The effect of plasma auto‐IgGs on CD4⁺ T cell apoptosis and recovery in HIV‐infected patients under antiretroviral therapy

Short abstract

Autoantibodies binding on CD4⁺ T cells may mediate CD4⁺ T cell death, and prevent CD4⁺ T cell recovery in aviremic HIV‐infected patients under ART.

Abstract

Although effective antiretroviral therapy (ART) suppresses HIV viral replication, prevents AIDS‐related complications, and prolongs life, a proportion of patients fails to restore the patients’ CD4⁺ T cell number to the level of healthy individuals. Increased mortality and morbidity have been observed in these patients. In the current study, we have investigated the role of auto‐IgGs in CD4⁺ T cell apoptosis and recovery in a cross‐sectional study. All HIV⁺ subjects were on viral‐suppressive ART treatment with a different degree of CD4⁺ T cell reconstitution. Total auto‐IgG binding on CD4⁺ T cell surfaces and its associated apoptosis and CD4⁺ T cell recovery were analyzed by flow cytometry ex vivo. Total IgGs from plasma were tested for their binding capacities to CD4⁺ T cell surfaces and their mediation to CD4⁺ T cell death through NK cell cytotoxicity in vitro. HIV⁺ subjects had increased surface binding of auto‐IgGs on CD4⁺ T cells compared with healthy controls, and IgG binding was associated with elevated CD4⁺ T cell apoptosis in HIV⁺ subjects but not in healthy controls. Plasma IgGs from HIV⁺ subjects bound to CD4⁺ T cells and induced cell apoptosis through NK cytotoxicity in vitro. Soluble CD4 (sCD4) preincubation prevented NK cell‐mediated CD4⁺ T cell death. Our results suggest that plasma autoantibodies may play a role in some HIV⁺ patients with poor CD4⁺ T cell recovery under viral‐suppressive ART.

Abbreviations

ADCC

antibody‐dependent cellular cytotoxicity

APC

allophycocyanin

ART

antiretroviral therapy

mCD

memory cluster of differentiation

nCD

naive cluster of differentiation

sCD

soluble cluster of differentiation

Introduction

The advent of ART has dramatically improved survival and disease progression in HIV‐infected individuals [1]. ART treatment suppresses HIV viral replication, improves immune function, restores peripheral CD4⁺ T cell counts, and decreases morbidity and mortality [2, –, 4]. However, long‐term ART‐treated patients exhibit an increased risk of cardiovascular diseases, cancer, osteoporosis, and other end‐organ diseases [5]. Incomplete CD4⁺ T cell reconstitution is mainly accounting for these aging‐like complications.

The mechanisms of incomplete CD4⁺ T cell recovery in HIV disease have been studied extensively, including direct viral cytopathogenicity, lymphoid fibrosis and thymic insufficiency [6, 7], indirect effects of persistent T cell activation and apoptosis [8, –, 10], gut mucosal dysfunction, and elevated levels of microbial translocation and inflammation [11, 12]. However, the exact mechanism is not fully understood.

In the current study, we examined the potential role of auto‐IgG binding on CD4⁺ T cell surfaces in HIV⁺ subjects after long‐term, viral‐suppressive ART treatment. We found that CD4⁺ T cell apoptosis was elevated in HIV⁺ subjects compared with healthy controls. The percentage of auto‐IgG binding on CD4⁺ T cell surfaces was inversely correlated with CD4⁺ T cell counts in HIV⁺ subjects. IgGs from plasma of HIV⁺ subjects induced CD4⁺ T cell death through NK cytotoxicity (ADCC) in vitro. sCD4 protein preincubation prevented IgG‐mediated CD4⁺ T cell death. Our results suggest that autoantibody‐mediated CD4⁺ T cell death may reveal an important mechanism of incomplete immune recovery in virologically suppressed HIV disease.

MATERIALS AND METHODS

Study subjects

Sixteen healthy controls and 26 HIV⁺ ART‐treated, aviremic, HIV‐infected subjects were evaluated in a cross‐sectional study. The clinical characteristics are shown in Table 1 . All HIV⁺ patients had been on ART treatment and had undetectable plasma HIV‐1 RNA (<50 copies/ml) for at least 2 yr. The clinical characteristics of patients were shown in our previous study [13]. This study was approved by the Institutional Review Board from the Medical University of South Carolina.

