Enhanced Activity of Akt in Teff Cells From Children With Lupus Nephritis Is Associated With Reduced Induction of Tumor Necrosis Factor Receptor–Associated Factor 6 and Increased OX40 Expression

Authors


Abstract

Objective

The breakdown of peripheral tolerance mechanisms is central to the pathogenesis of systemic lupus erythematosus (SLE). Although true Treg cells in patients with SLE exhibit intact suppressive activity, Teff cells are resistant to suppression. The underlying mechanisms are incompletely understood. This study was undertaken to examine the Akt signaling pathway and molecules that may alter its activity in T cells in lupus patients.

Methods

The Akt pathway and its regulators were analyzed in Teff and Treg cells from children with lupus nephritis and controls using flow cytometry and real-time quantitative polymerase chain reaction. T cell proliferation was assessed by analysis of 5,6-carboxyfluorescein succinimidyl ester dilution.

Results

CD4+CD45RA−FoxP3low and FoxP3− Teff cells from children with lupus nephritis expressed high levels of activated Akt, resulting in the down-regulation of the proapoptotic protein Bim and an enhanced proliferative response. The induction of tumor necrosis factor receptor–associated factor 6 (TRAF6) was impaired, and TRAF6 levels inversely correlated with Akt activity. Although the expression of OX40 was enhanced on Teff cells from children with lupus nephritis compared to controls, OX40 stimulation failed to significantly increase TRAF6 expression in cells from patients, in contrast to those from healthy controls, but resulted in further increased Akt activation that was reversed by blockade of OX40 signaling. Moreover, inhibition of Akt signaling markedly decreased the proliferation of Teff cells from lupus patients.

Conclusion

Our findings indicate that hyperactivation of the Akt pathway in Teff cells from children with lupus nephritis is associated with reduced induction of TRAF6 and up-regulation of OX40, which may cause Teff cell resistance to Treg cell–mediated suppression.

SLE (systemic lupus erythematosus) is characterized by an imbalance in T cell homeostasis and the aberrant activation and effector function of T cells, resulting in enhanced autoreactive T cell proliferation, enhanced helper function for B cells, and the production of multiple pathogenic antibodies ([1, 2]). T cells also play a critical role in renal inflammation and injury in lupus through direct effects ([3, 4]).

The incidence of nephritis is greater in children with SLE than in adults ([5]), and though the prognosis has improved over the past decades, lupus nephritis remains a major cause of mortality and morbidity ([6]). The disease course of lupus nephritis in children is more severe than that in adults, requiring intensive immunosuppressive therapy that is associated with severe side effects ([7, 8]). Moreover, in some patients the disease is refractory to conventional therapy ([9, 10]). Thus, novel drugs with greater specificity are highly needed.

The mechanisms by which T cells responsive to ubiquitous self antigens in SLE escape tolerance remain incompletely understood; although the central tolerance mechanisms appear intact ([11]), peripheral tolerance fails at various points ([12, 13]). The mechanisms of peripheral tolerance are diverse and include induction of anergy, activation-induced cell death, and suppression by Treg cells, each of which requires an appropriate intracellular signal transduction response of the responding T cell.

Previous studies showed controversial results regarding the frequency and function of Treg cells in patients with SLE ([14]). More recently, however, it has been shown that true Treg cells in patients with SLE exhibit an intact suppressive activity ([15]). In addition, recent findings indicate that CD4+CD45RA− T cells that express FoxP3, a key transcription factor for Treg cells, at low levels (CD4+CD45RA−FoxP3low T cells) are unable to suppress the proliferation of responder T cells, but produce proinflammatory cytokines such as interleukin-17 ([16]). Moreover, this CD4+CD45RA−FoxP3low effector-like non-Treg cell subset is significantly increased in adults with active SLE, suggesting a pathogenic significance ([16]).

Intriguingly, several recent studies have demonstrated that Teff cells are resistant to Treg cell–mediated suppression in murine models of SLE and in humans ([13, 15, 17, 18]). Moreover, in patients with juvenile idiopathic arthritis, hyperactivation of the Akt pathway in Teff cells from the site of inflammation causes these cells to become resistant to suppression ([18]). The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated (phosphorylated) downstream of many receptors on the T cell surface, including the T cell receptor and CD28, cytokine, adhesion, and chemokine receptors ([19-21]), and this pathway promotes various cell responses associated with survival, cell division, and proliferation ([22, 23]).

