SEARCH

SEARCH BY CITATION

Keywords:

  • Blimp-1;
  • CD4+ T cells;
  • HIV;
  • IL-2;
  • miR-9

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

The fine control of T-cell differentiation and its impact on HIV disease states is poorly understood. In this study, we demonstrate that B-lymphocyte-induced maturation protein-1 (Blimp-1/Prdm1) is highly expressed in CD4+ T cells from chronically HIV-infected (CHI) patients compared to cells from long-term nonprogressors or healthy controls. Stimulation through the T-cell receptor in the presence ofIL-2 induces Blimp-1 protein expression. We show here that Blimp-1 levels are translationally regulated by microRNA-9 (miR-9). Overexpression of miR-9 induces Blimp-1 repression, restoring IL-2 secretion in CD4+ T cells via reduction in the binding of Blimp-1 to the il-2 promoter. In CHI patients where IL-2 expression is reduced and there is generalized T-cell dysfunction, we show differential expression of both miR-9 and Blimp-1 in CD4+ cells compared with levels in long-term nonprogressors. These data identify a novel miR-9/Blimp-1/IL-2 axis that is dysregulated in progressive HIV 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

The fine control of T-cell differentiation and its impact on HIV disease states is poorly understood. It is generally agreed that virus-specific CD4+ T-cell responses are impaired early in the course of HIV disease. HIV-specific CD4+ T cells persist in patients with progressive disease, but the ability of these cells to proliferate and produce IL-2 in vitro is defective [1]. Studies in elite controllers showed that virus-specific CD4+ T cells produce more IL-2 and are more polyfunctional than those of chronic progressors [2]. In addition, in progressive HIV infection, there is a parallel increase of CD4+ T-cell dysfunction to antigenic stimuli in general [3]. Furthermore, it has been shown in both animal models and humans that in chronic viral infections, there is progressive suppression of immune functions through upregulation of inhibitory pathways, contributing to T-cell exhaustion [4-6]. Identifying the underlying mechanisms of T-cell dysfunction in HIV-1 infection will provide crucial information for novel targets and therapeutic interventions.

B-lymphocyte-induced maturation protein-1 (Blimp-1, originally designated PRDI-BF1 in humans) encoded by the Prdm1 gene is, a zinc finger-containing transcriptional repressor, necessary and sufficient for generation of terminally differentiated plasma cells [7]. In T cells, Blimp-1 is a master regulator of the terminal differentiation of CD8+ T-cell effectors [8, 9]. It has also a role in CD8+ T-cell exhaustion during chronic viral infection and as a repressor of memory differentiation. Exhausted CD8+ T cells have substantially higher expression of Blimp-1 than functional effector or memory CD8+ T cells generated after acute infection [10]. Furthermore, Blimp-1 directly represses the Il2 gene and attenuates IL-2 production upon antigen stimulation, while enhancing the expression of several molecules expressed by effector cells [7, 11, 12]. However, the molecular machinery modulating this axis needs to be determined.

Taking into account the fact that Blimp-1 contributes to T-cell dysfunction and represses IL-2 expression, two major characteristics of chronic HIV infection, we sought to examine Blimp-1 expression in T cells from infected patients and explore the mechanisms mediated by microRNAs to regulate the Blimp-1/IL-2 axis. We hypothesized that high levels of Blimp-1 in CD4+ T cells contribute to T-cell dysfunction and impaired IL-2 secretion during the course of HIV-1 infection.

Examination of Blimp-1 expression in samples from patients chronically infected with HIV-1 revealed that both Blimp-1/Prdm1 mRNA and protein were highly expressed in patients with progressive chronic HIV infection (CHI) compared to long-term nonprogressors (LTNPs) or healthy controls (HCs). Using different models including T-cell lines and primary CD4+ T cells, we report an increase in microRNA-9 (miR-9) expression in activated CD4+ T cells and show that miR-9 regulates Blimp-1/Prdm1 protein levels in T cells. Overexpression of miR-9 overcomes the Blimp-1 repression, partly restoring IL-2 secretion in CD4+ T cells. Furthermore, we found low-miR-9 expression in CD4+ T cells from CHI patients compared with that in LTNPs. These data identify miR-9/Blimp-1/IL-2 as a novel axis involved in T-cell dysfunction during chronic HIV infection.

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

Blimp-1 is highly expressed in CD4+ T cells during HIV infection, correlating with PD-1 expression

It is known that in chronic HIV disease, T-cell dysfunction is characterized by reduced IL-2 production [1], however, the mechanisms leading to this defect remain unclear. We used samples from HIV-1-infected patients including those with chronic progressive infection (CHI) and LTNPs, as well as uninfected HCs (Table 1), to study Blimp-1 protein and mRNA expression. LTNPs were infected for more than 8 years, had a CD4+ T-cell count more than 500 cells/μL, and were naive to antiretroviral therapy. CHI patients were defined as individuals who were therapy naïve, who either had CD4+ T-cell count more than 350 cells/μL or who had been diagnosed at primary infection, and who required commencement antiretroviral therapy within 2 years of that diagnosis on the basis of high set point viral load and symptoms or signs of progressive disease.

Table 1. Characteristics of healthy control and HIV-infected patient groups
Patient IDAge (years) /sexCD4 (count/μL)Plasma viral load (cpy/mL)Time since disease diagnosis (years)
  1. a

    Patients’ samples used for qRT-PCR experiments (Fig. 1C and D).