Table 1.

Clinical characteristics

Characteristic	Healthy control	HIV+/ART treated	P
Total no. of subjects	16	26
Sex, male/female	5/16	17/26	0.07
Age	38 (32–52)	43 (34–52)	0.99
CD4+ T cell counts	765 (523–936)	540 (366–720)	0.02
Nadir CD4+ T cell counts		294 (193–458)
Years of ART		9 (6–11)

CD4⁺ T cell counts (cells per microliter). Data are medians (interquartile ranges).

Flow cytometry

The fluorochrome‐labeled mAb used in this study included the following: anti‐human CD3 (OKT3), anti‐human CD4 (RPA‐t4), anti‐human CD8 (RPA‐t8), anti‐human CD27 (M‐t271), anti‐human CD45RA (HI100), annexin V (BD Pharmingen; BD Biosciences, San Jose, CA, USA), Ghost Dye Red 780 (Tonbo Biosciences, San Diego, CA, USA), and isotype control antibodies (BD Pharmingen; BD Biosciences). Cells were collected by a BD FACSVerse flow cytometer (BD Biosciences), and data were analyzed by FlowJo software (version 10.0.8; Ashland, OR, USA).

CD4 surface‐bound IgG ex vivo

Plasma was isolated by centrifugation of EDTA‐contained blood, aliquoted, and stored at −80°C. PBMCs were isolated over a Ficoll‐Paque, and freshly isolated PBMCs were used for annexin V assays (see Fig. 1). Thawed PBMC samples were used for detecting surface‐bound IgG on CD4⁺ T cells (see Fig. 2B and C). Fluorescent‐labeled antibodies were incubated with PBMCs at 4°C for 30 min for surface staining, and the cells were washed and stained with annexin V and then analyzed by flow cytometry immediately.

Figure 1.

The absolute CD4⁺ T cell counts and percentages of CD4⁺ T cell apoptosis ex vivo.

The median absolute counts of total CD4⁺ T cells (A) and nCD4⁺ (CD3⁺CD4⁺CD45RA⁺CD27⁺), and mCD4⁺ (CD3⁺CD4⁺CD45RA⁻CD27^+/−) T cells (B) in healthy controls and HIV⁺ subjects ex vivo. The median percentages of annexin V binding on total CD4⁺ T cells (C) and nCD4⁺ and mCD4⁺ T cells (D) in healthy controls and HIV⁺ subjects ex vivo. Mann‐Whitney U test (nonparametric).

Figure 2.

Surface‐bound IgG on CD4⁺ T cells and its association with cell apoptosis and recovery in HIV ex vivo.

The percentages of IgG⁺ on CD4⁺ T cells and annexin V binding were analyzed by flow cytometry. (A) Representative dot plots showing the gating strategies were used to assess gating strategies of anti‐IgG antibody surface binding and cell apoptosis (annexin V) on mCD4⁺ T cells and nCD4⁺ T cells. (B) The median frequencies of surface‐bound IgG on mCD4⁺ T cells and nCD4⁺ T cells among healthy controls and HIV⁺ subjects ex vivo. (C) The median frequencies of annexin V binding on IgG⁺ and IgG⁻ CD4⁺ T cell subsets in HIV⁺ subjects (50 percentile above the frequencies of auto‐IgG binding on CD4⁺ T cells in patients). (D) The correlations between surface‐bound IgG and peripheral CD4⁺ T cell counts in HIV⁺ subjects and healthy controls. Mann‐Whitney U test (nonparametric) and Spearman correlation tests. SSC‐A, Side‐scatter‐area; FSC‐A, forward‐scatter‐area; FSC‐H, forward‐scatter‐height; SSC‐H, side‐scatter‐height.

CD4 surface‐bound IgG detection using plasmas in vitro

PBMCs from a healthy control donor were cultured with PHA (2 μg/ml) at 37°C for 24 h, and plasma from HIV⁺ subjects or healthy controls was inactivated at 56°C for 30 min. Then, PHA‐stimulated PBMCs (5 × 10⁵ cells) were treated with 2.5 μl plasma in 50 μl buffer at 4°C for 60 min. After washing 3× with PBS, 50 μl aqua blue (Thermo Fisher Scientific, Waltham, MA, USA) was used at 4°C for 20 min to exclude dead cells. Next, 50 μl antibody cocktail containing anti CD3‐PerCP (OKT3), CD4‐BV421 (RPA‐t4), CD8‐PE‐Cy7 (RPA‐t8), CD27‐APC‐Cy7 (M‐t271), CD45RA‐FITC (HI100), IgM‐APC (G20‐127), and IgG‐PE (G18‐145) was surface stained at 4°C for 30 min. The cells were washed and analyzed by flow cytometry.

