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Keywords:

  • CD8+ T cell;
  • Cellular immunity;
  • Immune activation;
  • Interleukin-10 (IL-10);
  • Monocyte

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Interleukin-10 (IL-10) plays a key role in regulating proinflammatory immune responses to infection but can interfere with pathogen clearance. Although IL-10 is upregulated throughout HIV-1 infection in multiple cell subsets, whether this is a viral immune evasion strategy or an appropriate response to immune activation is unresolved. Analysis of IL-10 production at the single cell level in 51 chronically infected subjects (31 antiretroviral (ART) naïve and 20 ART treated) showed that a subset of CD8+ T cells with a CD25neg FoxP3neg phenotype contributes substantially to IL-10 production in response to HIV-1 gag stimulation. The frequencies of gag-specific IL-10- and IFN-γ-producing T cells in ART-naïve subjects were strongly correlated and the majority of these IL-10+ CD8+ T cells co-produced IFN-γ; however, patients with a predominant IL-10+/IFN-γneg profile showed better control of viraemia. Depletion of HIV-specific CD8+ IL-10+ cells from PBMCs led to upregulation of CD38 on CD14+ monocytes together with increased IL-6 production, in response to gag stimulation. Increased CD38 expression was positively correlated with the frequency of the IL-10+ population and was also induced by exposure of monocytes to HIV-1 in vitro. Production of IL-10 by HIV-specific CD8+ T cells may represent an adaptive regulatory response to monocyte activation during chronic infection.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Interleukin-10 (IL-10) plays a critical role in limiting proinflammatory immune responses that might otherwise cause damage to the host. During infection, the timing and cellular source of IL-10 production are essential to the balance between successful pathogen clearance by innate and adaptive responses and the prevention of immune pathology. Mistimed or excessive IL-10 production can interfere with elimination or control of various bacteria, viruses, and protozoa [1]. For example, in the murine lymphocytic choriomeningitis virus model, blockade of IL-10 signalling resulted in clearance of a chronic viral infection by host and vaccine-induced cell-mediated immune responses [2, 3]. It was noted nearly two decades ago that IL-10 is upregulated from an early stage of HIV-1 infection and this was proposed to underlie Th cell dysfunction [4, 5]. More recent studies reporting enhancement of HIV-specific effector T-cell responses following in vitro depletion of virus-specific IL-10-producing ‘suppressor’ cells or antibody-mediated blockade of IL-10 support this notion [6, 7]. However, IL-10 gene transcription is upregulated in multiple cell types in the peripheral blood during chronic HIV-1 infection [7]. Whether the reported immune suppressive effects are limited to a specific cell subset is unresolved [8]. This is of critical importance for the development of new therapeutic interventions aiming to ameliorate CD8+ and CD4+ T-cell dysfunction in chronic viral infections including HIV-1. An additional consideration is that IL-10 induction in HIV-1 infection may protect the host from excessive immune activation, since diverse pathogens that cause chronic infections drive the expansion of IL-10-producing adaptive or induced T regulatory (Treg) cells in the periphery [9-11]. In support of this notion, rapid induction of strong Treg-cell responses, together with TGF-β and IL-10, was observed in primary SIV infection of African green monkeys, which is typically nonpathogenic, while these responses were delayed in pathogenic SIV infection in macaques [12]. Furthermore, the presence of an IL-10 promoter polymorphism conferring increased cytokine expression was associated with delayed CD4+ T-cell decline in HIV-1 infection [13].

We sought to elucidate the consequences of IL-10 upregulation in HIV-infected individuals by examining HIV-1-driven IL-10 secretion at the single cell level using a highly sensitive but robust assay [14] and by investigating the function of HIV-specific IL-10-producing cells under physiological conditions. Our results show that HIV-specific CD8+ T cells contribute significantly to IL-10 production in the peripheral blood and that this subset modulates monocyte activation.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

HIV-specific CD8+ T cells contribute substantially to IL-10 production in viraemic subjects