  2. b

    These patients were recruited and had positive serology for HIV diagnosis at least 1 year prior to study entry.

  3. LTNP: Long-term nonprogressor; CHI: chronic progressive HIV infection; NA: not applicable.

LTNP
148/M60929318
257/M672<5016
352/M70321515
441/M1014710010
558/M107320 80014
660/M552460019
753/M868180011
853/M560420015
959/M812<20013
1057/M567<20010
1155/M1150100011
12a57/M754110010
13a48/M644<20012
14a50/M544360011
15a-/M13103208
16a52/M936<508
17a29/M1550<508
Mean51.8587269211.05
CHI
149/M528174 5002
241/M35250 4006
345/M45683 8002
432/M20924 8002
532/M384976 7307
642/M24249 8003
744/M24013 2002
849/M160673 0002
949/M72321 0001
10a30/M18254 9001
11a40/M15229 7001
12a48/M108698 0001
13a-/M49623 200>1b
14a-/M312231 200>1b
15a-/M19236 500>1b
16a-/M->20 000>1b
Mean41.8272321 623>1
Healthy controls
144/MNANANA
229/M   
331/F   
438/M   
525/F   
626/F   
730/M   
824/M   
923/M   
1026/M   
1155/M   
1228/M   
1327/M   
14-   
15-   
1655/M   
Mean33   

We determined ex vivo Blimp-1 protein expression by flow cytometry using two anti-Blimp-1 antibodies (C21 Blimp-1 Ab and 6D3 mAb) and mRNA by qRT-PCR in purified CD4+ T cells. CD3+CD4+CD45RO+ cells from CHI patients had significantly increased Blimp-1+ cell frequency compared to HCs (p < 0.0001) and LTNPs (p < 0.0001) (Fig. 1B). Similar profiles of Blimp-1 expression were obtained with both monoclonal and polyclonal 6D3 and C21 antibodies (Fig. 1A). The gating strategy for CD45RO+Blimp-1+ cells and the cut-off for determining whether a cell is Blimp-1+ has been set on CD45RO cells, as they are negative for Blimp-1 (Supporting Information Fig. 1).

image

Figure 1. Blimp-1 and PD-1 expression in CHI patients, long-term nonprogressors(LTNPs), and healthy controls (HCs). Blimp-1 and PD-1 expression were measured by flow cytometry in 16 CHI patients, 17 LTNPs, and 16 HCs. (A) Representative histograms of Blimp-1 expression in HCs and HIV-positive patients (HIV+) in CD4+CD45RO+ T cells as measured by flow cytometry using both anti-Blimp-1 C21 Ab (left) and 6D3 mAb (right) are shown. Dotted lines represent IgG isotype controls. (B) Blimp-1 expression in CD4+CD45RO+ T cells from CHI patients, LTNPs, and HCs are shown. Each symbol represents an individual donor and bars represent the means. (C) Blimp-1 mRNA expression in HCs, CHI patients, and LTNPs. Values were normalized β-actin. Data are shown as mean ± SEM for two independent experiments each performed in duplicate. (D) Correlation between Blimp-1 protein and mRNA in HCs, CHI patients, and LTNPs. (E) PD-1 expression in CD4+CD45RO+ T cells from CHI patients, LTNPs, and HCs is shown. (F) PD-1 and Blimp-1 co-expression in CD4+CD45RO+ T cells is shown. Each symbol represents an individual donor and bars represent the means. (G) Correlation is shown between percent CD4+PD-1+CD45RO+ and percent CD4+Blimp-1+CD45RO+ in CHI patients, LTNPs, and HCs. All data shown are representative of three independent experiments. Statistical significance was determined by nonparametric Krustal–Wallis test (B, C, E, and F) and Wilcoxon paired t-test (D and G).

Download figure to PowerPoint

Moreover, Blimp-1 protein expression correlated significantly with mRNA levels (p = 0.002) (Fig. 1C and D).

Blimp-1 has been identified as a transcriptional regulator associated with CD8+ T-cell exhaustion during chronic viral infection in a mouse model of lymphocytic choriomeningitis virus [10]. In order to assess whether Blimp-1 expression correlates with exhaustion, we determined Programmed cell death protein 1 (PD-1) expression, one of the best described markers of T-cell exhaustion [4-6], in CD4+ T cells from the same groups of patients. As expected, the proportion of PD-1+CD45RO+CD4+ T cells in CHI patients was significantly elevated compared with that of HCs (p < 0.0001) or LTNPs (p = 0.02) (Fig. 1E). Importantly, the extent of PD-1 and Blimp-1 coexpression in CD4+RO+ T cells was higher in CHI patients compared with that in HCs and LTNPs (p < 0.0001 and p = 0.01, respectively; Fig. 1F). The gating strategy for Blimp-1+PD-1+ cells is shown in Supporting Information Fig. 1. Moreover, there was a correlation between the size of PD-1+RO+ and Blimp-1+RO+ subsets across all patients and controls (p < 0.0001; Fig. 1G). These results support the idea that a common regulatory pathway links Blimp-1 and PD-1 expression, consistent with previously reported murine data [10].