NK‐mediated ADCC

CD4⁺ T cells and NK cells were isolated from aviremic, ART‐treated HIV⁺ subjects or healthy controls for cytolysis and apoptosis assay. In brief, NK cells were isolated from PBMC using an NK cell enrichment kit (Stemcell Technologies, Vancouver, BC, Canada) and CD4⁺ T cells were isolated from PBMC using a CD4 cell enrichment kit (Stemcell Technologies). The purities of CD4⁺ T cells were above 93%, and the purities of NK cells were above 93%.

We pretreated CD4⁺ T cells with sCD4 (Progenics Pharmaceuticals, New York, NY, USA) at a concentration of 25 μg/ml at 4°C for 60 min and stained with anti‐CD4 antibody eBioscience eFluor 670 (Thermo Fisher Scientific). CD4⁺ T cells were pretreated with sCD3 (Abcam, Cambridge, MA, USA) at a concentration of 25 μg/ml as Control 1. Anti‐CD4 mAb (zanolimumab, 6G5) was cultured with CD4⁺ T cells for 15 min and then treated with sCD4 (the concentration of 6G5:sCD4 is 1:5) as Control 2. 6G5 (5 μg/ml), cultured with CD4⁺ T cells without sCD4 or sCD3, was set as a positive control. Next, CD4⁺ T cells were cultured with autologous NK cells at a 3:1 ratio in Corning 96‐well, V‐bottom plates (Millipore‐Sigma, St. Louis, MO, USA). The CD4⁺ T cell cultures, in the absence of 6G5, sCD4, sCD3, and NK cells, were served as the additional negative controls. After incubation, CaCl₂ buffer and annexin V were added to the medium, which contained a constant number of flow cytometry particles (5 × 10⁴/ml; AccuCount blank particles, 5.3 μm; Spherotech, Lake Forest, IL, USA). A constant number of particles (2.5 × 10³) were counted during cytometry acquisition to normalize the number of CD4⁺ T cells. The percentage of cytolysis was calculated using the following formula: %cytolysis = [(number of CD4⁺ T cells of negative control) − (number of CD4⁺ T cells in the presence of anti‐CD4 IgGs, sCD4, or sCD3)]/(number of CD4⁺ T cells of negative control) × 100. Cell apoptosis was analyzed by annexin V binding.

Statistical analysis

All data were analyzed and graphed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA) and SPSS (Version 23; IBM, Armonk, NY, USA). Statistical significance between 2 groups was determined by the Mann‐Whitney U test (nonparametric) and the ANOVA test (paired test) for 3 or more groups. Associations between pairs of continuous variables were analyzed by Spearman correlation tests.

RESULTS

CD4⁺ T cells are highly apoptotic and depleted in viral‐suppressed, ART‐treated HIV⁺ subjects ex vivo

The absolute count and frequency of CD4⁺ T cell subsets were assessed by flow cytometry. Total CD4⁺ T cell, mCD4⁺ T cell (CD3⁺CD4⁺CD45RA⁻CD27^+/−), and nCD4⁺ T cell (CD3⁺CD4⁺CD45RA⁺CD27⁺) absolute counts were still not fully recovered, even after long‐term ART treatment in some HIV⁺ subjects compared with healthy control ( Fig. 1A and B ; P < 0.05). T Cell apoptosis is an important immunologic parameter for HIV disease progression [14]. In untreated HIV patients, T cells undergo apoptosis, leading to an eventual T cell decline [15]. In this study, we analyzed CD4⁺ T cell apoptosis using fresh blood samples. Consistent with the decline of CD4⁺ T cell counts, CD4⁺ T cells, including both nCD4⁺ and mCD4⁺ T cells from HIV⁺ subjects, experienced increased frequencies of T cell apoptosis compared with those from healthy controls (Fig. 1C and D). These results suggest that CD4⁺ T cells are not fully recovered, and their function is not normal, even after long‐term, viral‐suppressive ART treatment.

