Irreversible phenotypic perturbation and functional impairment of B cells during HIV-1 infection

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection can damage humoral immunity. The knowledge of B cell perturbations during chronic HIV-1 infection and their recovery after combined antiretroviral therapy (cART) is not complete yet, and thus attempts to further improve humoral immunity are impeded. In this study, an HIV-1 chronically infected cohort with similar demographics, infection history, genetic background, and HIV-1 genotype was established to probe B cell perturbations. Results showed that the B cells from this cohort were highly activated and prone to cell death, and B cell compartments were altered significantly. Notably, although cART partially reversed the hyperactivation and reduced tissue-like memory B cells, other B cell perturbations, including impaired expression of survival factor Bcl-2, costimulatory molecules, and shrunken resting memory B cells, were irreversible. Further functional characterization revealed that the influenza HA-specific antibody-secreting cells were significantly lower during HIV-1 infection, whereas the recalled antibody response to HIV-1-specific antigens was decreased after cART. Finally, CpG plus R848 treatment increased the survival of B cells and memory B cells in vitro from HIV-1-infected patients. In conclusion, this study identified irreversible B cell immune perturbations in chronic HIV-1 infections regardless of cART and proposed the potential strategy to enhance B cell functions through the improvement of cell survival.

Keywords

B cell / HIV-1 / phenotype / functionality / antiretroviral therapy

Introduction

The hallmark of human immunodeficiency virus type 1 (HIV-1) chronic infection is characterized by the progressive depletion and dysfunction of CD4⁺ T cells during virus replication, leading to profound damage to cellular immunity. In parallel, HIV-1 infection leads to extensive defects in the humoral immune arm. HIV-1-induced perturbations of B cells were first reported in 1983 [1]. In patients with acquired immunodeficiency syndrome (AIDS), these perturbations are manifested by hypergammaglobulinemia and inability to develop a proliferative response to the B cell mitogen. Subsequent studies indicated increased polyclonal B cell activation in advanced disease [2,3], as manifested by the increased number of B cells reactive with nonviral antigens, such as DNA, myosin, actin, trinitrophenylated keyhole limpet hemocyanin, and ovalbumin.

Antibody response is important for viral control in human immunity. However, antibody response against HIV-1 is ineffective. Early response is largely directed against non-neutralizing epitopes, and subsequent B cell responses are surpassed by rapidly escaping HIV-1 mutations [4,5]. Moreover, chronic HIV-1 infection systemically perturbs B cell subpopulations, manifested as more activated, exhausted, and terminally differentiated B cells [3,6–8]; more immature B cells [9,10]; and less CD27⁺ memory B cells [11–14] in the different stages of HIV-1 infection. These studies shed light on B cell abnormalities and impaired antibody responses [15]. Besides, B cells are more apoptotic during HIV-1 infections. Activated memory B cells with high levels of CD95 death receptor are significantly increased and highly sensitive to apoptosis induced by CD95L [6,16] in HIV-1-infected patients. Meanwhile, immature B cells that increase during HIV-1 infections are prone to intrinsic apoptosis because of their low antiapoptotic molecule Bcl-2 levels [16].

Some reports showed that effective combined antiretroviral therapy (cART) can reverse the perturbations of B cells. Suppressed HIV-1 viremia is associated with a significant increase in B cell counts, especially naive B cells, and significant decreases in apoptosis-prone B cells. Restoration of resting memory B cells is possible when the initiation time of cART is in early stages of HIV-1 infections [12]. However, many reports found that cART often leads to incomplete recovery of the humoral immunity, even after the long-term suppression of HIV-1 viral load [17]. Complicated factors may contribute to the persistent impairment of B cell immune compartments, and the underlying mechanisms are not thoroughly understood yet. The parts of the B cell immunity that are irreversibly dampened and the parts that are restored early after cART are still unclear. A strategy to improve B cell immunity in the cART era is also urgently needed.