Several studies in genetically altered mice have linked the PI3K/Akt pathway with autoimmune susceptibility ([24]). Transgenic mice that express an active form of PI3K in T cells exhibit enhanced PI3K activation, resulting in the activation of Akt that is sufficient to enhance CD4+ memory T cell survival and to produce lymphoproliferative disorder and autoimmune renal disease ([25]). Moreover, CD4+ T cells from MRL-lpr mice display elevated levels of activated Akt compared to wild-type mice, and the inhibition of PI3Kγ reduces glomerulonephritis and prolongs survival in the MRL-lpr mouse model of SLE ([26]).

Tumor necrosis factor receptor–associated factor 6 (TRAF6), an important signaling adaptor that functions downstream of tumor necrosis factor receptor (TNFR) superfamily members, contributes to the maintenance of peripheral tolerance by rendering T cells responsive to Treg cell–mediated suppression ([27]). Findings in a transgenic animal model show that the T cell–specific loss of TRAF6 results in multiorgan inflammatory disease; moreover, these T cells show hyperactivation of the PI3K/Akt pathway and are resistant to Treg cell–mediated suppression ([27]).

Furthermore, OX40 (CD134), a member of the TNFR superfamily, promotes the activation of Akt, acts as an important regulator of T cell effector functions ([28]), and augments cell survival and expansion ([29]). In contrast, OX40 signaling inhibits Treg cell differentiation ([30]) and activity ([29]) and counteracts the suppression of CD4+CD25− T cells by Treg cells ([31]).

In patients with SLE, the molecular mechanisms that underlie the resistance of Teff cells to Treg cell–mediated suppression are poorly known. This prompted us to investigate the levels of activated Akt, Bim, TRAF6, and OX40 expression in Teff cells from children with lupus nephritis. In this study, we show that CD4+CD45RA−FoxP3low and FoxP3− T cells from children with lupus nephritis display elevated levels of activated Akt, a down-regulation of the proapoptotic protein Bim, and an enhanced proliferative capacity compared to controls. The hyperactivation of the Akt pathway in CD4+CD45RA−FoxP3low and FoxP3− T cells is associated with the reduced induction of TRAF6 and an increased expression of OX40. Moreover, inhibition of Akt activity significantly reduces the proliferative response of Teff cells from lupus patients.

MATERIALS AND METHODS

Patients

Peripheral blood samples were obtained from 17 pediatric patients with definite lupus nephritis (mean ± SEM age 15.1 ± 0.9 years), 5 patients with frequently relapsing nephrotic syndrome (minimal-change glomerulonephritis or focal segmental glomerulosclerosis) (mean ± SEM age 9.3 ± 1.6 years), and 22 age-matched healthy controls (mean ± SEM age 14.2 ± 0.4 years). The pediatric patients with lupus nephritis were recruited from the Departments of Pediatrics in Innsbruck (Austria) and Heidelberg, Rostock, and Bremen (all in Germany). The patients with nephrotic syndrome visited the Department of Pediatrics in Innsbruck, and the age-matched healthy controls underwent blood testing for elective surgery at the Department of Pediatric Surgery of Innsbruck Medical University. None of the patients or controls had an infectious disease.

Patients were diagnosed as having pediatric SLE according to the revised classification criteria of the American College of Rheumatology for definite SLE with disease onset prior to 16 years of age ([32, 33]). Renal biopsy samples were subjected to histologic analysis and classified according to the International Society of Nephrology/Renal Pathology Society criteria ([34]). Three patients had class II, 5 patients had class III, and 9 patients had class IV nephritis. Disease activity was determined using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) ([35, 36]). Eight patients had active lupus nephritis (median SLEDAI-2K score 14 [range 10–26]; median renal SLEDAI-2K score 12 [range 8–12]), and 9 had inactive lupus nephritis (median SLEDAI-2K score 0 [range 0–2]; median renal SLEDAI-2K score 0). At the time of sampling, patients with lupus nephritis were receiving no treatment (n = 3), mycophenolate mofetil (MMF) plus prednisone (n = 6), prednisone alone (n = 2), MMF plus prednisone plus hydroxychloroquine (n = 2), prednisone plus hydroxychloroquine (n = 1), MMF plus cyclosporine (n = 2), or MMF alone (n = 1). Patients with nephrotic syndrome were treated with prednisone (n = 1), MMF (n = 2), or cyclosporine (n = 2). Blood samples were obtained twice from 7 patients with lupus nephritis (4 with active lupus nephritis and 3 with inactive lupus nephritis).