Increased surface‐bound IgG on CD4⁺ T cells is associated with increased CD4⁺ T cell apoptosis in HIV⁺ subjects under viral‐suppressed ART treatment

Increased surface binding of IgG on CD4⁺ T cells and elevated levels of apoptotic IgG⁺CD4⁺ T cells have been reported in HIV‐infected patients with hemophilia, suggesting that attachment of IgG to CD4⁺ T cells may be associated with cell apoptosis [16]. To determine whether IgG‐bound CD4⁺ T cells could be detected in aviremic, ART‐treated patients, we analyzed the percentages of surface IgG binding on CD4⁺ T cells using anti‐total IgG antibodies. Notably, we observed increased frequencies of IgG surface binding on both mCD4⁺ and nCD4⁺ T cells in HIV⁺ subjects compared with healthy controls ( Fig. 2B ; P < 0.05). Moreover, nCD4⁺ T cells had a higher frequency of IgG surface binding compared with mCD4⁺ T cells in both healthy controls and HIV⁺ subjects (Fig. 2B; P < 0.05).

Next, we chose samples with relatively higher frequencies of auto‐IgG binding on total CD4⁺ T cells (above 5 percentile in patients) and tested annexin V binding. We found that apoptosis was elevated in IgG⁺CD4⁺ T cells compared with IgG−CD4⁺ T cells (Fig. 2C). Importantly, the percentage of IgG binding on CD4⁺ T cells was inversely correlated with the absolute CD4⁺ T cell counts in HIV⁺ subjects (r = −0.476, P = 0.016) but not in healthy controls (r = 0.093, P = 0.74; Fig. 2D). These results suggest that auto‐IgG binding on CD4⁺ T cells may contribute to CD4⁺ T cell apoptosis and depletion in ART‐treated HIV disease in vivo.

Furthermore, to investigate the role of auto‐IgGs from plasma of HIV⁺ subjects in CD4⁺ T cell apoptosis and recovery, total IgGs from plasma were tested for their binding abilities on CD4⁺ T cell surfaces in vitro. Consistent with the amount of auto‐IgGs on CD4⁺ T cell surfaces ex vivo, the amount of auto‐IgGs bound to CD4⁺ T cells was increased significantly in plasma from ART‐treated HIV⁺ subjects compared with healthy controls ( Fig. 3A ). To determine if the presence of auto‐IgGs antibodies was represented specifically on CD4⁺ T cells, we assayed auto‐IgGs on CD8⁺ T cells. Notably, plasma levels of IgG binding on anti‐CD8⁺ T cells were not significantly different between healthy controls and HIV⁺ subjects (Fig. 3B; P > 0.05). Moreover, the frequencies of auto‐IgG binding on CD8⁺ T cells were 0.93 ± 0.47 vs. 1.35 ± 0.94 in healthy controls and HIV⁺ subjects separately (means ± sd), which were much lower than those on CD4⁺ T cells (3.34 ± 1.11 vs. 4.45 ± 1.84 in healthy controls and HIV⁺ subjects separately; Fig. 3A and B).

Figure 3.

Binding abilities of auto‐IgGs from plasma to CD4⁺ T cell surfaces in vitro.

Total IgGs from plasmas of healthy controls and HIV⁺ subjects were cocultured with PBMCs from the same donor; surface IgG binding was tested by flow cytometry. (A) The median frequencies of IgG binding on nCD4⁺ and mCD4⁺ T cells of plasmas from healthy controls or HIV⁺ subjects. (B) The median frequencies of IgG binding on CD8⁺ T cells of plasmas from healthy controls or HIV⁺ subjects. Mann‐Whitney U test (nonparametric).

NK cells purified from ART‐treated HIV⁺ subjects exhibit cytotoxicity to CD4⁺ T cells via surface‐bound auto‐IgGs

To analyze further the potential impact of surface autoantibodies on CD4⁺ T cells, we purified NK cells and CD4⁺ T cells from ART‐treated, HIV‐infected subjects that had high amounts of autoantibody on the CD4⁺ T cell surface (above 5 percentile; Fig. 3A) or from healthy controls. Anti‐CD4 IgGs (zanolimumab 6G5, a human mAb) served as the positive control. Surprisingly, coculture NK cells with autologous CD4⁺ T cells from HIV⁺ subjects resulted in the increases of CD4⁺ T cell apoptosis and cytolysis compared with cells from healthy controls ( Fig. 4A and B ).

