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Introduction

Human immunodeficiency virus type 1 (HIV-1) preferentially destroys CD4+ T lymphocytes and leads to disturbed T cell homeostasis, characterized by the depletion of CD4+ T cells (1). The sustained deletion and dysfunction of CD4+ T cells caused by HIV-1 infection can result in opportunistic infections and tumors, diagnosed as acquired immune deficiency syndrome (1–3). The majority of attempts to manage HIV-1 infection have focused on CD4+ T cell recovery, whereas changes in CD8+ T cells have received less attention. However, the overall course of HIV-1 infection is largely shaped by CD8+ T cell responses. The CD4/CD8 ratio has been reported to be a useful marker for clinical outcome, immune dysfunction and viral reservoir size in HIV-1-infected patients (4,5). Cytotoxic T lymphocytes (CTLs), the critical effector CD8+ T cells, are vital in host defense against HIV-1 by impeding viral replication through cytolytic and non-cytolytic pathways (6). The dynamics of effector CD8+ T cell expansion in acute HIV-1 infection is similar to that in other viral infections (7). However, CD8+ T cells in patients with chronic HIV-1 infection exhibit characteristics of exhaustion and immunosenescence (7,8). Although may patients with HIV-1 maintain high circulating CD8+ T cell counts, the suppression of HIV-1 replication is attenuated by the disturbed differentiation and homeostasis of the CD8 T cell compartment (9).

The molecular mechanisms controlling peripheral CD8+ T cell differentiation in humans remain to be fully elucidated. Several microRNAs are critical in the development of hematopoietic cells (10,11). A study by Zhang and Bevan demonstrated that CD8+ T cells with knockout of dicer are defective in cell accumulation and survival (12). Compared with naïve CD8+ T cells, miRNA (miR)-21 and miR-155 were found to be upregulated in effector CD8+ T cells (13). The sustained expression of miR-155 has been associated with effector and effector memory cells, whereas lower expression levels of miR-155 have been associated with central memory cells (14). miR-155 has also been shown to be an important regulator for the differentiation of T helper cells in HIV-1-infected individuals, particularly Th17 and regulatory T cells (15,16). However, the profile of miR-155 in HIV-1-infected individuals and its association with CD8+ T cell differentiation remain to be fully elucidated.

In the present study, the expression levels of miR-155 were investigated in CD8+ T cells from patients with HIV-1, with or without highly active antiretroviral therapy (HAART), and correlation between the levels of miR-155 and percentages of CD8+ T cell subsets was examined. It was found that the expression of miR-155 in CD8+ T cells of patients with HIV-1 was increased, particularly in the discord controllers and HAART-naïve patients. The level of miR-155 in CD8+ T cells was positively correlated with the percentages of effector and effector memory subsets, and negatively correlated with the percentages of naïve and central memory subsets.

Subjects and methods

Subjects

Patients with HIV-1 were recruited from the First Affiliated Hospital, School of Medicine, Zhejiang University (Hangzhou, China) between June 2015 and December 2015. HIV-1 infection was diagnosed on the basis of positive results from serological and HIV-1 RNA detection assays. The subjects were excluded if they had received systemic antibiotics, vaccination or any immunomodulatory drug in the previous 3 months. A total of 35 apparently healthy uninfected control subjects from the community clinics were also recruited. The present study received approval from the Ethics Review Boards of the First Affiliated Hospital, School of Medicine, Zhejiang University (approval no. 2015–06103). All subjects were volunteers and provided written informed consent prior to involvement in the study.

Flow cytometry

The subsets of CD8+ T cells were analyzed using four-color flow cytometry with a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and a commercial flow cytometry assay kit (BD Biosciences). Cell staining was performed at room temperature using a cocktail of the following fluorochrome-conjugated antibodies, at the manufacturer's recommended dilution: Anti-CD3-PerCP-CY5.5 (cat. no: 561478), anti-CD8-APC-Cy7 (cat. no: 561967), anti-CD45RA-PE (cat. no: 560975) and anti-CD62L-FITC (cat. no: 561914) (BD Biosciences). Briefly, 50 µl whole blood was added to the antibody cocktail, mixed and then incubated for 20 min in the dark at room temperature. The red cells in whole blood were lysed with red cell lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), washed three times with 1 ml phosphate-buffered saline, and subsequently detected using FACScan flow cytometry. The percentages of CD3+CD8+CD45RA+CD62L+, CD3+CD8+CD45RA−CD62L−, CD3+CD8+CD45RA+CD62L− and CD3+CD8+CD45RA−CD62L+T cells were determined.