Optimal Blimp-1 protein expression depends on TCR stimulation and IL-2

We attempted to modulate Blimp-1 protein expression in dividing CD4+ T cells and explore the determinants of this expression by stimulating PBMCs in vitro for 3–5 days with a CD3-specific mAb in the presence or absence of IL-2. Using flow cytometry, we assessed Blimp-1 expression and observed increased levels that correlated with increased cell division. Importantly, increased Blimp-1 expression, as measured by mean fluorescence intensity, was greater when IL-2 was added to the culture in both HCs and HIV-infected patients (Fig. 2A and B). We have also observed increased surface expression of CD25 and CD38, two markers of activation, in parallel to cell division, indicating that Blimp-1 expression increases when the cells show an activated phenotype (data not shown).

image

Figure 2. Blimp-1 expression in activated and proliferating CD4+ T cells. (A) Representative cytometry plots from PBMCs from five healthy individuals stained with CFSE and cultured in the presence of 1 μg/mL CD3-specific mAb with or without IL-2 for 5 days. Cells were stained for assessing Blimp-1 expression during CD4+ T-cell proliferation. (B-C) Blimp-1 expression in dividing cells, indicated by CFSE dilution from (B) five healthy individuals and (C) four HIV-positive patients, is shown as mean fluorescence intensity (MFI). CFSE+++ indicates nondivided cells, which are represented by 0 in (A). CFSE++, CFSE+, CFSE− and CFSE−− represent division 1, 2, 3, and 4, respectively in (A). Data are shown as mean ± SEM of five pooled samples for HCs and four pooled samples for CHI patients. Data shown are representative of three experiments performed. Statistical significance was determined by Wilcoxon paired t-test (B and C).

Download figure to PowerPoint

miR-9 expression in CD4+ T cells

Two computer algorithms (TargetScan and PicTar), and published data [11], suggested that miR-9 has three complementary binding sites within Prdm1 3′ untranslated region (UTR) (Fig. 3A).

image

Figure 3. Computer prediction of miRNAs target sites in the 3′ untranslated region (UTR) of Prdm1 mRNA. (A) Putative target sites for miR-9 in the 3′ UTR of human Prdm1 mRNA were identified using TargetScan (version 5.1). (B) Hela cells were co-transfected with Blimp-1 3′UTR firefly luciferase reporter construct and either a negative pre-miRNA control, pre-let-7b, pre-miR-9 or none in equimolar amounts. Differences are shown as intensity of luminescence. (C) RNA was extracted from sorted CD4+ T cells from healthy individuals either directly after, or after 2, 24, 48, or 72 h in vitro stimulation using CD3-specific mAb. miR-9 expression was assessed by RT-PCR. mRNA results were normalized to RNU44. Data are shown as mean + SEM and results pooled from two independent experiments from two different healthy individuals each performed in duplicate. Statistical significance was determined by nonparametric Mann–Whitney U test.

Download figure to PowerPoint

To directly demonstrate the interaction between miR-9 and Blimp-1, we cotransfected Hela cells with a Prdm1 3′UTR firefly luciferase reporter construct, and precursors to miR-9 (pre-miR-9). Luciferase activity was significantly reduced in pre-miR-9-treated cells (Fig. 3B), but not when treated with pre-Let7b, demonstrating that miR-9 directly binds to the Prdm1 3′UTR.

We assessed miR-9 expression in ex vivo purified CD4+ T cells from HCs and attempted to modulate this expression by stimulating the cells in vitro for 3–5 days with or without IL-2. miR-9 expression was low in ex vivo CD4+ T cells from HCs, but increased with TCR stimulation and peaked at 48 h (Fig. 3C).

Increased miR-9 levels correlate with Blimp-1 protein downregulation and IL-2 upregulation

In order to link IL-2, Blimp-1, and miR-9 during CD4+ T-cell activation, we first assessed whether miR-9 levels modulate Blimp-1 protein expression by transfecting HUT78 cells with pre-miR-9 at 10 and 30 nM (pre-miR-155 and pre-Let7b were used in parallel as specificity controls as they do not have recognized targets within the Prdm1 3′UTR). As expected, miR-9, miR-155, and Let-7b levels were significantly increased at 24 and 48 h posttransfection (≈ 150-fold, p < 0.05; Fig. 4A). The transfection rates were similar in the study and control populations (see Materials and methods). Importantly, pre-miR-9, but not pre-miR-155 or pre-Let-7b, specifically reduced Blimp-1 protein expression in HUT78 cells by approximately 45% (p < 0.05; Fig. 4B). Endogenous IL-2 protein secretion was increased (38%) in pre-miR-9-transfected HUT78 (Fig. 4C), consistent with miR-9 levels downregulating Blimp-1 expression and consequently upregulating IL-2. Importantly, in pre-miR-9-transfected primary CD4+ T cells, Blimp-1 protein decreased 42% and IL-2 secretion increased 44% (Fig. 4D and E). Similarly, a 48% decrease in Blimp-1 mRNA and 40% increase in IL-2 mRNA levels was observed in pre-miR-9-transfected Jurkat T cells (Fig. 4F and G). These results suggest that miR-9 contributes directly to Blimp-1 downregulation and indirectly to increased IL-2 production.