Written informed consent was obtained from all patients and parents prior to inclusion. The study was performed according to the 1964 Declaration of Helsinki and its later amendments with the approval of local ethics committees.

Cell isolation, stimulation, and culture

Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque Plus (GE Healthcare Biosciences) density-gradient centrifugation either immediately after the blood withdrawal for immediate fluorescence-activated cell sorting analysis or within 24 hours of blood collection. For the time-course experiments examining phospho-Akt expression, PBMCs were incubated with anti-CD3 (3 μg/ml) and anti-CD28 (1 μg/ml) on ice for 20 minutes, followed by incubation with goat anti-mouse Ig (BD Biosciences). Then, after washing, cells were incubated at 37°C for 5, 10, or 30 minutes. Alternatively, PBMCs were cultured in 96-well round-bottomed plates (BD Biosciences) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin (all from Gibco Life Technologies). For the dose-response experiments, PBMCs were stimulated with increasing concentrations of anti-CD3 (0.5, 1, and 3 μg/ml) and anti-CD28 (0.5 and 1 μg/ml) for 24 hours. After resting, T cells from lupus patients regain intrinsic hyperactivity and intrinsic defects can be detected ([37]). Thus, after 3 days of resting, cells were stimulated with functional grade purified anti-CD3 (OKT3; 1 μg/ml) (eBioscience) and functional grade purified anti-CD28 (CD28.6; 1 μg/ml) (eBioscience) for 24 hours (for analysis of pAkt, TRAF, and Bim) or for 48 hours (for analysis of OX40) and cultured at 37°C with 5% CO2. In some experiments, recombinant human soluble OX40 ligand (sOX40L) (100 ng/ml; PeproTech) was added to the cultures. For costimulation blockade experiments, PBMCs were activated with anti-CD3/CD28 in the presence of recombinant human sOX40L (100 ng/ml) for 48 hours and a neutralizing antibody (100 ng/ml; Abcam or 5 μg/ml; R&D Systems) or isotype-matched control antibodies.

For those experiments that were performed without a resting period (time-course and dose-response experiments analyzing pAkt expression, TRAF6 expression analysis following OX40 stimulation, and T cell proliferation assays of cells stimulated for 4 days), cells were prepared from 1 patient with current early reactivation because of medication noncompliance who was not currently receiving therapy, 1 patient with inactive lupus nephritis who was receiving low-dose MMF therapy, 1 patient with inactive lupus nephritis who was not currently receiving therapy, and control subjects.

Flow cytometric analysis

After 5, 10, or 30 minutes or 24 or 48 hours of T cell activation, or immediately after blood collection and PBMC preparation, cells were fixed and permeabilized with Fixation/Permeabilization Buffer set (for analysis of pAkt, Bim, and TRAF; eBioscience) or Cytofix/Cytoperm kit (for analysis of OX40; BD Biosciences). Cells were stained with different combinations of monoclonal antibodies specific for CD4 (SFCI12T4D11; Beckman Coulter), CD25 (M-A251; BD PharMingen), CD8a (RPA-T8), FoxP3 (236A/E7), CD45RA (HI100), CD134 (ACT35) (all from eBioscience), Akt (5G3), phospho-Akt (Ser 473) (D9E), Bim (C34C5), TRAF6 (D21G3), IgG Fab2 anti-rabbit (all from Cell Signaling Technology), or isotype-matched control antibodies. Samples were acquired on a Beckman Coulter Cytomics FC500 system and analyzed with CXP software version 2.2.

Proliferation assay

PBMCs were labeled with 5 μM 5,6-carboxyfluorescein succinimidyl ester (CFSE; eBioscience), washed, recounted, and stimulated with anti-human CD3 and anti-human CD28 (1 μg/ml each; eBioscience) either for 7 days after a 3-day resting period or for 4 days immediately after PBMC preparation. In some experiments, Akt inhibitor VIII (0.2 μM; Calbiochem [EMD Millipore]) or diluent was added to the culture. Proliferation of CFSE-labeled cells was assessed by flow cytometry after 4 or 7 days. The proliferation index was calculated using ModFit software (Verity Software House).