Detection of miR-155

Peripheral blood mononuclear cells (PBMCs) were isolated from all patients from 5 ml venous whole blood samples, using a density gradient centrifugation method with Ficoll-Paque PLUS (GE Healthcare Life Sciences, Marlborough, MA, USA) at 2,000 × g for 10 min at room temperature. The CD8+ T lymphocytes were purified from the PBMCs using MACS human CD8 microbeads for positive selection (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The purity of the CD8+ T lymphocytes was >90%. The total RNA was extracted from the CD8+ T lymphocytes using TRIzol (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The quantitative analysis of miR-155 was performed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis with a Bulge-Loop™ miRNA qRT-PCR Starter kit (Guangzhou RiboBio, Co., Ltd., Guangzhou, China) and thehsa-miR-155 qRT-PCR primer set (Guangzhou RiboBio, Co., Ltd.). A U6 small nuclear RNA primer set (Guangzhou RiboBio, Co., Ltd.) was used as the internal control. The experiments were performed according to the protocol provided in the manufacturer of the kit using a 10 µl reaction system. Briefly, the miRNA RT reaction mix included 1 µl RNA template (68 ng/µl), 1 µl miRNA RT primer, 2 µl 5X reverse transcription buffer, 2 µl RTase mix and 4 µl RNase-free water. The mix was incubated at 42°C for 60 min followed by incubation at 70°C for10 min. The miRNA qPCR reaction system contained 10 µl SYBR-Green Master mix, 0.8 µl miRNA forward primer, 0.8 µl miRNA reverse primer and 2 µl RT product; the final volume was normalised to 20 µl with DNase-free water. Real-time PCR was performed for 40 cycles of denaturation (95°C, 45 sec), annealing (62°C, 30 sec) and extension (72°C, 30 sec). Double-stranded DNA was measured at 86°C following each cycle. Each sample was repeated three times. The relative expression levels of miRNA were calculated using the 2ΔΔCq method (17).

Statistical analysis

Statistical analyses were performed using SPSS for Windows version 20.0 (IBM SPSS, Armonk, NY, USA). Student's t-test was used to compare between two groups and one-way analysis of variance was used when comparing more than three groups. χ2 was used for categorical variables. The correlation was tested using Spearman's correlation test. All tests were two-tailed. P<0.05 was considered to indicate a statistically significant difference.

Results

Clinical information of subjects

A total of 94 HIV-1-infected patients were recruited to the present study, including 31 HAART-naïve patients and 63 patients receiving HAART. The patients receiving HAART were divided into two groups according to the recovery of their CD4+ T counts and viral suppression: 34 typical controllers had a CD4+ T count >500 cells/ml and a viral load <400 copies/ml; 29 discord controllers had a CD4+ T count <500 cells/ml or a viral load >400 copies/ml. The three groups of HIV-1-infectedpatientsand normal controls were all appropriately age- and sex-matched. The mean durations of HIV-1 infection for the typical and discord controllers were 6.38±2.51 and 4.96±1.36 years, respectively, which were significantly longer, compared with that for the HAART naïve patients (2.12±1.43 years; P<0.05). The durations of HAART for the typical and discord controllers were 4.17±1.13 and 3.84±1.62 years, respectively. The typical controllers had a significantly lower viral load, and a higher CD4+ T cell count and CD4/CD8 ratio, compared with the other HIV-1 patients (P<0.05). The regimens for patients receiving HAART were primarilyd 4T+3TC+NVP and AZT+3TC+NVP (49 cases; 78%). The detailed participant data is presented in Table I.

Table I. Clinical data from the study participants.

Table I.

Clinical data from the study participants.

Characteristic	Typical controller (n=34)	Discord controller (n=29)	HAART-naïve (n=31)	Control (n=35)	P-value
Males, n (%)	21 (62%)	17 (59%)	19 (61%)	27 (77%)	0.317
Age (years)	41.57±10.01	38.35±15.36	36.41±13.47	39.63±11.79	0.254
Years with HIV-1	6.38±2.51	4.96±1.36	2.12±1.43	NA	0.027
Years on HAART	4.17±1.13	3.84±1.62	NA	NA	0.184
Viral load (log 10)	1.631±0.75	3.91±0.49	4.32±1.54	NA	<0.001
CD4+ cells (/µl)	655.45±263.81	353.68±187.95	431.77±284.56	798.32±261.19	0.001
CD8+ cells (/µl)	618.76±330.17	785.35±296.54	801.52±272.21	518.23±241.36	0.008
CD4/CD8 ratio	1.21±0.42	0. 54±0.22	0.57±0.30	1.63±0.41	0.017
HAART regimen, n (%)					0.116
d4T+3TC+NVP	11 (32%)	9 (31%)	NA	NA	–
AZT+3TC+NVP	17 (50%)	12 (41%)	NA	NA	–
Other	6 (18%)	8 (28%)	NA	NA	–

[i] Data are presented as the mean ± standard deviation. HIV-1, human immunodeficiency virus; HAART, highly active antiretroviral therapy; NA, not applicable.