image

Figure 4. Modulation of Blimp-1 expression and IL-2 secretion in T cells after transfection with pre-miR-9. (A) HUT78 cells were transfected with 0, 10, or 30 nM pre-miR9, pre-miR-155, or pre-Let-7b. (B) Mean fluorescence intensity (MFI) of Blimp-1 measured in HUT78 cells transfected with pre-mir9, pre-mIR-155, or pre-miR-Let-7b using anti-Blimp-1 PE antibody is shown. (C) IL-2 secretion in transfected HUT78 with mock control or with pre-miR-9. (D) MFI was measured in transfected primary CD4+ T cells, using anti-Blimp-1 PE antibody. Data shown are from two different healthy individuals, each experiment performed in duplicate. (E) Fold difference comparison in IL-2 secretion in pre-miR-9 or pre-Let7b-transfected primary CD4+ T cells. (F) and (G) Blimp-1 and IL-2 mRNA expression (normalized to GAPDH) in pre-miR-9-transfected Jurkat cells. (H) Binding of Blimp-1 to il2 promoter. Chromatin immunoprecipitation (ChIP) assays were performed on Jurkat cells and the results are presented as Blimp-1 ChIP enrichment ratio. All data are shown as mean ± SEM for at least two independent experiments. Statistical significance was determined by nonparametric Mann–Whitney U test.

Download figure to PowerPoint

To directly demonstrate that miR-9 by binding to Blimp-1 prevents il2 gene repression, we performed Blimp-1 chromatin immunoprecipitation (ChIP) assays in miR-9 transfected Jurkat T cells. In the presence of miR-9, there is a significant decrease in Blimp-1 binding to the il2 promoter (Fig. 4H) and a decrease in the repressive mark, di-methylation of lysine-9 on histone H3 (H3K9me2) in the same region (data not shown), correlating with increased gene transcription. These data demonstrate that Blimp-1 binds directly to the il2 promoter, confirming previous observations in mice [7], and that miR-9 modulates this interaction. We also observed Blimp-1 binding to the il2 promoter in primary CD4+ T cells (data not shown).

Blimp-1, IL-2 and miR-9 expression levels in HIV-infected patients and HCs

As the Il2 locus is a direct target of Blimp-1 repression in CD4+ T cells [12, 13], we reasoned that if Blimp-1 is highly expressed in CD4+ T cells of CHI patients, this could explain, at least in part, the low levels of IL-2 production in these patients [1] and that miR-9 might be involved in regulating these linked processes.

We simultaneously assessed Blimp-1 and miR-9 levels in ex vivo CD4+ T cells from the same patients. We found that miR-9 was significantly decreased in CHI patients compared with that in LTNPs (p < 0.05, Fig. 5B), and its expression inverse to that of the Blimp-1 protein (Fig. 5A). miR-9 levels in HCs were very low in ex vivo CD4+ T cells compared with that in LTNPs, suggesting that miR-9 is induced and functionally regulated under stimulatory conditions (Fig. 3C), such as immune activation. This is in line with our previously published data showing that pre-miR-9 transfected T cells significantly increased CD69 expression, a marker of recently activated cells [14].

image

Figure 5. Blimp-1, IL-2, and miR-9 expression in CD4+ T cells from CHI patients, LTNPs, and HCs. (A) Blimp-1, (B) miR-9, and (C) IL-2 expression was determined by flow cytometry (A, C) and qRT-PCR (B), in patients and controls. Intracellular IL-2 production was determined in anti-CD3 stimulated PBMCs for 3 days followed by PMA/ionomycin boost 6 h before cell staining and analysis by flow cytometry. Data are shown as mean + SEM for two to three independent experiments. (D) Correlation between Blimp-1 and IL-2 expressions as shown in (A) and (C). Statistical significance was determined by nonparametric Krustal–Wallis test (A–C) and Wilcoxon paired t-test (D).

Download figure to PowerPoint

Next, we assessed intracellular IL-2 expression in anti-CD3 stimulated cells followed by PMA/ionomycin boost. As expected, we found that IL-2 levels were very low in CHI patients compared with those in HCs and LTNPs (p = 0.01 and p = 0.0006 respectively; Fig. 5C), suggesting that CD4+ T cells from CHI patients are unable to produce IL-2 even in optimal in vitro conditions. Importantly, when these data were correlated to Blimp-1 levels, we found an inverse correlation in the three groups of patients and controls (Fig. 5D, p < 0.0001), with high Blimp-1 levels in CHI patients compared with that in HCs and LTNPs (p = 0.0002 and p = 0.0007, respectively; Fig. 5A).

Altogether, these data suggest that miR-9 may have a direct role controlling Blimp-1 expression and indirectly controlling IL-2 secretion (Fig. 6).

image

Figure 6. Model for the Blimp-1/miR-9/IL-2 axis in CD4+ T cells. Blimp-1 upregulation represses IL-2 transcription. In the presence of miR-9, which is able to bind the Blimp-1 site 3′UTR, Blimp-1 expression is downregulated and IL-2 transcription is increased.

Download figure to PowerPoint

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 demonstrate that Blimp-1/Prdm1 is highly and preferentially expressed in human CD4+ T cells from chronically HIV-infected patients but not in LTNPs or HCs. Interestingly, these high levels of Blimp-1 correlated with PD-1 co-expression in CD4+ T cells, confirming recent reports on the involvement of Blimp-1 in T-cell exhaustion [10]. Given that CD4+ T-cell impairment translates into anergy and incapability of the cells to produce IL-2, we propose that taking into account Blimp-1 expression when studying CD4+ T-cell responses in HIV-1 infection is critical because this transcription factor is directly involved in IL-2 regulation by both direct and indirect Il2 gene repression [12, 13]. However, it should be noted that the molecular machinery modulating this mechanism is unknown despite the established role of Blimp-1 in IL-2 regulation.