Constitutive IL-10 gene transcription was reported to be upregulated in multiple cell subsets among PBMCs in chronically HIV-infected individuals but there is uncertainty as to whether this is universally reflected in increased spontaneous or antigen-driven cytokine production [7]. We therefore analysed the fractions of IL-10-producing cells among circulating CD4+ T cells, CD8+ T cells, CD19+ B cells and CD14+ monocytes ex vivo, after stimulation with either 0.05% DMSO or HIV-1 gag peptides, in three subject groups: patients who were antiretroviral (ART) naïve (n = 31, median viral load – 17 964 copies/mL) or fully suppressed on ART for >1 year (n = 20) and HIV-uninfected healthy controls (n = 5). Study participants’ characteristics are described in Table 1. The gating strategy used to identify IL-10-producing cells is shown in Supporting Information Fig. 1. Constitutive IL-10 release (0.05% DMSO control) was detected in all cell subsets analysed; the proportion of IL-10-producing cells was highest among CD19+ B cells and CD14+ monocytes in all three groups but there were no significant differences among the groups for each cell subset analysed, suggesting that constitutive IL-10 expression was not increased at the protein level in this patient cohort (Supporting Information Fig. 2). By contrast, we observed significant IL-10 secretion in response to HIV-1 gag stimulation, predominantly in CD8+ T cells. These IL-10+ CD8+ T cells were rare but reproducibly detected in ART-naïve viraemic individuals, at a median frequency of 0.01% (range 0–0.13%, tenfold greater than the 0.05% DMSO control). Frequencies among ART-treated and uninfected subjects were <0.001 and 0%, respectively (p < 0.01, Fig. 1A and B). Although the proportion of IL-10+ cells was lower among CD8+ T cells than monocytes, CD8+ T cells were the major contributors to IL-10 production in response to HIV-1 gag, due to the higher absolute numbers of CD8+ T cells in the peripheral blood (Fig. 1C).

Table 1. Characteristics of study participants
 ART naïveaART treated
  1. a

    Values represent median (IQR).

  2. b

    Values at time of sampling.

  3. NRTI: nucleoside reverse transcriptase inhibitor; NNRTI: non-nucleoside reverse transcriptase inhibitor; PI: protease inhibitor.

Number per group3120
Age33 (29–43)45 (39–49)
Males, n (%)19 (61)12 (60)
Time since HIV diagnosis (years)2 (1–4.5)6.5 (4.75–13)
Plasma viral load (copies/mL)b17 964 (4119–45 757)<40 (N/A)
CD4+ cell count (cells/μL)b450 (380–650)410 (312–602)
CDC stage:  
A2914
B24
C02
Duration of ART (years)N/A5 (4–7.5)
ART regimen: 2 NRTI + NNRTI (n) 18
ART regimen: 2 NRTI + PI (n) 2
image

Figure 1. Characterisation of CD8+ T cells with the capacity to produce IL-10 rapidly upon short-term stimulation with HIV-1 gag peptides. (A) Representative flow cytometry plots showing HIV-1 gag-specific IL-10-secreting cells among PBMCs from subjects who were aviraemic (ART-treated), viraemic (ART-naïve) or HIV-negative. A minimum of 9 × 104 PBMCs were enriched for IL-10 expression by magnetic bead selection after cytokine capture. (B) Frequency of HIV-1 gag-specific IL-10+ cells among PBMCs from ART-treated (n = 20), ART-naïve (n = 24, with analysis of CD8+ T cells only in a further seven patients) and HIV-negative control subjects (n = 5). Horizontal lines represent median values. (C) Contribution of CD4+ T cells, CD8+ T cells, B cells and monocytes to total gag-specific IL-10 production in ART-naïve individuals. Data are shown as mean + SD. (B, C) Statistical significance was determined by Kruskal–Wallis test with Dunn's multiple comparison post-test. (D) Representative histograms (left) show FoxP3, CD25 and CXCR3 expression in HIV-1 gag-specific IL-10+ CD8+ T cells; the percentages of HIV-1 gag-specific IL-10+ CD8+ T cells expressing FoxP3, CD25 and CXCR3 among ART-naïve subjects (n = 6–10) are also shown (right). Data are shown as mean + SD. (E) Representative plot showing beta-7 expression in HIV-1 gag-specific IL-10+ CD8+ T cells (left); the frequencies of beta-7+ cells among HIV-1 gag-specific IL-10+ CD8+ T cells (white bar) and IL-10neg CD8+ T cells (gray bar) in ART-naïve subjects are also shown (right, n = 10). Data are shown as mean + SD.

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Phenotypic analysis of HIV-specific IL-10+ CD8+ T cells revealed that the majority were CD25- and FoxP3-negative and a substantial minority expressed CXCR3, a ligand for inflammatory chemokines that promotes migration to sites of inflammation and differentiation towards an effector phenotype [15] (Fig. 1D). We also investigated the expression of the gut-homing integrin alpha-4/beta-7 on HIV-specific IL-10+ CD8+ T cells, since IL-10 expression is upregulated in gut-associated lymphoid tissue (GALT) during acute HIV-1 infection [16]. Beta-7 integrin expression was detected in the majority of HIV-specific IL-10+ CD8+ T cells (mean, SD: 59 ± 18%), indicating that a substantial proportion were capable of homing to GALT, a major site of viral replication (Fig. 1E).