Subsets of circulating CD8+ T cells are altered in patients with HIV-1. Based on the expression of CD45RA and CD62L, human CD8+ T cells can be divided into four subsets with distinct homing and functional properties: Naïve (CD45RA+CD62L+), central memory (CD45RA−CD62L+), effector memory (CD45RA−CD62L−) and effector (CD45RA+CD62L−) cells (18,19). In the present study, the CD8+ T cell subsets were determined in patients with HIV-1. Compared with the normal controls and typical controllers, the discord controllers and HAART naïve patients showed higher percentages of effector and effector memory CD8+ T cells, and a lower percentage of naïve CD8+ T cells (P<0.05; Fig. 1). The typical controllers had a similar composition of CD8+ T cell subsets to the normal controls, but had a higher percentage of central memory CD8+ T cells (P<0.05; Fig. 1).

Figure 1. Percentage of CD8+ T cell subsets in patients with human immunodeficiency virus type 1 and healthy controls. The subsets of CD8+ T cells were analyzed using four-color flow cytometry. Based on the expression of CD45RA and CD62L, CD8+ T cells were subdivided into four major subsets: Naïve (CD45RA+CD62L+), central memory (CD45RA−CD62L+), effector memory (CD45RA−CD62L−) and effector (CD45RA+CD62L−) cells. *P<0.05; **P<0.01. Data are presented as the mean ± standard deviation. HAART, highly active antiretroviral therapy.

Expression of miR-155 is elevated in CD8+ T cells of patients with HIV-1. The present study compared the expression levels ofmiR-155 in CD8+ T cells of typical and discord controllers with HAART and HAART-naïve patients with normal levels. It was found that the levels of miR-155 in CD8+ T cells of all three groups of HIV-1 patients were significantly higher, compared with that in the normal controls (P<0.05; Fig. 2). Although increased, the expression levels of miR-155 in the CD8+ T cells of typical controllers were almost normal, and were significantly lower, compared with the levels in the discord controllers and HAART-naïve patients, in which viremia was not suppressed (P<0.01; Fig. 2).

Figure 2. Relative levels of miR-155 in CD8+ T cells of patients with human immunodeficiency virustype1 and healthy controls. CD8+ T lymphocytes were purified from peripheral blood mononuclear cells using MACS human CD8 microbeads. Total RNA was isolated and miR-155 was detected using reverse transcription-quantitative polymerase chain reaction analysis. Data are presented as the mean ± standard deviation. *P<0.05; **P<0.01. miR, microRNA; HAART, highly active antiretroviral therapy.

Correlation between levels of miR-155 and percentages of CD8+ T cell subsets in patients with HIV-1. To determine whether miR-155 is associated with CD8+ T cell differentiation in HIV-1-infected individuals, the present study analyzed the correlation between levels of miR-155 and percentages of CD8+ T cell subsets. In all patients with HIV-1, the expression of miR-155 in CD8+ T cells was positively correlated with the percentages of effector and effector memory CD8+ T cells (r=0.692 and 0.803 respectively; P<0.01; Fig. 3), and was negatively correlated with the percentage of naïve and central memory CD8+ T cells (r=−0.457 and −0.522 respectively; P<0.01; Fig. 3).

Figure 3. Correlation between levels of miR-155 and percentages of CD8+ T cell subsets inpatients with human immunodeficiency virus1 infection. Spearman's correlation test was used. miR, microRNA.