In this study, we identify, both in vitro and in vivo, a role for miR-9 in the direct regulation of Blimp-1 expression, and thereby an indirect role in IL-2 production. miRNAs are emerging as key regulators, fine tuning gene expression [15] by binding to mRNA via target sequences within the 3′ UTR and directing posttranscriptional repression by translational inhibition and/or mRNA degradation [15].

We show a clear association between Blimp-1 expression and miR-9 levels by demonstrating that: (i) Blimp-1 protein expression in human CD4+ T-cell subsets is induced upon TCR stimulation in the presence of IL-2 and this is associated with a significant decrease in miR-9 levels; and (ii) Blimp-1 protein expression decreases significantly after miR-9 over-expression in HUT78 cells, with similar changes seen in transfected primary human CD4+ T cells.

Few studies have reported a role for miR-9 in cell function and development [11, 16]. In the study by Bazzoni et al. [16], the authors used monocytes and neutrophils to show that Toll-Like Receptor 4-activated NF-κB, rapidly increases the expression of miR-9 that operates a feedback control of the NF-κB-dependent responses. Interestingly, these data suggest that miR-9 expression is important in regulating the release of pro-inflammatory cytokines by monocytes and during infections such as HIV. Moreover, a very recent study by Thiele et al. [17] showed that miR-9 abundance increases upon T-cell activation and binds to the 3′UTR of Prdm1, which results in a reduction of Blimp-1 abundance by degrading the Prdm1 mRNA and a subsequent increase in production of IL-2 and IFN-γ. This study, based on the use of T cells from healthy individuals, is in line with our data that were obtained from both healthy individuals and HIV-infected patients [17]. Furthermore, Bignami et al. [18] evaluated the expression of 377 miRNAs in CD4+ T cells from HIV-1 infected elite LTNPs, HIV-1 infected treatment naive patients, multiply exposed but uninfected (MEU) individuals and HCs. They observed that miR-9 levels were significantly different between the groups [18]. By using larger numbers of patients in our three groups (CHI patients, LTNPs, and HCs), we confirm and complement these studies by identifying miR-9 as one of the key regulators of Blimp-1 and IL-2 expressions in CD4+ T cells during HIV infection.

The miR-9-Blimp-1/Prdm1 axis appears to be an important regulatory component impacting on IL-2 secretion in activated CD4+ T cells. The biological significance of this axis is further supported by the observations made in lymphocytes from HIV-1 infected patients. miR-9 levels in CD4+ T cells from CHI patients were low, Blimp-1 levels were high, and IL-2 secretion was low when compared with that in LTNPs. Levels of Blimp-1 and IL-2 expression in LTNPs who have chronic HIV infection without significant disease progression are similar to those seen in HCs. One possible interpretation of these data is that LTNPs have some inherent difference in their regulation of miR-9 expression, which might function as a modulator to fine-tune and maintain low Blimp-1 expression and consequently high IL-2 production. Recently, the miRNA profiles from a number of lymphocyte subsets have been shown to reveal distinct miRNA signatures [19], which further supports the argument that differences in T-cell subsets in HIV-1 infection may be a cause of why there are differences in miRNA levels between various HIV-1 infected populations. Given the failure of genome-wide association studies and mRNA arrays to explain the majority of the factors determining LTNP status [20], understanding Blimp-1 regulation including the role of miR-9 could lead to new types of intervention modulating CD4+ T-cell differentiation.

It is generally agreed that CD4+ T cells specific for HIV and for other antigens persist to varying degrees in patients with progressive disease, but the ability of these cells to proliferate and produce IL-2 in vitro is impaired [1, 21]. Our finding that Blimp-1 and PD-1 levels correlate and are co-expressed in HIV-positive patients, is consistent with the finding that Blimp-1 upregulates expression of inhibitory receptors PD-1, LAG-3, 2B4, and CD160 [8]. It has been reported that PD-1 expression on CD4+ T cells positively correlates with viral load and that HIV-specific CD8+ T cells expressing high amounts of PD-1 are functionally exhausted and less able to respond to cognate antigen in vitro [4-6]. Consequently, our data showing correlation between Blimp-1 and PD-1 expression in HIV-infected patients, potentially reveal a critical role for Blimp-1 and miR-9 in T-cell dysfunction because the dysregulation of these two molecules appears to be involved in the IL-2 repression [12], the reduced proliferative capacity, and/or the reduced effector/memory responses [8] in HIV disease.

Recent efficacy trials of recombinant (r)IL-2 therapy with ART for HIV-infection (SILCAAT and ESPRIT) showed no clinical benefit despite substantial, sustained increases in CD4+ T-cell count [22]. It is possible that high exogenous IL-2 levels induced increased Blimp-1 expression, potentially explaining the ineffective immune reconstitution observed in rIL-2 recipients [22, 23]. This hypothesis is under investigation. Lastly, given the importance of Blimp-1 on Treg-cell differentiation and function [24], further work will be aimed at understanding the relationship between Blimp-1 expression in Treg cells in HIV-1 infected individuals compared with HCs.

Taken together, our results strongly suggest that the Blimp-1/IL-2/miR-9 axis impacts on CD4+ T-cell function in pathological conditions such as HIV, therefore deserves further investigation.