Emergence of IL-10-producing CD8+ T cells linked to HIV-specific IFN-γ production and virus control

As HIV-specific IL-10+ CD8+ T cells lacked natural Treg-cell markers but expressed CXCR3, which is a characteristic of Th1 cells and recently activated cells [17, 18], we hypothesised that their emergence in chronically infected ART-naïve individuals was related to the effector T-cell response to HIV-1. The frequencies of gag-specific IL-10+ CD8+ T cells, as measured by cytokine secretion, and gag-specific IFN-γ+ T cells determined by ELISpot using PBMCs from the same bleed from each subject were strongly correlated (r = 0.74, p < 0.0001) (Fig. 2A). In view of this observation, we investigated whether gag-specific IL-10+ CD8+ T cells co-expressed IFN-γ, a phenotype identified in human CD4+ IL-10+ Tr1 cells with regulatory functions [19]. Dual IL-10/IFN-γ-secreting cells were detected in all ART-naïve individuals tested and outnumbered the IL-10+ IFN-γneg subset in the majority (mean, SD – 54 ± 20% HIV-specific IL-10+ CD8+ T cells; Fig. 2B and C). There were no notable phenotypic differences, in terms of CD25, FoxP3 or CXCR3 expression, between the HIV-specific CD8+ T cells that co-produced IL-10 and IFN-γ and those that produced IL-10 alone (data not shown). However, we observed a significant inverse relationship between the fraction of the latter subset and plasma viral load (r = −0.62, p = 0.018; Fig. 2D). By contrast, the frequency of HIV-specific IL-10+ CD8+ T cells (IFN-γ+ and IFN-γneg combined) did not correlate with viraemia (r = 0.02, p = 0.97). This suggested that shifting of the balance of HIV-specific IL-10-producing CD8+ T cells away from IFN-γ co-production was associated with spontaneous control of HIV-1.

image

Figure 2. Co-production of IFN-γ by HIV-1-specific IL-10+ CD8+ T cells. (A) Relationship between the frequency of HIV-1 gag-specific IL-10+ CD8+ T cells (determined by cytokine secretion assays as in Fig. 1B) and gag-specific IFN-γ+ T cells (determined by ELISpot assays) in 26 ART-naïve subjects. (B) Representative plot showing IFN-γ expression in sorted HIV-1 gag-specific IL-10+ CD8+ T cells. (C) Histogram showing the proportion of HIV-1 gag-specific IL-10+ CD8+ T cells that co-produce IFN-γ+ (gray bar) in 15 ART-naïve subjects. (D) Correlation between plasma viral load and the proportion of gag-specific IL-10+ CD8+ cells that do not express IFN-γ. Correlation was determined by Spearman's rank test.

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HIV-specific IL-10+ CD8+ T cells are distinct from IL-10+ CD8+ T cells induced by other viruses

Next, we investigated whether antigen-specific CD8+ T cells with a similar phenotype could be induced in other chronic viral infections such as CMV and HCV, or whether the IL-10-producing CD8+ T-cell population we identified was unique to HIV-1 infection. As CMV co-infection is highly prevalent in HIV-infected populations, we first studied HIV-positive individuals with detectable IFN-γ responses to CMV. In addition, we selected HCV-mono-infected individuals with responses to HCV antigens for analysis, as HCV-specific IL-10-producing CD8+ T cells have been detected within the liver in chronically infected patients [9]. Responders were identified by either IFN-γ secretion assays (CMV, Fig. 3A) or ELISpot assays (HCV) as described previously [20]. These individuals were then tested for virus-specific IL-10 responses using cytokine secretion assays (Fig. 3B). Using the criterion that a response had to be twofold greater than the unstimulated control to be considered positive, virus-specific IL-10-producing CD8+ T cells were detected in eight of ten subjects defined as CMV IFN-γ responders and six of ten HCV IFN-γ responders, albeit at low frequencies (Fig. 3B and C). Among subjects with detectable virus-specific IL-10+ CD8+ T cells, co-production of IFN-γ was observed in the majority of CMV-specific cells, while only a minority of HCV-specific IL-10+ CD8+ T cells co-produced IFN-γ (Fig. 3D). In contrast to HIV-1, the expression of FoxP3 and CD25 in these CMV- and HCV-specific populations was heterogeneous (Fig. 3E).