In the previous study, we established an HIV-1-infected cohort consisting of unregulated commercial former plasma donors (FPDs), who acquired HIV-1 between 1992 and 1995 in central China. The practice of using contaminated blood collection equipment or reinfusing pooled blood cells back to the donors caused a rapid HIV-1 spread among those FPDs within a narrowed period [18]. In addition, they had similar demographics and genetic backgrounds, and the majority of them were infected with HIV-1 subtype B. Therefore, this cohort represented a unique population to probe the B cell perturbations during chronic HIV-1 infection.

Taking advantage of this HIV-1-infected cohort, we comprehensively investigated the B cell perturbations, including their phenotype, subsets constitutions, and immune functions, in both cART-naive and cART-treated patients. We identified both reversible and irreversible B cell immune perturbations after cART and proposed a novel strategy to enhance B cell functions through enhancing the cell survival in chronic HIV-1-infected patients.

Materials and methods

Human study subjects

In this study, 66 HIV-1-infected individuals were enrolled. Of these individuals, 32 were naive to cART and 34 were receiving cART, and 12 HIV-negative individuals were recruited as controls. The demographic information and clinical characteristics of the subjects are listed in Table 1. Human subject protocols were followed as previously described [17,19]. Written informed consents were provided by all participants. The overall study was reviewed and approved by the Ethics Committee of Shanghai Public Health Clinical Center. After enrollment, the patients were monitored quarterly, and the absolute CD4⁺ T cell counts were measured in every visit and viral loads were tested semiannually.

Sample collection

Whole blood specimens were collected in heparinized sterile tubes. All samples were transferred to the laboratory within 12 h after collection. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation and then were prepared for multicolor flow cytometry analyses or functionality detection. Fresh whole blood samples were also used for CD4⁺ T and B cell counting, and plasma samples were stored at −80 °C for HIV-1 viral load measurement.

B cell subpopulations and phenotype characterization

B cell subpopulations were defined by the following fluorochrome-conjugated antihuman monoclonal antibodies: ECD (PE-Texas Red) anti-CD19 (Beckman Coulter, Indianapolis, IN, USA); Phycoerythrin-Cy7 (PE-Cy7) anti-CD10, PerCp anti-CD20, PE anti-CD21, APC-Cy7 (Allophycocyanin-Cy7) anti-CD27 (BioLegend, San Diego, CA, USA); and APC anti-CD21 (eBioscience, San Diego, CA, USA).

For the characterization of the phenotypes of B cell subpopulations, PBMCs were stained with additional fluorochrome-conjugated antihuman monoclonal antibodies: FITC anti-CD38, Pacific Blue anti-CD95, and APC anti-PD-1 (BioLegend, San Diego, CA, USA); FITC anti-CD40 (eBioscience, San Diego, CA, USA); and PE anti-CD70 (BD Biosciences, Franklin Lakes, CA, USA). For intracellular stains, cells were first stained for cell surface markers and then fixed and permeabilized with Fixation/Permeabilization Solution (BD Biosciences, Franklin Lakes, CA, USA). The antibody for intracellular stains was V450 anti-Bcl-2 and PE anti-Ki-67 (BD Biosciences, Franklin Lakes, CA, USA). The Live/Dead blue dye was used to exclude dead cells. A total of 200 000 events were collected and analyzed using FACSAria II flow cytometer (BD Biosciences, Franklin Lakes, CA, USA). The data were analyzed with FlowJo version 10.0.7 software (Tree Star Inc., Ashland, OR, USA).

B cell stimulation

B cells were enriched using B cell negative selection kit (Human B cell Enrichment kit, STEMCELL Technologies, Vancouver, Canada), according to the manufacturer’s specifications. The purity of B cells was consistently>90%. Enriched B cells were stimulated with 5 mg/mL of CpG-B ODN 2006 (InvivoGen, San Diego, CA, USA), 1 mg/mL of R848 (Mabtech, Nacka Strand, Sweden), and 10 ng/mL of IL-15 (PeproTech, Rocky Hill, NJ, USA) [20,21] for 4 days. At day 4, the cell culture supernatants and the cells were collected separately for functionality analysis.