Figure 4.

Autoantibody‐dependent, NK cell‐mediated cytolysis of primary CD4⁺ T cells from HIV⁺ subjects in vitro.

CD4⁺ T cells and NK cells were isolated from healthy controls or HIV⁺ subjects. CD4⁺ T cells were cultured with autologous NK cells at a ratio of 1:3 in the presence or absence of 6G5 or sCD4 protein, and the percentage of CD4⁺ T cell apoptosis and cytolysis was analyzed by flow cytometry. The median percentages of annexin V binding (A) and cytolysis (B) of CD4⁺ T cells from healthy controls or HIV⁺ subjects cocultured with autologous NK cells in vitro. Mann‐Whitney U test (nonparametric). The median percentages of IFN‐γ⁺ (C) and CD107a⁺ (D) in NK cells and CD4⁺ T cell apoptosis (E) were shown in culturing cells either from healthy control or HIV⁺ subjects who had low auto‐IgGs on CD4⁺ T cells in the presence of total IgGs at 50 μg/ml from healthy controls or HIV⁺ subjects with high frequency of auto‐IgGs on CD4⁺ T cells and zanolimumab (a positive control) at 5 μg/ml in vitro. Mann‐Whitney U test (nonparametric). The median percentages of CD4⁺ T cell apoptosis (F) or CD4⁺ T cells cytolysis (G) from HIV⁺ subjects cultured with autologous NK cells, with or without sCD4 (25 μg/ml), sCD3 (25 μg/ml), and zanolimumab‐6G5 antibody (5 μg/ml) in vitro. ANOVA, paired.

To determine if the increased CD4 apoptosis is the result of greater proportions of auto‐IgG antibodies, NK activation, or CD4⁺ T cell susceptibility, we cultured NK cells and autologous CD4⁺ T cells with purified total IgGs (50 μg/ml); the cells were from low auto‐IgG HIV⁺ subjects or from healthy controls. Total IgGs were isolated from plasma of high auto‐IgG HIV⁺ patients or healthy controls. Zanolimumab‐6G5 antibody (5 μg/ml) was used a positive control. The CD4⁺ T cells and NK cells from HIV⁺ subjects were more sensitive than those from healthy controls in the presence of patients’ IgGs (Fig. 4C and D; P = 0.0002). Notably, there was no significant induction of CD4⁺ T cell cytolysis in autologous NK cells and CD4⁺ T cells from healthy controls, cultured with total IgG either from controls or from high auto‐IgG HIV⁺ subjects. There was also no significant induction of CD4⁺ T cell cytolysis in autologous NK cells and CD4⁺ T cells from HIV⁺ patients in the presence of total IgG from healthy controls (Fig. 4E). However, when autologous CD4⁺ T cells and NK cells from HIV⁺ subjects were cultured with total IgG from high auto‐IgG HIV⁺ subjects, the induction of CD4⁺ T cell cytolysis was observed (Fig. 4E). These results may suggest that NK activation, CD4⁺ T cell susceptibility, and concentration of autoantibodies are all important for the induction of CD4⁺ T cell death.

To determine the antibody‐binding specificity that mediated CD4⁺ T cell death from HIV⁺ subjects and whether this process is specific to CD4⁺ T cells, we assessed CD4⁺ T cell apoptosis and cytolysis after treatment with sCD4. Our results showed that the positive control anti‐human mAb 6G5 mediated ADCC against CD4⁺ T cells, and sCD4 completely inhibited this effect (Fig. 4F and G). Furthermore, coculture of autologous NK cells and CD4⁺ T cells from HIV⁺ subjects resulted in NK‐mediated CD4⁺ T cell death, and sCD4 significantly reduced this effect (Fig. 4F and G). Unlike sCD4, sCD3 failed to protect the CD4⁺ T cells of HIV⁺ subjects from death (Fig. 4F and G). These results implied that NK cell‐mediated CD4⁺ T cell death in HIV disease is through CD4 binding.