image

Figure 3. Phenotype of virus-specific IL-10+ CD8+ T cells in subjects with CMV or chronic HCV infection. Representative plots of (A) CMV-specific IFN-γ+ CD8+ T cells in a CMV-seropositive subject and (B) virus-specific IL-10+ CD8+ T cells in subjects with CMV (HIV-co-infected) or chronic HCV infections (mono-infected). PBMCs were stimulated for 3 h with the relevant viral antigens, after which the frequency of IL-10+ and/or IFN-γ+ cells was determined using the cytokine secretion assay. Antigen-specific CD8+ IL-10+ responses were considered positive if more than double the frequency in the 0.05% DMSO control. (C) Frequencies of CMV-specific and HCV-specific IL-10+ CD8+ T cells from ten CMV/HIV-1 co-infected and ten HCV-infected subjects; frequencies of HIV-1 gag-specific IL-10+ CD8+ T cells (n = 31, as in Fig. 1B) are shown for comparison. (D) Histogram showing the proportion of CMV-specific IL-10+ CD8+ T cells (left) and HCV-specific IL-10+ CD8+ T cells (right) that co-produce IFN-γ (gray bars). (E) Expression of FoxP3 and CD25 expression by CMV- and HCV-specific IL-10+ CD8+ T cells. Data are shown as mean + SD.

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HIV-specific IL-10+ CD8+ T cells modulate CD14+ monocyte activation and IL-6 production in vitro

HIV-1-specific IL-10+ T cells have been defined as immunosuppressive on the basis of the effects of their depletion on other HIV-specific T-cell populations, such as enhancement of cytolytic, proliferative and IL-2-producing capacities in vitro [6, 21]. However, interpretation of these data could be confounded by the method of depletion used, which would have led to removal of spontaneous IL-10-producing cells (monocytes and B cells) in addition to virus-specific T cells (Supporting Information Fig. 1). To address this, we examined the effects of selectively depleting HIV-specific IL-10+ CD8+ T cells on the responses of other T cells and of peripheral blood monocytes following stimulation with HIV-1 gag peptides (see Materials and methods and schema in Fig. 4A). We confirmed that removal of the CD8+ IL-10-producing T-cell population resulted in a decrease in IL-10 accumulating in the supernatant during subsequent culture (Fig. 4B). The depletion of IL-10+ CD8+ cells led to a small but statistically significant increase in the frequency of activated (CD38+ HLA-DR+) CD8+ T cells after subsequent culture (Fig. 4C) but had no effect on the activation of CD4+ T cells, as indicated by expression of CD38 and HLA-DR (Supporting Information Fig. 3), or on the T-cell effector functions, indicated by production of IL-2, IL-4, IFN-γ and TNF-α during an 18-h culture (data not shown). However, levels of IL-6, which is predominantly secreted by innate cells including peripheral blood monocytes in both HIV-infected and -uninfected individuals [22-24], were upregulated by a median 1.4-fold (range 0.6- to 3.4-fold, p = 0.013) (Fig. 4D). Using intracellular cytokine staining, we confirmed that CD14+ monocytes were the predominant source of IL-6 in gag-stimulated PBMCs in ART-naïve individuals; this population accounted for more than 85% of IL-6+ cells in the majority of subjects tested (Fig. 4E). In addition to augmenting IL-6 production, depletion of HIV-1 gag-specific IL-10+ CD8+ T cells led to a modest yet significant upregulation of CD38 in CD14+ monocytes (p = 0.001), and the magnitude of the change in CD38 expression was directly correlated with the magnitude of IL-10+ CD8+ T-cell population that was depleted (r = 0.91, p = 0.0005, Fig. 4C). In contrast to CD8+ T cells, increased CD38 expression in monocytes was not accompanied by a significant change in cell surface HLA-DR expression (data not shown).

image

Figure 4. Depletion of HIV-1 gag-specific IL-10+ CD8+ T-cells increases monocyte activation and IL-6 production. (A) Schematic representation of the method used to remove HIV-1 gag-specific IL-10+ CD8+ T cells from PBMCs, described in the Materials and methods. (B) IL-10 concentrations in supernatants from HIV-1 gag stimulated PBMCs, either containing or depleted of HIV-1 gag-specific IL-10+ CD8+ T cells, after 18 h culture (n = 6). (C) Effect of depletion of gag-specific IL-10+ CD8+ T cells on the expression of CD38 and HLA-DR by CD8+ T cells stimulated with HIV-1 gag peptides (n = 8). (D) Effect of depletion on the expression of CD38 by CD14+ monocytes and the relationship between the frequency of gag-specific IL-10+ CD8+ T cells depleted and the magnitude of change in CD38 expression by monocytes (n = 13). (E) Effect of depleting HIV-1 gag-specific IL-10+ CD8+ T cells on the accumulation of IL-6 in culture supernatants (n = 7). (B–D) Statistical significance of paired analyses was determined by Wilcoxon matched-pairs test and correlation by Spearman's rank test. (F) Representative flow cytometric plots showing IL-6 production by CD14+ monocytes, B cells, CD4+ T cells and CD8+ T cells in PBMCs stimulated for 6 h with HIV-1 gag peptides. The IL-6+ monocyte population is highlighted by the gray box (left). The contribution of each cell type to total IL-6 production in ART-naïve individuals is also shown (right).