B cell functionality analysis

The ability to secrete antigen-specific antibodies was measured using optimized ELISpot assay, which was described in a published study by Dosenovic et al. [22]. Eight-well nitrocellulose filtration strips (EMD Millipore, Billerica, Massachusetts, USA) were coated with 15 mg/mL of anti-IgG (Mabtech) and then incubated overnight at 4 °C. After the plates were blocked, 2 × 10⁵ stimulated B cells per well were plated and incubated for 20 h at 37 °C. For the detection of spots, the cells were removed by washing the plates with phosphate buffer saline (PBS) containing 0.05% Tween20 (PBST). A total 100 ng/well biotinylated-gp120 or biotinylated-HA antigens diluted in PBS containing 5% FBS was added. After 2 h of incubation, plates were washed before the addition of 100 mL of ALP-conjugated streptavidin (Mabtech). Plates were incubated for 1 h at room temperature and then washed. BCIP/NBT-plus substrate (100 mL; Mabtech) was added and incubated for 10 min at room temperature. Plates were then washed extensively with water and air-dried. Spots were counted in an ImmunoSpot analyzer (Cellular Technology, Kennesaw, Georgia, USA).

The ability to secrete cytokines was evaluated using a multiplexed bead array system (Cytometric Bead Assay kit and Flex Sets, BD Biosciences). The cell culture supernatants were incubated with cytokine capture beads and PE-conjugated detection reagent and then measured using FACSAria II flow cytometer. The data were analyzed with FCAP Array software (BD Biosciences) according to the manufacturer’s specifications. The cytokines assayed were interlukin-1b (IL-1b), interlukin-6 (IL-6), interlukin-8 (IL-8), interlukin-10 (IL-10), interlukin-12p70 (IL-12p70), tumor necrosis factor-a (TNF-a), lymphotoxin-a (LT-a), chemokine (C-C motif) ligand 3 (CCL-3), and transforming growth factor-b (TGF-b).

B cell survival assay upon TLR ligand treatment

The B cell immune reconstruction was evaluated using the survival assay, which was manifested by Annexin V staining (eBioscience, San Diego, CA, USA). CD19⁺CD27⁺ memory B cells were isolated using a flow cytometer after being stained with ECD-anti-CD19 and APC-Cy7-anti-CD27 and cultured at 37 °C in the presence or absence of 5 mg/mL of CpG and 1 mg/mL of R848 for 24 h. Annexin V staining was performed to assess the survival assay.

Statistical analysis

Nonparametric Mann–Whitney U test was conducted for intergroup comparisons, and Spearman rank correlation tests were performed with GraphPad Prism version 6.0 (GraphPad Software, La Jolla, CA, USA) to evaluate the data. A P-value of less than 0.05 was considered significant.

Results

Characteristics of the studied cohort

The participants consisted of three groups, namely, the HIV-infected ART-naive group, ART-treated group, and HIV-negative control group. The demographic data are shown in Table 1. The patients acquired HIV-1 infection from unregulated blood/plasma donation, and HIV-1 strains in this cohort were all subtype B. Majority of the patients had the infection for more than seven years, and all the ART-treated patients showed HIV-1 viral load lower than the detection limit (HIV RNA 50 copies/mL).

We observed that CD4⁺ T cell and B cell counts were significantly declined in HIV-infected ART-naive individuals compared with those of the HIV-negative controls (P<0.001 and P = 0.05, respectively; Fig. 1A and 1B). The ART-treated group showed a trend of increase in the B cell counts but not in the CD4⁺ T cell counts. In addition, B cell counts were positively correlated with CD4⁺ T cell counts in ART-naive patients (P = 0.037, Fig. 1C).

Compared to the HIV-infected groups, the HIV-negative group was significantly younger. We agreed that the age difference might be a confounding factor. However, recent report found no significant gene deregulation in B cells from donors below 60 years [23]. In addition, another study in China with a large sample size showed that old age affected the T cell numbers and phenotype; however, B cells remained stable in aging individuals [24]. Therefore, we considered that the differences between the HIV-negative controls and HIV-infected patients were mainly related to HIV or cART, not to age difference.