Real-time quantitative polymerase chain reaction (PCR).

RNA isolation and complementary DNA synthesis were performed using a TaqMan Gene Expression Cells-to-CT kit according to the recommendations of the manufacturer (Ambion). Gene expression was analyzed using TaqMan Gene Expression Master Mix (Ambion) and specific TaqMan Gene Expression Assays (Applied Biosytems) and analyzed with a Bio-Rad iCycler iQ Multicolor Real-Time PCR Detection System. The following probes were used: Bim (Hs00197982_m1), TRAF6 (Hs00377558_m1), and CD134 (Hs00533968_m1). Messenger RNA (mRNA) levels were analyzed in PBMCs following 24 hours of T cell stimulation with anti-CD3/CD28 and in PBMCs that were rested without T cell stimulation. The mRNA expression levels were normalized to GAPDH expression (Hs99999905_m1) and are reported as fold changes relative to the expression in unstimulated PBMCs.

Statistical analysis

Except where indicated otherwise, data are the mean ± SD. Mann-Whitney U test, Kruskal-Wallis test, and Spearman's rank correlation test were used for statistical analyses. P values less than 0.05 were considered significant.

RESULTS

Elevated frequencies of CD4+CD45RA−FoxP3low and FoxP3− T cells, and increased Akt activity in CD4+CD45RA−FoxP3low and FoxP3− T cells in response to T cell stimulation, in pediatric patients with lupus nephritis

The molecular mechanisms of Teff cell resistance to suppression in patients with lupus nephritis are largely unknown. Therefore, we analyzed the expression of activated or pAkt, a kinase shown in mice to be crucial in autoimmune T cell responses and renal injury ([25-27]), in effector and Treg cells from children with lupus nephritis and controls (patients with nephrotic syndrome and healthy subjects). Moreover, in transgenic models, constitutively active Akt renders Teff cells resistant to suppression ([27, 38]). We analyzed the levels of pAkt in CD4+CD45RA−FoxP3low and FoxP3− Teff cells that were gated on CD4+ T cells, then based on the expression of CD45RA, CD25, and FoxP3 (Figures 1A and B), as previously described by Miyara et al ([16]). We found that the frequencies of CD4+CD45RA−FoxP3low and FoxP3− T cells were significantly higher in children with lupus nephritis than in controls (Figure 1A). The non–regulatory T cell nature of the CD4+CD45RA−FoxP3low population was confirmed by analyzing the coexpression of CD25 (Figure 1B). CD4+CD45RA−FoxP3low T cells express intermediate levels of CD25 ([16]).

Figure 1.

Elevated frequencies of CD4+CD45RA−FoxP3− and FoxP3low T cells in children with lupus nephritis (LN). Peripheral blood mononuclear cells were activated with anti-CD3/CD28 for 24 hours. A, Top, Representative density plots of FoxP3 and CD45RA expression by gated CD4+ T cells within the lymphocyte population from a patient with lupus nephritis and a healthy control (HC). Numbers indicate the percentage in each gate (FoxP3− and FoxP3low). Bottom, Dot plots showing the percentages of CD4+CD45RA−FoxP3− and FoxP3low T cells in patients with lupus nephritis (n = 17) and healthy controls (n = 19). Symbols represent individual patients; horizontal lines show the mean. ∗∗∗ = P < 0.0001 by Mann-Whitney U test. B, Expression of CD25 by gated CD4+CD45RA− FoxP3high, FoxP3low, and FoxP3− T cells.

Moreover, as shown in Figure 2A, in fresh CD4+CD45RA−FoxP3− T cells from children with active lupus nephritis, the levels of pAkt (mean ± SD median fluorescence intensity [MFI] 8.0 ± 2.3; n = 3) were higher than in those with inactive lupus nephritis (mean ± SD MFI 1.2 ± 0.3; n = 2) or healthy controls (mean ± SD MFI 0.6 ± 0.2; n = 2) and correlated with the disease activity as assessed by the SLEDAI-2K (r = 0.9; P = 0.0417). Following short-term stimulation with anti-CD3/CD28, Teff cells from lupus patients displayed higher levels of Akt activation than those from controls after 5, 10, and 30 minutes, indicating that activation of Akt is exaggerated and prolonged in Teff cells from lupus patients (Figure 2B). In addition, stimulation with anti-CD3/CD28 resulted in a dose-dependent increase in Akt activity that was markedly higher in Teff cells from lupus patients than in those from controls (data not shown). Furthermore, the levels of pAkt were significantly increased in anti-CD3/CD28–activated CD4+CD45RA−FoxP3low and FoxP3− T cells from children with lupus nephritis compared to controls (Figure 2C) and correlated with SLE disease activity (r = 0.74; P = 0.0007 and r = 0.72; P = 0.001, respectively) (Figure 2D). These findings indicate that activation of Akt in Teff cells from lupus patients is exaggerated and prolonged.