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To confirm that HIV-1 infection could modulate CD38 in CD14+ monocytes, we examined the effect of in vitro infection of CD4+ cells (CD8-depleted PBMCs) from healthy donors with HIV-1BaL, a laboratory-adapted CCR5-tropic clade B isolate. CD38 mean fluorescence intensity (MFI) increased not only in CD14+ monocytes that became infected but also in monocytes in the same cultures that remained uninfected (Supporting Information Fig. 4). These results indicate that exposure to HIV-1 is sufficient to cause upregulation of CD38 in peripheral blood monocytes in vitro and, taken together with the observed effects of depleting HIV-specific IL-10+ CD8+ T cells, suggest that the latter could protect monocytes from activation by HIV-1 in vivo.

Finally, we investigated whether the effects of HIV-specific IL-10+ CD8+ T cells on monocyte CD38 expression were IL-10-dependent. Treatment of CD8-depleted PBMCs with an IL-10 receptor (IL-10R) blocking antibody prior to co-culture overnight with CD8+ T cells led to a marginal increase in monocyte CD38 expression, when compared with the effect of depleting HIV-specific IL-10+ CD8+ T cells. This could reflect incomplete receptor blockade on monocytes; alternatively, it could indicate that this population may not mediate its effects solely through IL-10 production (Supporting Information Fig. 5).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

In this study, we have shown that a distinct subpopulation of HIV-specific CD8+ T cells contributes substantially to IL-10 production by PBMCs in chronic uncontrolled HIV-1 infection. The magnitude of this population was positively correlated with the magnitude of the IFN-γ response to the same HIV-1 antigens and the majority of the CD8+ T-cell subset co-produced IL-10 and IFN-γ upon short-term HIV-1 gag stimulation. However, a shift towards lone IL-10 production was associated with better virological control. Together, these observations suggest that a subset of HIV-1 gag-specific IFN-γ-secreting CD8+ T cells may have acquired the capacity to produce IL-10 in response to chronic viral replication, possibly as a protective response to inflammation in the context of ongoing antigenic stimulation. Their virtual absence in patients treated with an effective ART regimen is consistent with this notion, although this remains to be confirmed in a longitudinal study. Furthermore, their lack of a conventional Treg-cell phenotype contrasted with CMV-specific IL-10+ CD8+ T cells that were detected in some co-infected individuals and suggests that these populations have distinct ontogenies.

Co-expression of IL-10 and IFN-γ by tissue-homing virus-specific T cells has been extensively reported in murine viral infection models and in human CD4+ T cells [11, 19, 25-28]. By contrast, human virus-specific CD8+ T-cell populations with dual IL-10-/IFN-γ-secreting capacity appear to be rare: to our knowledge, the only precedent for this is in Epstein-Barr virus (EBV) infected solid organ transplant recipients, in whom CD8+ Treg type 1 cells expressing FoxP3 could be induced in vitro by type-1-polarising DCs [11]. This may reflect challenges in detecting such populations in the peripheral blood or true rarity, possibly due to preferential trafficking to tissues. HIV-specific IL-10+ CD8+ T cells were present at low frequencies in the peripheral blood in our study cohort (median 0.01% in ART-naïve individuals), whereas the dual IL-10-/IFN-γ-secreting CD4+ Tr1-like cells described by Haringer et al. [19] comprised approximately 1% of antigen-experienced (CD45RAneg) CD4+ T cells. The size of this population reflected its composition of many different antigen specificities, whereas the population we identified was specific for a single HIV-1 antigen and its frequency was expressed as a percentage of the entire CD8+ T-cell subset (as opposed to antigen-experienced cells only). Furthermore, the expression of beta-7 integrin and CXCR3 would endow this population with the capacity to home to GALT and other sites of inflammation. This suggests that they could play a role in limiting virus-driven immune activation, as GALT is a major site of HIV-1 replication throughout infection [29]. It should also be noted that the contribution of HIV-specific CD8+ T cells to overall IL-10 production is considerable, despite a recent report finding that CD14+ monocytes were the major source of spontaneous IL-10 production in uncontrolled HIV-1 infection [8], as the data reported by Kwon et al. did not take into account the greater (typically approximately fivefold) absolute numbers of lymphocytes than monocytes in the peripheral blood.