Increased expression of activated and apoptotic markers and impaired costimulatory signal on B cells during HIV-1 infection

HIV-1 infection leads to the chronic activation of both innate and adaptive immunity. The hyperactivation of B cells is described as hypergammaglobulinemia and polyclonal activation [1,2,25]. However, the expression of various molecules on B cells is controversial [26,27]. In this study, we investigated the expression levels of activation marker CD38 and cell turnover marker Ki-67 on CD19⁺CD20⁺ B cells. The HIV-infected ART-naive patients showed significantly higher levels of CD38 than HIV-negative controls. Between the ART-naive and ART-treated patients, we found that ART treatment partially decreased CD38 (Fig. 2B). However, Ki-67 in B cells was also increased in the ART-naive and ART-treated patients (Fig. 2C).

B cells isolated from HIV-1-infected patients are more susceptible to apoptosis. To investigate whether the lower counts of B cells in HIV-infected individuals were caused by apoptosis, the expression of the proapoptotic molecule CD95 and antiapoptotic molecule Bcl-2 was assessed using flow cytometry. CD95 expression significantly increased, whereas Bcl-2 expression decreased on B cells from the ART-naive patients, compared with HIV-negative individuals (Fig. 2D and 2E). In addition, ART decreased the CD95 expression though it remained higher than HIV-negative controls. The perturbed Bcl-2 molecules were not restored by ART.

To characterize the interaction molecules between T and B cells, we examined the expression of exhausted molecule PD-1 and costimulatory molecules CD40 and CD70 in the HIV-1-infected patients. The level of PD-1 showed a trend of increase, whereas CD40 and CD70 decreased significantly on B cells from the ART-naive patients compared to the HIV-negative individuals (Fig. 2F–2H). Moreover, ART cannot fully restore these impaired alterations.

Perturbation of B cell subpopulations in HIV-1 infection

We examined the frequencies of B cell subpopulations. As shown in Fig. 3A, B cell subsets were defined as immature B (CD10⁺CD27⁻), naive B (CD10⁻CD27⁻CD21⁺), resting memory B (CD10⁻CD27⁺CD21⁺), activated memory B (CD10⁻CD27⁺CD21⁻), tissue-like memory B (CD10⁻CD27⁻CD21⁻), and plasmablasts (CD10⁻CD27⁺⁺CD21⁻CD20⁻) [7,12,28]. In contrast to HIV-negative controls, naive and resting memory B cells shrank significantly, whereas tissue-like memory B cells expanded considerably during HIV-1 infection (Fig. 3B). In other minor subsets, the frequency of immature B cells and plasmablasts decreased whereas activated memory B cells increased in HIV-1 infections. After ART, naive B cells recovered, whereas those of tissue-like and activated memory B cells decreased. However, the frequences of immature and resting memory B cells in the ART group remained significantly lower compared to those in the HIV-negative controls (Fig. 3B).

A high level of CD38 was found on immature B, naive B, resting memory B, and tissue-like memory B cells in HIV-infected ART-naive patients. After ART, CD38 levels significantly decreased on immature B and naive B cells (Fig. 4A). Ki-67 expression on each B cell subpopulation was comparable among the three groups (Fig. 4B). All B cell subpopulations from HIV-1-infected ART-naive patients showed increased CD95 and decreased Bcl-2 levels, compared with those from the HIV-negative individuals (Fig. 4C and 4D). PD-1 expression on resting memory B cells and plasmablasts was higher in the ART-naive patients than in the HIV-negative individuals (Fig. 4E). ART decreased the CD95 expression on resting memory B and activated memory B cells but not on other subsets, whereas ART did not affect the Bcl-2 and PD-1 expression on any B cell subpopulation.

The expression levels of costimulatory molecule CD40 on each B cell subpopulation were decreased in the HIV-infected ART-naive patients (Fig. 4F). For CD70, resting memory B and activated memory B cells showed decreased CD70 expression during HIV-1 infection (Fig. 4G). ART did not restore CD40 and CD70 expression.

