells express PD-1 in HIV-1-infected patients than HIV-1⁻ individuals. Also, as reported by other groups, CD8 T_EM numbers are increased during HIV-1 infection [13] and express more PD-L1 [10,24,25], which is also increased in the CD8 T_EMRA subset.

These results confirm previous findings obtained by other groups using different markers to define CD8 T cell memory subsets [24,25], showing that up-regulation of PD-1 is present not only on the anti-HIV-1-specific CD8 T cell but also in the general CD8 T cell and CD4 T cell populations, despite patients being on successful HAART regimens with suppressed viral load. Previously published results from other groups have also shown the role of PD-1 in CD4 T cell apoptosis during HIV-1 infection [26].

The importance of PD-1 and PD-L1 in affecting T cell functionality, in particular the specific ability to generate responses against HIV-1, is demonstrated by the fact that specific blocking antibodies against the two molecules increased T cell proliferation upon stimulation against Gag.

Blocking PD-1 resulted in increased anti-HIV-1-specific proliferative responses in one of eight patients. It was noted that anti-Gag responses were already present in this patient. A possible explanation for this is the fact that PD-1 expression is normalized by HAART [7].

When PD-L1 was blocked, T cells from four of eight of the HIV-1⁺ patients were able to proliferate against Gag; only one of the four patients had proliferative responses before the blockade. This concurs with results from other groups [7,9]. The higher efficacy of PD-L1 blockade compared to PD-1 blockade might be explained by the fact that PD-L1 expression is less affected by HAART than PD-1 expression [10]. Furthermore, while anti-PD-1 antibody would be expected to block only the PD-1/PD-L1 interaction, blocking PD-L1 could have a broader stimulatory effect by preventing the binding of PD-L1 to other receptors such as the CD28 receptor B7-1 on both lymphocytes and APC [27]. This leads to the conclusion that, together with HAART, which seems able to restore subset composition (especially in the CD4⁺ T cell compartment), novel strategies or therapies are needed in order to reverse anergy and rescue the ability of CD8⁺CTL to respond to HIV-1-specific stimuli. PD-1 and PD-L1 seem to be interesting targets for intervention, bearing in mind that modulation of these molecules will have to be achieved while avoiding the risk of generating autoimmune responses. Longitudinal studies will be required to define more clearly the expression profile of PD-1 and PD-L1 during the course of HIV-1 infection. Further detailed investigations are needed into the interlinked signalling cascade regulating the different co-stimulatory and co-inhibitory molecules and their receptors.

Acknowledgments

We thank Adriano Boasso for helpful discussion and critical review of the manuscript. The authors thank patients and staff at the Chelsea & Westminster Hospital who participated in this study. We are also grateful to the staff of the Clinical Laboratory (Department of Immunology, Imperial College London, Chelsea & Westminster Hospital) for lymphocyte subsets and viral load measurement. This study was supported by funding from AVIP EU Program Grant (no. LSHP-CT-2004-503487). N. I. and F. G. are also funded by the MRC (grant no. G0501957).

Disclosures

The authors have no financial conflict of interest.

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PERMALINK

Programmed death (PD)-1 molecule and its ligand PD-L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus-1-infected individuals

G Rosignoli

C H Lim

M Bower

F Gotch

N Imami

Abstract

Introduction