The capacity to secrete IL-10 suggested that HIV-specific CD8+ T cells may have an immunoregulatory role. Conventionally, this is demonstrated by the capacity to inhibit the proliferation or cytokine secretion of other T-cell populations in vitro. However, such assays typically employ non-physiological suppressor/responder ratios. An alternative approach that has been used previously is to deplete the putative regulatory population and examine the effects of its removal on responder cells [30, 31]. In view of the low frequencies of HIV-specific IL-10+ CD8+ T cells, we considered the latter approach to be more physiological. The enhanced proinflammatory responses by monocytes that were revealed by selective depletion of HIV-specific IL-10+ CD8+ T cells suggested that IL-10 production by HIV-specific CD8+ T cells could constitute an adaptive response to virus-driven monocyte activation. The simultaneous upregulation of CD38 and increased IL-6 production is intriguing and may reflect induction of IL-6 in monocytes as a direct result of CD38-mediated signalling, possibly triggered by a viral ligand [32]. Recently, Andrade and colleagues [33] demonstrated that antibody blockade of IL-10 signalling in PBMCs from HIV-infected individuals resulted in increased expression of IL-6 following stimulation with HIV-1 envelope protein peptides. Our data extend these findings by suggesting that a specific population of HIV-specific CD8+ T cells may have the capacity to alter IL-6 expression in this way. IFN-γ has also been reported to enhance CD38 and IL-6 expression by human monocytes [34, 35]. While this appears to be contradictory to our findings, it is unlikely that the effects we observed were mediated by IFN-γ, since the selective depletion of IL-10+ cells removed only small fraction (typically <1%) of the total IFN-γ+ CD8+ T-cell population. Previously, Almeida et al. [36] found that expression of CD38 on monocytes was increased in HIV-infected individuals, and only partially declined after suppression of viral replication following the initiation of ART. When taken together with our data showing that infection of PBMCs with HIV-1 in vitro increased CD38 expression on monocytes, these results suggest that monocyte CD38 expression reflects virus-driven immune activation in HIV-infected individuals. Our findings extend a previously reported observation that monocytes from chronically infected subjects express high levels of innate immune activation markers [37]. We propose that HIV-specific IL-10+ CD8+ T cells control inflammation by modulating the expression of CD38 and IL-6 in monocytes, and may thus influence virological control and HIV-1 pathogenesis. The shift towards lone IL-10 production that we observed in ART-naïve patients with low viral loads supports this hypothesis. However, as our study was cross-sectional, cause and effect cannot be distinguished with certainty, and this needs to be tested in a prospective study.

The lack of a discernible effect of depletion of HIV-specific IL-10+ CD8+ T cells from viraemic individuals on other HIV-specific T cells, other than increased co-expression of CD38 and HLA-DR on CD8+ T cells, was unexpected. This could reflect the short duration of the culture (18 h) and an effect on T-cell function might have become apparent during a longer culture period [8, 21]. Furthermore, since viraemic individuals generally have higher frequencies of CD38/HLA-DR double-positive CD8+ T cells than CD4+ T cells, the former may have a lower threshold for activation [38, 39]. The failure of IL-10R blockade to recapitulate the effects on monocytes of depletion of IL-10-producing CD8+ T cells may also be due to technical limitations in our study, although we cannot rule out the possibility that IL-10R blockade had opposing effects on other cellular targets, such as enhanced effector functions of HIV-specific CD8+ and CD4+ T cells [4, 7, 40].

In summary, our findings highlight the importance of understanding IL-10 regulation at the single cell level before embarking on cytokine modulatory strategies; we caution that manipulation of IL-10 signalling could have potential adverse effects on immune activation in chronic HIV-1 infection that might outweigh any beneficial enhancement of virus-specific effector T-cell responses.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Study subjects

Adults with chronic HIV-1 infection were recruited from clinics in Oxford and London, UK. Blood samples from healthy HIV-uninfected donors were obtained from laboratory volunteers or from blood donors (Oxford University Hospitals Blood Transfusion Service). The study was approved by local ethics committees (the Oxfordshire Research Ethics Committee (Ref. 08/H0607/51) and the Camden and Islington Community Local Research Ethics Committee (Ref. 98/60) and all subjects gave written informed consent. Adults with chronic untreated HCV infection were recruited from clinics in Oxford and London, UK, with approval from the Oxfordshire Research Ethics Committee (Ref. 04.OXA.010). PBMCs were isolated from blood samples by density gradient centrifugation and cryopreserved within 4 h of sampling. Cell viability upon thawing was consistently greater than 90%.