Impaired immune responses of B cells from HIV-1-infected individuals

Whether phenotypic differences in HIV-1 infection were associated with alterations in B cell function was considered. HIV/gp120-specific antibody-secreting cells (ASCs) and influenza HA ASCs were examined. First, total IgG ASCs frequencies were significantly higher in the HIV-negative than those in the HIV-1-infected ART-naive group (P = 0.05, Fig. 5B), whereas a slight restoration was observed after ART. For antigen-specific ASCs, influenza HA-specific ASCs were significantly higher in the HIV-negative controls than those in the HIV-1-infected patients (P = 0.028, Fig. 5B). However, HIV/gp120-specific ASCs in the ART-naive patients were significantly higher than those in the ART-treated patients (P<0.01, Fig. 5B) possibly because of the lack of antigen stimulation after ART treatment [12,29,30]. In addition, we found that CD95 expression on B cells was negatively correlated with the frequency of hemagglutinin (HA)-specific ASCs, whereas no correlation was found with gp120-specific ASCs (Fig. 5B).

The effector properties of B cells following polyclonal stimulation were also evaluated for cytokine secretion (Fig. 5C). The levels of LT-a, IL-10, IL-6, IL-8, and CCL-3 were significantly lower in the HIV-1-infected ART-naive patients, compared with those in the HIV-negative controls. After ART, LT-a, IL-10, and IL-6 were restored, whereas other cytokines were not.

Enhanced survival of B cells by TLR stimulation

The perturbed phenotype and function of B cells from the HIV-1-infected patients can only be partially restored by ART. We hypothesized that B cell function might be improved by preventing cell death. Considering that CpG was reported to promote survival of B cells and pDC [31], we tested the effect of TLR ligands on CD27⁺ classical memory B cells isolated from the HIV-1-infected individuals. Our data showed that the treatment of CpG plus R848 significantly reduced cell death of total B cells and CD27⁺ classical memory B cells (Fig. 6A and 6B).

Discussion

Patients with HIV-1 infection exhibit highly activated and apoptotic B cell phenotypes [16,32]. Our findings confirmed the increased proportions of CD38⁺ B cells in HIV-infected patients. In addition, B cells showed significantly increased CD95 and decreased Bcl-2 levels. However, although successful viral suppression by ART increased the counts of CD4⁺ T cells, CD38 and CD95 expression remained higher and Bcl-2 expression was lower than those in HIV-negative controls, suggesting a persistence of highly activated and apoptotic status even after ART.

The importance of PD-1 has been emphasized in the development of hyperactivation and exhaustion of T cells during chronic viral infections, including HIV-1 [33–35]. We were interested in the expression of PD-1 on B cells during HIV-1 infection because previous studies demonstrated that PD-1 expression on activated memory B cells in SIV infection is associated with rapid disease progression [36]. In this study, an elevated PD-1 expression on B cells was observed, and ART could not reduce it, suggesting a persistent exhaustion of B cells even after ART.

Regarding the interaction molecules of T and B cells during HIV-1 infection, we found that the expression of costimulatory molecules CD40 and CD70 was significantly decreased on B cells during HIV-1 infection. The defect of CD40 expression on B cells along with the low expression of CD40L on T cells with HIV-1 infection [37] seemed to contribute to the impaired B and T cell interactions. The decreased levels of CD70 on B cells were also consistent with a previous study in asymptomatic HIV-infected individuals [27], indicating the impaired costimulatory axis. Importantly, ART was not able to reverse these alterations in our study. Overall, the current data indicated an irreversible dysfunction of costimulatory signals of B cells during HIV-1 infection, regardless of ART.