Figure 2.

Enhanced Akt activity in CD4+CD45RA−FoxP3− and FoxP3low T cells from children with lupus nephritis (LN). A, Histogram showing pAkt fluorescence intensity in fresh CD4+CD45RA−FoxP3− T cells from children with active lupus nephritis (n = 3), children with inactive lupus nephritis (n = 2), and healthy controls (HC; n = 2). B, Histograms showing pAkt fluorescence intensity in gated CD4+CD45RA−FoxP3− T cells from a patient with lupus nephritis and a healthy control. Cells were stimulated with anti-CD3/CD28 with Fc crosslinking antibody for the indicated times. Results are representative of 3 experiments. C, Median fluorescence intensity (MFI) of pAkt upon anti-CD3/CD28 stimulation in the different CD4+ T cell populations from patients with lupus nephritis (n = 17), patients with nephrotic syndrome (NS; n = 5), and healthy controls (n = 19). Data are shown as box plots, where the boxes represent the 25th to 75th percentiles, the lines inside the boxes represent the median, and the whiskers represent the minimum and maximum. D, Correlation between pAkt levels and Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) scores in the indicated T cell populations from children with lupus nephritis.

Consequently, the induction of the downstream target Bim was impaired in CD4+CD45RA−FoxP3low and FoxP3− T cells from lupus patients (Figure 3A) and correlated with the SLEDAI-2K scores (r = 0.61; P = 0.001 and r = 0.66; P = 0.004, respectively). Furthermore, the induction of Bim mRNA following 24 hours of T cell stimulation with anti-CD3/CD28 was reduced in PBMCs from patients with lupus nephritis compared to healthy controls (Figure 3B). In contrast, in CD4+CD45RA+FoxP3low resting Treg cells and CD4+CD45RA−FoxP3high activated Treg cells, the expression of pAkt and Bim was comparable in patients with lupus nephritis and controls (Figures 2C and 3A).

Figure 3.

Impaired Bim expression in CD4+CD45RA−FoxP3− and FoxP3low T cells from children with lupus nephritis. A, Bim expression and MFI in peripheral blood mononuclear cells (PBMCs) activated with anti-CD3/CD28 for 24 hours. Top, Representative fluorescence-activated cell sorting histograms illustrating the expression of Bim in gated CD4+CD45RA−FoxP3−, FoxP3low, and FoxP3high T cells and CD4+CD45RA+FoxP3low T cells from a patient with lupus nephritis, a healthy control, and a patient with nephrotic syndrome. Bottom, MFI of Bim in the indicated CD4+ T cell subsets from patients with lupus nephritis (n = 17), patients with nephrotic syndrome (n = 5), and healthy controls (n = 19). Data are shown as box plots, where the boxes represent the 25th to 75th percentile, the lines inside the boxes represent the median, and the whiskers represent the minumum and maximum. B, Real-time quantitative polymerase chain reaction analysis of Bim mRNA isolated from anti-CD3/CD28–activated PBMCs from patients with lupus nephritis (n = 17) and healthy controls (n = 19). Relative mRNA expression levels were normalized to GAPDH. Bars show the mean ± SD fold change in Bim mRNA expression relative to the expression in unstimulated PBMCs. P values were determined by Mann-Whitney U test. See Figure 2 for other definitions.

Reduced induction of TRAF6 in CD4+CD45RA−FoxP3low and FoxP3− T cells from pediatric patients with lupus nephritis

To investigate possible mechanisms involved in the induction of Akt hyperactivation in Teff cells from lupus patients, we next examined whether the levels of TRAF6, a T cell–intrinsic negative regulator essential for the maintenance of peripheral tolerance ([39]), are altered in CD4+CD45RA−FoxP3low and FoxP3− T cells from pediatric patients with lupus nephritis. Previous findings demonstrated that TRAF6-deficient T cells exhibit Akt hyperactivation, resulting in resistance to Treg cell–mediated suppression ([39]).