Discussion

The cellular immune response is critical in controlling the viral replication of HIV-1 (6–8). The majority of circulating CD8+ T cells in healthy individuals are naïve and central memory cells. When combined with antigens, CD8+ T cells become activated and undergo a program of clonal expansion (20). The majority of effector cells die through apoptosis whereas certain surviving CD8 T cells become long-lasting virus-specific memory CD8+ T cells, forming a protective mechanism upon antigen re-exposure (21,22). In the present study, higher percentages of effector and effector memory cells, and a lower percentage of naïve CD8+ T cells were found in discord controllers and HAART-naïve patients, in which viral replication was not suppressed. In typical controllers with higher CD4+ T counts and suppressed viremia, the CD8+ T cell subsets were restored almost to normal levels. These results are consistent with previous studies, which reported that the majority of circulating CD8+ T cells in individuals with chronic HIV-1 infection were mature effector and effector memory CD8+ T cells (23,24). However, in a cross sectional study by Groves et al (19), discord controllers and typical controllers had higher numbers of naive CD8+ T cells and reduced CD8+ T cell activation, compared with the patients with rapidly progressing disease. Notably, Groves et al defined discord controllers as patients with a viral RNA load <2,000 copies/ml and <450 CD4+ T cells/mm3, with the viral load of discord controllers ranging between 100.3 and 1,043.0 copies/ml. In the present study, the viral load of the discord controllers ranged between 1,491 and 23,386 copies/ml. The higher naïve CD8+ T cells and lower CD8+ T cell activation may be associated with lower virus replication, although no direct association between CD8+ T cell subsets and HIV-1 RNA load was found. These results suggested that a more preserved CD8+ T cell compartment is associated with the control of plasma viremia.

The expansion of effector CD8+ T cells is associated with certain microRNAs, which can control gene expression at the post-transcriptional level. miR-155 is an important microRNA, which regulates the immune response and is important in controlling lymphocyte differentiation at multiple levels (25). miR-155 is essential for normal B cell differentiation and antibody production (26), and controls the differentiation of CD4+ T cells into the Th1, Th2, and Th17 subsets of helper T cells (15,27). In addition, miR-155 is essential for efficient antigen presentation by dendritic cells (28). miR-155 is also reported to regulate CD8+ T cell differentiation. Antigen-specific CD8+ effector T cells express high levels of miR-155 (29). Naïve and central memory cells express low levels of miR-155, and effector memory cells express intermediate levels of miR-155 (13). However, the association between miR-155 and CD8+ T cell differentiation in HIV-1-infected individuals has not been reported. In the present study, increased levels of miR-155 were found in CD8+ T cells of HIV-1-infected patients, which was higher in the discord controllers and HAART-naïve patients. The expression of miR-155 in CD8+ T cells was positively correlated with the percentages of effector and effector memory CD8+ T cells, and negatively correlated with the percentages of naïve and central memory CD8+ T cells. These results were consistent with those of previous studies on other pathogen infections. Lind et al (30) demonstrated that miR-155 was essential for optimal CD8+ T cell responses against influenza virus and Listeria infection, and was crucial for the generation of CD8+ T cell memory against pathogens. Tsai et al (14) showed that miR-155 was an important regulator in effector and memory virus-specific CD8+ T cell responses in murid herpes virus 68-infected mice. Compared with the wild-type mice, chimeric mice lacking miR-155 in CD8+ T cells showed a weaker effector response and a skewing toward memory precursor cells with significantly higher viral titers (14). However, the present study found no correlation between the levels of miR-155 levels and virus replication.

The expression of miR-155 has been reported to be associated with HIV infection. miR-155 can affect disease progression by regulating the transformation of naïve Tregs and naïve CD4 subsets into activate subsets (31,32). In addition, miR-155 exerts an anti-HIV-1 effect by targeting several HIV-1 dependency factors involved in post-entry and pre-integration events, for example, DC-SIGN and TRIM32, leading to severely reduced HIV-1 infection (33). The present study suggested that miR-155 may be an important regulator of the CTL response in patients with HIV. However, how the expression of miR-155 is altered by HIV-1 infection remains to be elucidated, as does the causal association between the expression of miR-155 and virus replication. The mechanisms underlying altered levels of miR-155 in the CD8+ T compartment of HIV-1-infected patients requires further investigation. A limitation of the present study is that miR-155 was detected in total CD8+ T cells, but not in subsets. Further investigations on the expression of miR-155 in subsets of CD8+ T cells in patients with HIV-1 may provide additional information onmiR-155 and the differentiation of CD8+ T cells.

Acknowledgements

This study was supported by the Zhejiang Provincial Science and Technology Foundation (grant no. 2014C33252 & 2015C33183), the National Key Technologies R&D Program for the 12th Five-year Plan of China (grant no. 2012ZX10001-004) and the National Natural Science Foundation of China (grant no. 81402726).

Glossary

Abbreviations

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References