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

Samples

Buffy coats were obtained from the Australian Red Cross Blood Service. Studies were approved by Institutional Ethics committees and an informed consent was provided. Peripheral blood, collected as part of the observation studies, was obtained from antiretro-viral naïve patients with chronic progressive HIV-1 (CHI) infection, LTNPs, and HCs. CD4+ T-cell count, plasma HIV RNA levels of each of these groups were determined by the NSW HIV Reference laboratory located at St Vincent's Hospital, Sydney. The LTNPs were recruited as previously described [25]. Minimal criteria for entry to the study was more than 8 years since diagnosis, CD4 count more than 500 cells/μL, and antiretroviral therapy naïve. CHI patients were defined as individuals who were therapy naïve who either had CD4+ T-cell count less than 350 cells/μL or who having been diagnosed at primary infection within 2 years of that diagnosis had high set point viral load and symptoms or signs of progressive disease.

Mononuclear cell preparation

Buffy coat from HCs were prepared as previously described [26].

RT-PCR

Real-time PCR (RT-PCR) was performed using an IQ5 Real-Time PCR Detection System (Bio-Rad). For some experiments, ABI PRISM 7900 sequence detector (PerkinElmer/PE Applied Biosystems) system was used. Taqman primers/probes were: Prdm1 (Accession number: Hs01068508_m1), and β-Actin (Cat#401846) from Applied Biosystems. Primers and probes for amplification and detection of miRNAs were supplied by Applied Biosysytems, accession numbers are as follows: RNU44 (cat # 4373384), miR-9 (hsa-miR-9), let7b (hsa-let-7b), and miR-155 (hsa-miR-155).

Human genomic primer sequences covering the il2 promoter (−380 to −158 base pairs from the transcription start site): 5′-CTTGCTCTTGTCCACCACAA-3′ and 5′-ACCCCCAAAGA CTGACTGAA-3′.

Flow cytometry

MAbs used were: anti-CD3-PerCP-Cy5.5; CD4-PECy7 and FITC; CD8-allophycocyanin-Cy7; IL-2FITC; CD25-allophycocyanin; CD38-FITC, CD62L-allophycocyanin-Cy7; anti-PD-1-FITC; (BD); CD45RO-ECD (Beckman Coulter), CD127-PE (Immunotech); CD127-Pacific Blue (eBiosciences). C21 Blimp-1-PE (sc-13206) is a polyclonal antibody and normal goat IgG-PE (sc-3992), were from Santa Cruz. 6D3 purified Blimp-1 mAb is a generous gift from L. Corcoran (Walter and Eliza Hall Institute, Melbourne). PE AffiniPure F(ab’)2 Fragment Goat Anti-Rat IgG (Jackson ImmunoResearch). Intracellular staining was performed 5 h after reactivation with PMA/ionomycin. Cells were fixed, permeabilized, and stained for surface CD3, CD4, and CD45RO, and intracellular Blimp-1 and/or IL-2 in the presence of Brefeldin A.

PBMCs from adults were cultured with 1 μg/mL CD3-specific mAb (Hit3a, Pharmingen) plus or minus 50 U/mL IL-2 (National Institute of Health, NIH) for 5 days.

MicroRNA quantitation

miR-9, miR-155, and Let7b levels in T-cell subsets and cell lines were determined using TaqMan MicroRNA Assays (Applied Biosystems) following the protocols recommended by the manufacturer. A 10 ng of fresh or frozen extracted total RNA was used in a total volume of 15 μL per reaction during reverse transcription, which was further diluted 1/10 with sterile PCR grade water and 3 μL of reverse transcription product was used for subsequent RT-PCR reactions. For each microRNA of interest, individual reverse transcription reactions were performed using different stem loop primers (Applied Biosystems) and each reaction was assayed in triplicate by RT-PCR. The levels of miRNAs were normalized with RNU44 (small nucleolar RNA) and the relative levels were calculated using the ΔCt method.

Computer prediction of miRNAs target sites in the 3′UTR of Prdm1 mRNA

Putative target sites for miRNAs in the 3′UTR of human Blimp-1/Prdm1 mRNAs were identified using the PicTar (http://pictar.mdc-berlin.de/cgi-bin/PicTar_vertebrate.cgi) and TargetScan (version 5.1) http://www.targetscan.org).

Cell transfection with pre-miRNAs

Optimized transfection protocols generated by Amaxa (Lonza) were used to transfect HUT78 and Jurkat cell lines. Different concentrations of 10, 25, 30, or 100 nM of pre-miRNA were first used for transfection after which cell viability was assessed by flow cytometry based on forward and side scatter. The best cell viability was obtained with a concentration of 30 nM, which was then chosen for further experiments. A concentration of 10 nM was also included for comparison. Cells were collected at 24–48 h after electroporation and subject to mAbs staining and flow cytometry analysis for determination of Blimp-1 expression. Independent transfection experiments were repeated two to seven times.

For primary CD4+ T cells from two healthy donors were negatively selected using beads (untouched human CD4 Dynabeads, Invitrogen). After transfection using AMAXA system, cells were seeded at 2 × 106 cells per well in 12-well plates in RPMI 10% AB serum (Cambrex) and stimulated with CD3-specific mAb (1 μg/mL) and CD28-specific mAb (2 μg/mL) for 48 h.

FAM3™ Dye-Labeled Pre-miR Negative Control #1 (Ambion, Catalogue number AM17121) was used to assess transfection efficiency and was electroporated into primary CD4+ T cells or HUT78 cells using Amaxa. The percentage of fluorescent-labeled cells was measured using a flow cytometer and in HUT78 cells, the transfection efficiency was 54% and in CD4+ T cells, it was at least 20%.