Detection and phenotyping of cytokine-producing cells

IL-10-secreting cells were detected using a bispecific antibody to capture IL-10 in the immediate vicinity of the secreting cell and then enriched by magnetic bead selection according to the manufacturer's instructions (Miltenyi Biotec, Germany). Briefly, cryo-preserved PBMCs were thawed, rested overnight in RPMI supplemented with 10% human AB serum, penicillin/streptomycin and l-glutamine (H10 medium), and stimulated for 3 h at 37°C with a pool of 123 overlapping 15-mer peptides (2.5 μg/mL) based on the HIV-1 clade B consensus gag sequence (Research and Reference Reagent Program, Division of AIDS, NIAID, NIH). In all assays, 0.05% DMSO in H10 medium was used as a negative control (the same concentration of DMSO as used in the gag peptide pool) and a proprietary polyclonal activator Cytostim (Miltenyi Biotec) was used as a positive control. PBMCs were then labelled with a bispecific IL-10 capture antibody (Miltenyi Biotec) for 45 min at 37°C. IL-10-producing cells were enriched by labelling captured cells with a PE-conjugated anti-IL-10 antibody, followed by magnetic separation using anti-PE antibody-coated microbeads. The enriched cell fraction was stained with CD3-allophycocyanin-Cy7, CD4-FITC, CD8-allophycocyanin, CD14-Pacific Blue, CD19-PerCP (BD Biosciences) and LIVE/DEAD® fixable aqua dead cell stain (Invitrogen). In selected experiments, a second bispecific IFN-γ capture antibody was added and enriched IL-10+ cells were stained with the following: IFN-γ-FITC, IL-10-PE (Miltenyi Biotech), CD3-allophycocyanin-Cy7, CD8-PerCP, beta7-PE-Cy5 (BD Biosciences), CXCR3-Pacific blue or FoxP3-Pacific blue, CD25-Alexa Fluor 700 (Biolegend) and LIVE/DEAD® fixable aqua dead cell stain (Invitrogen). To confirm expression of alpha-4/beta-7 integrin, PBMCs from four ART naïve individuals were stained with CD3-allophycocyanin-Cy7, CD4-FITC, CD8-PerCP, beta-7-PE-Cy5 (BD Biosciences), LIVE/DEAD® fixable aqua dead cell stain (Invitrogen) and alpha-4-PE (Biolegend). In all four subjects, ≥95% of CD8+ T cells expressing beta-7 also expressed alpha-4 (data not shown). CMV- and HCV-specific IL-10+ cells were identified using the same assay, and phenotyping was performed using the same antibody panel as that described for HIV-specific IL-10+ cells. PBMCs from CMV- and HCV-infected individuals were stimulated for 3 h with peptides based on the CMV pp65 sequence and peptides spanning the HCV genotype 1 or 3 core, envelope and non-structural regions of the viral proteome, respectively. Cells were acquired on an LSRII flow cytometer and data were analysed using Flow-Jo software version 9.2.

IL-10-producing CD8+ T-cell depletion assay

Removal of IL-10-producing CD8+ T cells was achieved in two steps. First, CD8+ cells were isolated to >90% purity from PBMCs by anti-CD8 multi-sort microbead selection followed by enzymatic removal of the microbeads (Miltenyi Biotec). The CD8+ and CD8neg fractions were stimulated separately with HIV-1 gag peptides for 6 h, after which the CD8neg fraction was maintained at 4°C. The CD8+ fraction was split into two aliquots and IL-10-producing cells were depleted from one aliquot by cytokine capture and magnetic separation, as described in the previous section. The other aliquot was treated identically apart from addition of the IL-10 capture antibody. The CD8+ fractions containing or depleted of IL-10+ cells were each recombined with CD8neg cells (restoring the original ratio of CD8+ to CD8neg PBMCs) and incubated either overnight or for 3 days in H10 medium. In selected experiments, CD8neg PBMCs were incubated with an IL-10R blocking antibody (Biolegend) for 20 min at room temperature prior to co-culture with complete CD8+ T cells. Supernatants were harvested and stored at −20°C for determination of the following cytokines: IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α. Cells were stained with CD3-FITC, CD8-PerCP, CD38-PE, HLA-DR-allophycocyanin, CD14-Pacific blue (BD Biosciences) and LIVE/DEAD® fixable aqua dead cell stain (Invitrogen), and analysed as described earlier. Cytokines in culture supernatants were quantified by Luminex assay (Bio-Rad) according to the manufacturer's protocol. Data were acquired using Bio-Plex Manager software version 5.0.

Intracellular cytokine staining

Cryopreserved PBMCs were thawed, rested overnight in H10 medium, and then stained with CD3-allophycocyanin-Cy7, CD14-Pacific blue, CD8-allophycocyanin and CD19-PerCP antibodies (BD Biosciences) and LIVE/DEAD® fixable aqua dead cell stain (Invitrogen). They were then fixed and permeabilised using FACS™ Lysing Solution and FACS Permeabilizing Solution (BD Biosciences), according to the manufacturer's protocol and stained intracellularly with IL-10-PE and IL-6-FITC (Biolegend). Cells were acquired and analysed as described earlier.