TRAF6 expression was slightly higher in fresh CD4+CD45RA−FoxP3− T cells from children with active lupus nephritis who display activated T cells (mean ± SD MFI 1.4 ± 0.4; n = 3) than in those with inactive lupus nephritis (mean ± SD MFI 0.8 ± 0.5; n = 2) or healthy controls (mean ± SD MFI 1.1 ± 0.1; n = 2). However, since TRAF6 is rapidly up-regulated upon T cell activation ([27]), cells were stimulated with anti-CD3/CD28 for 24 hours and then analyzed by flow cytometry and quantitative real-time PCR. Following T cell activation, the expression of TRAF6 was reduced in CD4+CD45RA−FoxP3low and FoxP3− T cells from children with lupus nephritis compared to controls (Figure 4A). Additionally, the induction of TRAF6 mRNA following T cell activation was impaired in PBMCs from patients with lupus nephritis compared to controls (Figure 4B). Furthermore, TRAF6 correlated inversely with the levels of pAkt in CD4+CD45RA−FoxP3low and FoxP3− T cells from pediatric patients with lupus nephritis (r = −0.767; P = 0.0003 and r = −0.725; P = 0.001, respectively) (Figure 4C). These results suggest that impaired TRAF6 induction could contribute to the hyperactivation of the Akt pathway in Teff cells from lupus patients, although a direct causal link remains to be elucidated.

Figure 4.

Reduced tumor necrosis factor receptor–associated factor 6 (TRAF6) induction in CD4+CD45RA−FoxP3− and FoxP3low T cells from children with lupus nephritis. A, TRAF6 staining and MFI in cells stimulated with anti-CD3/CD28 for 24 hours. Top, Representative fluorescence-activated cell sorting histograms showing TRAF6 staining in gated CD4+CD45RA−FoxP3− and FoxP3low T cells from a patient with lupus nephritis, a healthy control, and a patient with nephrotic syndrome. Bottom, MFI of TRAF6 in the indicated cell populations from patients with lupus nephritis (n = 17), patients with nephrotic syndrome (n = 5), and healthy controls (n = 19). Data are shown as box plots, where the boxes represent the 25th to 75th percentiles, the lines inside the boxes represent the median, and the whiskers represent the minumum and maximum. B, Real-time quantitative polymerase chain reaction analysis of TRAF6 mRNA isolated from anti-CD3/CD28–activated peripheral blood mononuclear cells (PBMCs) from patients with lupus nephritis (n = 17) and healthy controls (n = 19). Relative mRNA expression levels were normalized to GAPDH. Bars show the mean ± SD fold change in TRAF6 mRNA expression relative to the expression in unstimulated PBMCs. C, Correlation of pAkt and TRAF6 expression in CD4+CD45RA−FoxP3− T cells from children with lupus nephritis (r = −0.725; P = 0.001). D, Histograms showing TRAF6 fluorescence intensity in gated CD4+CD45RA−FoxP3− T cells from a healthy control and a patient with lupus nephritis. Cells were stimulated with anti-CD3/CD28 for 48 hours in the presence or absence of soluble OX40 ligand (sOX40L). Results are representative of 3 experiments. See Figure 2 for other definitions.

Moreover, the addition of sOX40L to cultures stimulated for 48 hours with anti-CD3/CD28 resulted in the increased expression of TRAF6 in CD4+CD45RA−FoxP3low and FoxP3− T cells from healthy controls (mean fold increase in MFI 2.5 and 3.1, respectively; n = 3 for each cell type) (Figure 4D), as described for CD4+ T cells by Xiao et al ([40]). In contrast, sOX40L did not markedly induce TRAF6 expression in CD4+CD45RA−FoxP3low and FoxP3− T cells from children with lupus nephritis (mean fold increase in MFI 1.2 and 1.4, respectively; n = 3 each) (Figure 4D).