In some experiments (Fig. 4F–H), Jurkat T cells or primary CD4+ T cells were seeded at 1 × 105 cells per well in 24-well plates and transfected with the synthetic microRNAs, miR-9, miR-1, or the negative control pre-miRNA (Ambion) at a final concentration of 50 nM using Lipofectamine 2000 transfection reagent (11668–027, Invitrogen) according to the manufacturer's guidelines. Total RNA was subsequently extracted 2 days posttransfection following stimulation for 2 h for Jurkat cells and Taqman real-time PCR analysis was performed. Jurkat cells were stimulated with 20 ng/mL of PMA/ionomycin.

Pre-miR tranfection into Blimp-1 3′UTR reporter construct

One day prior to transfection, 50 × 104 HeLa cells were plated into each well of 24 wells. Twenty-four hours later, cells were co-transfected with the Blimp-1 3′UTR firefly luciferase reporter construct (Switchgear Genomics, California, USA) and appropriate pre-miRNA using Lipofectamine 2000 reagent. A total of 3.2 μL of pre-miRNA at a concentration of 6.25 μmol/L was used for the transfection experiments. Samples were lysed the following day using Passive Lysis Buffer from the Dual-Luciferase® Reporter Assay System (Promega). A total of 20 μL of lysate was plated and appropriate substrate was added for luciferase, and then read on a luminometer (Fluostar), followed by the substrate for Renilla firefly and then re-reading for luminescence.

ChIP assays

ChIP assays were performed as previously described [27]. Briefly, the soluble chromatin fraction was incubated overnight at 4°C with 20 μL (200 μg/mL) of anti-Blimp-1 mAb (sc-13206). In all ChIP assays, a corresponding no antibody IP control was included to ensure that only specific enrichment was monitored. ChIP samples were then subjected to SYBR real-time PCR amplification (Applied Biosystems). ChIP enrichment ratios were analyzed such that only enrichment above the negative control was calculated as specific protein binding [27, 28].

Statistical analyses

Prism 4.0 (GraphicPad) was used for statistical analyses. Analysis of differences between groups was by nonparametric 1-way ANOVA Mann–Whitney U test and Wilcoxon paired t-test for paired samples. Linear regression was calculated using Prism software. P values were considered significant when <0.05.

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

The authors would like to thank all the patients for their cooperation and B. Anderson, T. Men Soo, R. Hain, A. Carr, M. Kelly, K. Shroder, D. Smith, M. McMurchie, N. Roth, and B. Genn for recruiting patients to the long-term nonprogressor cohort study.

This study was funded from the following sources: the Australian Government Department of Health and Ageing; the NHMRC via a Program (510448) grant, a PhD Scholarship (SS), and a Practitioner Fellowship (ADK). The views expressed in this publication do not necessarily represent the position of the Australian Government. The Kirby Institute is affiliated with the Faculty of Medicine, University of New South Wales.