In vitro infection of PBMCs and intracellular staining for HIV-1 p24

CD8+ T cells were depleted from PBMCs using anti-CD8 microbeads followed by magnetic separation. CD8-depleted PBMCs were activated with PHA for 3 days, then infected with HIV-1BaL at a multiplicity of infection of 0.01 and incubated at 37°C. After 3 and 5 days culture, aliquots of the cells were stained with CD3-allophycocyanin-Cy7, CD4-PerCP, CD14-Pacific blue and CD38-PE antibodies (BD Biosciences) and LIVE/DEAD® fixable aqua dead cell stain (Invitrogen), followed by an intracellular HIV-1 gag p24 stain (KC57-FITC). Cells were acquired and analysed as described earlier.

IFN-γ ELISpot assay

Cryopreserved PBMCs were thawed, rested overnight in H10 medium, plated at a density of 106/mL in 96-well filter plates (Millipore Corporation, USA) pre-coated with anti-IFN-γ antibody 1-DIK (Mabtech, Sweden) and incubated overnight at 37°C in the presence of the pool of overlapping gag peptides described above. H10/0.05% DMSO was used as a negative control and PHA was used as a positive control. The following day, the cells were discarded and the plate was incubated with biotinylated anti-IFN-γ antibody (Mabtech) for 3 h at 37°C, followed by streptavidin-conjugated alkaline phosphatase (Mabtech) for 1 h at 37°C. The plate was developed with alkaline phosphatase conjugate substrate (Bio-Rad). Spots were counted using an automated ELISpot plate reader (AID Systems, Germany) and the frequencies of IFN-γ-producing cells were expressed as IFN-γ SFU per 106 PBMCs.

Statistical analysis

The Kruskal–Wallis test followed by Dunn's multiple comparisons post-test (multiple group comparisons), Wilcoxon matched-pairs test, Spearman's rank test and paired t-test were performed using GraphPad Prism version 5. p values of <0.05 were considered statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

This work was supported by the Oxford NIHR Biomedical Research Centre, UK. A.M., P.B. and L.D. are Jenner Investigators.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information
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Abbreviations
ART

antiretroviral therapy

IL-10R

IL-10 receptor

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

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FilenameFormatSizeDescription
eji2751-sup-0001-FigureS1.pdf310K

Supplementary Figure 1 Progressive gating strategy used to identify CD4+ T cells, CD8+ T cells and CD19+ B cells within the lymphocyte population and CD3- CD14+ cells within the monocyte population.

Supplementary Figure 2 Frequencies of CD4+ T cells, CD8+ T cells, CD19+ B cells and CD14+ monocytes that constitutively express IL-10 among ART-naive patients (n=25), ART-treated patients (n=20) and uninfected controls (n=5).

Supplementary Figure 3 Effect of depletion of HIV-1 gag-specific IL-10+ CD8+ T cells on HIV-1 gag-induced expression of HLA-DR and CD38 on CD4+ T cells.

Supplementary Figure 4 (A) CD38 expression on CD14+ monocytes from infected (p24 Ag+) and mock-infected PBMC (n = 4) after 3 and 5 days’ culture. CD8+ T cell-depleted PBMC from four HIV-negative subjects were activated for 3 days with phytohaemagglutinin and then infected with HIV-1BaL in the presence of IL-2, using a MOI to achieve infection of 5-10% CD4+ T cells, as indicated by expression of p24 antigen (p24 Ag). (B) Representative plots showing p24 Ag expression in monocytes from mock-infected PBMC cultures (left) and HIV-1BaL-infected PBMC cultures (right) after 3 days. (C) CD38 expression on CD14+ monocytes in mock-infected PBMC cultures (unexposed uninfected, UU), uninfected (p24 Agneg) monocytes in HIV-1BaL-infected PBMC (exposed uninfected, EU), or infected (p24 Ag+) monocytes in HIV-1BaL-infected PBMC (exposed infected, EI) (n = 6). Median values are indicated by horizontal bars.

Supplementary Figure 5 CD38 expression by monocytes in cultures where all CD8+ T cells were present (Undepleted), IL-10+ CD8+ T cells were depleted prior to co-culture of CD8+ and CD8neg fractions (“Depleted”) and where the CD8neg fraction was incubated with an IL-10R-blocking antibody prior to co-culture with undepleted CD8+ T cells (“Undepleted + αIL-10R”). Mean fluorescence intensity is expressed as arbitrary units. Three donors were tested; median values are indicated by horizontal bars.

eji2751-sup-0002-S1.pdf276KPeer review correspondence

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