DISCUSSION

In the current study, we found that elevated surface IgG binding on CD4⁺ T cells was inversely correlated with peripheral CD4⁺ T cell counts in aviremic, ART‐treated subjects, and autoantibodies from plasma of HIV⁺ subjects induced CD4⁺ T cell death through NK‐mediated ADCC and CD4 surface binding. These results suggest a possible role of these antibodies in incomplete immune reconstitution in HIV disease.

Previous studies in SIV and HIV have shown inverse correlations between plasma or serum autoantibody levels and peripheral CD4⁺ T cell counts, suggesting that autoantibodies against surface antigens on CD4⁺ T cells may play a role in CD4⁺ T cell decline [16, –, 19]. However, these studies only investigated autoantibodies of diverse surface proteins on CD4⁺ T cells in untreated HIV patients or animal models. In the current study, we show clear evidence that even after long‐term, viral‐suppressive ART treatment, autoantibodies that bind onto CD4⁺ T cell surfaces are not only present but also facilitate CD4⁺ T cell death by NK cell‐mediated ADCC. The different results between previous studies and ours can be a result of different patient populations (ART‐naïve patients in previous studies vs. aviremic, ART‐treated patients in our study). Although autoimmune diseases may present in the stage of acute HIV infection, they mainly occur in the immunologic reconstitution phase after ART [20, 21], suggesting that B cells may produce pathologic autoantibodies during immunologic recovery under ART.

The mechanism of NK cell‐mediated ADCC against CD4⁺ T cells in vitro (Fig. 4E–G) may be a result of auto‐IgG‐activated NK cytotoxicity against IgG⁺CD4⁺ T cells. In addition, the inhibition effect of sCD4 indicates CD4‐specific binding. Therefore, the percentages of NK cell‐induced CD4⁺ T cell death in vitro were low from HIV⁺ subjects (Fig. 4E–G; interquartile ranges, 2–5%); however, the long‐term effect of NK cells in CD4⁺ T cell recovery can be significant in vivo.

Unlike HIV patients, healthy individuals have a low level of autoantibody bound to the surface of CD4⁺ T cells, but their cocultured NK cells did not induce cell death of autologous CD4⁺ T cells in vitro, suggesting a nonpathologic role of autoantibodies in plasma of healthy controls [22]. In addition, CD4⁺ T cells from HIV⁺ subjects may be more susceptible to NK‐mediated cytotoxicity compared with controls [23]. Consistently, our recent study showed that NK cells are activated in HIV patients with CD4⁺ T cell counts ≤350 cells/μl compared with healthy individuals and that the percentages of activated NK cells were inversely correlated with CD4⁺ T cell counts [24]. Notably, sCD4 but not sCD3 inhibited CD4⁺ T cell death induced by coculturing with autologous NK cells and autoantibodies, indicating a CD4‐specific, autoantibody‐mediated ADCC effect. However, whether other autoantibodies against surface antigens on CD4⁺ T cells also play a role in the ADCC needs to be explored further.

The source of the autoantibodies against CD4⁺ T cells still needs to be addressed. Notably, in recent studies found in lymph nodes and tissues, HIV still actively replicates, even in patients with complete viral suppression under ART [25]. As a consequence, HIV may lead to CD4⁺ T cell death directly or indirectly. At the same time, the lower frequencies of macrophages and impaired macrophage function in HIV‐infected subjects [26] failed to clear the apoptotic debris and immune complexes [27, 28], which may result in an increased level of self‐antigens. Furthermore, CD4 antigens from apoptotic CD4⁺ T cells or released HIV protein‐bound CD4 may accumulate in the lymph nodes, providing the antigen stress for pathologic autoantibody generation in HIV patients after ART treatment.

In summary, we found that autoantibodies from HIV patients mediated ADCC against CD4⁺ T cells through NK cytotoxicity. This may lead to incomplete CD4⁺ T cell reconstitution from ART.

AUTHORSHIP

Z. Luo, H.W., L.H., and W.J. conceived of and directed the project. Z. Luo wrote the manuscript. Z.Z., E.O., T.Z., Z.Li, L. Martin, Z.W., J.Z., and L. Ma performed the laboratory sample testing and analyses. Z.Q., T.O., G.L., S.H., and W.J. were involved in critically revising the manuscript.

DISCLOSURES

The authors declare no conflicts of interest.