References

  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
  • 1
    Zaunders, J., Dyer, W., Munier, M., Ip, S., Liu, J., Amyes, E., Rawlinson, W. et al., CD127+CCR5+CD38+++ CD4+ Th1 effector cells are an early component of the primary immune response to vaccinia virus and precede development of interleukin-2+ memory CD4+ T cells. J. Virol. 2006. 80: 1015110161.
  • 2
    Porichis, F. and Kaufmann, D. E., HIV-specific CD4 T cells and immune control of viral replication. Curr. Opin. HIV AIDS 2011. 6: 174180.
  • 3
    McMichael, A. J., Borrow, P., Tomaras, G. D., Goonetilleke, N. and Haynes, B. F., The immune response during acute HIV-1 infection: clues for vaccine development. Nat. Rev. Immunol. 2010. 10: 1123.
  • 4
    Day, C., Kaufmann, D., Kiepiela, P., Brown, J., Moodley, E., Reddy, S., Mackey, E. et al., PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 2006. 443: 350354.
  • 5
    Petrovas, C., Casazza, J., Brenchley, J., Price, D., Gostick, E., Adams, W., Precopio, M. et al., PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection. J. Exp. Med. 2006. 203: 22812292.
  • 6
    Trautmann, L., Janbazian, L., Chomont, N., Said, E., Gimmig, S., Bessette, B., Boulassel, M. et al., Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat. Med. 2006. 12: 11981202.
  • 7
    Martins, G. and Calame, K., Regulation and functions of Blimp-1 in T and B lymphocytes. Annu. Rev. Immunol. 2008. 26: 133169.
  • 8
    Rutishauser, R., Martins, G., Kalachikov, S., Chandele, A., Parish, I., Meffre, E., Jacob, J. et al., Transcriptional repressor Blimp-1 promotes CD8(+) T cell terminal differentiation and represses the acquisition of central memory T cell properties. Immunity 2009. 31: 296308.
  • 9
    Kallies, A., Xin, A., Belz, G. and Nutt, S., Blimp-1 transcription factor is required for the differentiation of effector CD8(+) T cells and memory responses. Immunity 2009. 31: 283295.
  • 10
    Shin, H., Blackburn, S., Intlekofer, A., Kao, C., Angelosanto, J., Reiner, S. and Wherry, E., A role for the transcriptional repressor Blimp-1 in CD8(+) T cell exhaustion during chronic viral infection. Immunity 2009. 31: 309320.
  • 11
    Nie, K., Gomez, M., Landgraf, P., Garcia, J., Liu, Y., Tan, L., Chadburn, A. et al., MicroRNA-mediated down-regulation of PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg cells: a potential pathogenetic lesion in Hodgkin lymphomas. Am. J. Pathol. 2008. 173: 242252.
  • 12
    Gong, D. and Malek, T., Cytokine-dependent Blimp-1 expression in activated T cells inhibits IL-2 production. J. Immunol. 2007. 178: 242252.
  • 13
    Martins, G., Cimmino, L., Liao, J., Magnusdottir, E. and Calame, K., Blimp-1 directly represses Il2 and the Il2 activator Fos, attenuating T cell proliferation and survival. J. Exp. Med. 2008. 205: 19591965.
  • 14
    Sutcliffe, E. L., Bunting, K. L., He, Y. Q., Li, J., Phetsouphanh, C., Seddiki, N., Zafar, A. et al., Chromatin-associated protein kinase C-theta regulates an inducible gene expression program and microRNAs in human T lymphocytes. Mol. Cell. 2011. 41: 704719.
  • 15
    Lodish, H., Zhou, B., Liu, G. and Chen, C., Micromanagement of the immune system by microRNAs. Nat. Rev. Immunol. 2008. 8: 120130.
  • 16
    Bazzoni, F., Rossato, M., Fabbri, M., Gaudiosi, D., Mirolo, M., Mori, L., Tamassia, N. et al., Induction and regulatory function of miR-9 in human monocytes and neutrophils exposed to proinflammatory signals. Proc. Natl. Acad. Sci. USA 2009. 106: 52825287.
  • 17
    Thiele, S., Wittmann, J., Jack, H. M. and Pahl, A., miR-9 enhances IL-2 production in activated human CD4(+) T cells by repressing Blimp-1. Eur. J. Immunol. 2012. 42: 21002108.
  • 18
    Bignami, F., Pilotti, E., Bertoncelli, L., Ronzi, P., Gulli, M., Marmiroli, N., Magnani, G. et al., Stable changes in CD4+ T-lymphocyte microRNA expression following exposure to HIV-1. Blood 2012. 119: 62596267.
  • 19
    Rossi, R. L., Rossetti, G., Wenandy, L., Curti, S., Ripamonti, A., Bonnal, R. J., Birolo, R. S. et al., Distinct microRNA signatures in human lymphocyte subsets and enforcement of the naive state in CD4+ T cells by the microRNA miR-125b. Nat. Immunol. 2011. 12: 796803.
  • 20
    Rotger, M., Dang, K. K., Fellay, J., Heinzen, E. L., Feng, S., Descombes, P., Shianna, K. V. et al., Genome-wide mRNA expression correlates of viral control in CD4+ T-cells from HIV-1-infected individuals. PLoS Pathog. 2010. 6: e1000781.
  • 21
    Harari, A., Vallelian, F., Meylan, P. R. and Pantaleo, G., Functional heterogeneity of memory CD4 T cell responses in different conditions of antigen exposure and persistence. J. Immunol. 2005. 174: 10371045.
  • 22
    Abrams, D., Levy, Y., Losso, M., Babiker, A., Collins, G., Cooper, D., Darbyshire, J. et al., Interleukin-2 therapy in patients with HIV infection. N. Engl. J. Med. 2009. 361: 15481559.
  • 23
    Sereti, I., Imamichi, H., Natarajan, V., Imamichi, T., Ramchandani, M., Badralmaa, Y., Berg, S. et al., In vivo expansion of CD4CD45RO-CD25 T cells expressing foxP3 in IL-2-treated HIV-infected patients. J. Clin. Invest. 2005. 115: 18391847.
  • 24
    Cretney, E., Xin, A., Shi, W., Minnich, M., Masson, F., Miasari, M., Belz, G. T. et al., The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. Nat. Immunol. 2011. 12: 304311.
  • 25
    Stewart, G. J., Ashton, L. J., Biti, R. A., Ffrench, R. A., Bennetts, B. H., Newcombe, N. R., Benson, E. M. et al., Increased frequency of CCR-5 delta 32 heterozygotes among long-term non-progressors with HIV-1 infection. The Australian long-term non-progressor study group. AIDS 1997. 11: 18331838.
  • 26
    Seddiki, N., Santner-Nanan, B., Tangye, S., Alexander, S., Solomon, M., Lee, S., Nanan, R. et al., Persistence of naive CD45RA+ regulatory T cells in adult life. Blood 2006. 107: 28302838.
  • 27
    Sutcliffe, E. L., Parish, I. A., He, Y. Q., Juelich, T., Tierney, M. L., Rangasamy, D., Milburn, P. J. et al., Dynamic histone variant exchange accompanies gene induction in T cells. Mol. Cell. Biol. 2009. 29: 19721986.
  • 28
    Pokholok, D. K., Zeitlinger, J., Hannett, N. M., Reynolds, D. B. and Young, R. A., Activated signal transduction kinases frequently occupy target genes. Science 2006. 313: 533536.
Abbreviations
Blimp-1

B-lymphocyte-induced maturation protein-1

ChIP

chromatin immunoprecipitation

HC

healthy control

LTNP

long-term nonprogressor

miR-9

microRNA-9

pre-miR-9

precursors to miR-9

UTR

untranslated region

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

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
eji2494-sup-0001-FigureS1.pdf361KFigure S1

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.