Disturbances in the differentiation and function of B cells occurred during HIV-1 infection, and often these impairments correlated with the loss of CD4⁺ T cells and the increase of HIV-1 load [25,38]. In the present study, we demonstrated a correlation of CD4⁺ T cell counts and B cell counts in HIV-1-infected ART-naive patients. Among the decreasing B cells, not all B cell subpopulations were diminished. In particular, the tissue-like memory B cells were expanded, whereas the frequencies of naive and resting memory B cells were decreased in the ART-naive compared to those in the HIV-negative controls. These findings were consistent with previous studies [28]. Noticeably, immature B cells and plasmablasts were not increased but decreased in HIV-1 infection in our study. Immature B cells were previously found to be inversely correlated with CD4 counts in HIV-1-infected patients with advanced-stage diseases, and they might appear following the large reduction of CD4 T cell counts [7,10]. Plasmablasts can increase significantly after acute HIV-1 infection, but the frequency of plasmablasts decreased in chronic phases [39]. In addition, most of the patients in our cohort were slow progressors (the drop of CD4 counts every year was lower than 30%). Thus, the profiles of their plasmablasts may be different from those reported in previous studies. In this study, ART can partially normalize naive B cells and decrease tissue-like memory B cells, whereas other subsets were still not restored. Overall, our study showed that the perturbations of B cell subpopulations during HIV-1 infection could not be fully restored by ART.

Functionally, we examined the abilities of B cells to secrete antibodies and cytokines. The lower frequencies of total IgG and HA-specific ASCs from the HIV-1-infected ART-naive group indicated their impaired recall antigen responses. With regard to HIV-1-specific responses, high levels of HIV/Env-specific ASCs were observed in the ART-naive patients. Moreover, negligible ASCs were detected after ART, indicating that Env-specific antibodies were possibly secreted by short-lived plasma cells. In addition, many reports have demonstrated that the maintenance of HIV-specific antibodies and memory B cells was dependent on the presence of HIV-1 antigen [30,40–42]. For cytokines secretion, B cells from the HIV-1-infected ART-naive individuals showed severely suppressed production of LT-a, IL-10, IL-6, IL-8, and CCL-3. The loss of LT-a production is striking and may have important implications given the fact that B cells are the main source of LT-a and that germinal centers do not form properly and antibody responses are defective in the absence of LT-a [43]. Of note, after ART, the levels of total IgG and the secretion of LT-a, IL-10, and IL-6 were increased to comparable levels of the HIV-negative controls, suggesting that some functions of B cells could indeed be restored after ART.

In this study, the ART-naive and ART-treated groups, although having similar CD4⁺ T cell counts, differed in viral load only, and thus the viral effects on B cell perturbations can be compared. We found that ART can decrease the expression of CD38 and CD95 but spared Ki-67, Bcl-2, PD-1, CD40, and CD70. Regarding the B cell compartments, only naive B and tissue-like memory B cells were significantly affected, whereas other subsets had no obvious changes. Moreover, the total IgG ASCs and the production of cytokines, such as LT-a, IL-10, and IL-6, were improved after ART. These findings demonstrated that ART can significantly decrease B cell activation and extrinsic apoptosis and improve the cytokine secretion. However, ART failed to reverse the enhanced intrinsic apoptosis and improve costimulatory molecule expressions and recall responses to antigens. Therefore, additional strategies were urged to remedy the perturbed B cells. Interestingly, a negative correlation of CD95 expression on B cells and HA-specific ASCs was observed, suggesting that the survival rate enhancement of B cells may increase the specific response to HA antigens. Indeed, TLR ligands, CpG plus R848, increased the survival of memory B cells, indicating that TLR ligands could be utilized to improve B cell perturbations.

In summary, B cells from the HIV-1-infected patients showed hyperactivation and apoptosis, impaired costimulatory signals, perturbed subset distributions, and decreased responses to recall antigens. ART initiation can partially reverse the high activation and extrinsic apoptosis, increase naive B cells, reduce tissue-like memory B cells, and benefit cytokines secretion. However, increased intrinsic apoptosis and exhaustion, impaired costimulatory signals on B cells, reduced resting memory B cells, and impaired antigen-specific ASCs are unlikely to be restored by only ART. Therefore, additional immune intervention strategies may correct these persistent B cell perturbations. In this study, we proposed that CpG plus R848, ligands of TLR9 and TLR7, may assist B cell recovery during HIV-1 infections through enhancing the survival of B cells, especially classical memory B cells.

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