Up-regulation of expression of OX40 in CD4+CD45RA−FoxP3low and FoxP3− T cells from pediatric patients with lupus nephritis upon T cell stimulation

OX40 (CD134), a TNFR family member, promotes the activation of Akt upon T cell activation ([28]), critically regulates T cell effector functions ([29]), and counteracts Treg cell–mediated suppression ([31]). We were therefore interested in whether the expression of OX40 is up-regulated in CD4+CD45RA−FoxP3low and FoxP3− T cells from pediatric patients with lupus nephritis, which were found to express high levels of pAkt. The expression of OX40 was markedly higher in fresh CD4+CD45RA−FoxP3− T cells from patients with active lupus nephritis (mean ± SD MFI 15.9 ± 10.8; n = 3) than in those with inactive lupus nephritis (mean ± SD MFI 1.4 ± 0.3; n = 2) or healthy controls (mean ± SD MFI 0.4 ± 0.01; n = 2) (Figure 5A).

Figure 5.

Increased OX40 expression in Teff cells from pediatric patients with lupus nephritis. A, Histogram showing OX40 fluorescence intensity in fresh CD4+CD45RA−FoxP3− T cells from children with active lupus nephritis (n = 3), children with inactive lupus nephritis (n = 2), and healthy controls (n = 2). B, OX40 fluorescence intensity in cells activated with anti-CD3/CD28 for 48 hours. Left, Histogram showing OX40 fluorescence intensity in CD4+CD45RA−FoxP3− T cells from a patient with lupus nephritis, a healthy control, and a patient with nephrotic syndrome. Right, MFI of OX40 in CD4+CD45RA−FoxP3− T cells from patients with lupus nephritis (n = 17), patients with nephrotic syndrome (n = 5), and healthy controls (n = 19). Data are shown as box plots, where the boxes represent the 25th and 75th percentiles, the lines inside the boxes represent the median, and the whiskers represent the minumum and maximum. C, Real-time quantitative polymerase chain reaction analysis of OX40 mRNA levels in anti-CD3/CD28–stimulated peripheral blood mononuclear cells (PBMCs) from patients with lupus nephritis (n = 17) and healthy controls (n = 19). Relative mRNA expression levels were normalized to GAPDH. Bars show the mean ± SD fold change in OX40 mRNA expression relative to the expression in unstimulated PBMCs. D, Flow cytometric analysis of pAkt levels in CD4+CD45RA−FoxP3− T cells. PBMCs were activated with anti-CD3/CD28 alone or with anti-CD3/CD28 in the presence of soluble OX40 ligand (sOX40L), sOX40L and a neutralizing antibody (anti-OX40), or sOX40L and IgG control. Top, Histogram showing pAkt levels in CD4+CD45RA−FoxP3− T cells from a patient with lupus nephritis and a healthy control. Bottom, MFI of pAkt in CD4+CD45RA−FoxP3− T cells from patients with lupus nephritis (n = 7) and healthy controls (n = 3). Bars show the mean ± SD. ∗∗ = P < 0.01, anti-CD3/CD28 + sOX40L versus anti-CD3/CD28 alone and anti-CD3/CD28 + sOX40L + anti-OX40 versus anti-C3/CD28 + sOX40L, by Mann-Whitney U test. See Figure 2 for other definitions.

In addition, after resting and T cell stimulation for 48 hours, CD4+CD45RA−FoxP3low and FoxP3− T cells from children with lupus nephritis had enhanced levels of OX40 expression compared to those from controls (Figure 5B). The increase in OX40 expression in CD4+CD45RA−FoxP3low and FoxP3− T cells correlated clinically with the SLEDAI-2K scores (r = 0.827; P < 0.0001 and r = 0.801; P = 0.0001, respectively). In addition, up-regulation of OX40 at the mRNA level upon T cell stimulation was higher in PBMCs from children with lupus nephritis than in those from healthy controls (Figure 5C). Addition of recombinant sOX40L further increased Akt activity in CD4+CD45RA−FoxP3− T cells, whereas blockade of OX40 signaling significantly reduced the levels of activated Akt in children with lupus nephritis and to a lesser degree in controls (Figure 5D). However, it should be noted that OX40-independent mechanisms also underlie the increase in Akt activity in Teff cells from lupus patients, because enhanced levels of activated Akt were observed in the absence of OX40 stimulation, as outlined above.

Enhanced proliferation of CD4+CD45RA−FoxP3low and FoxP3− T cells from pediatric patients with lupus nephritis

One of the mechanisms by which Teff cells from lupus patients can escape tolerance is resistance to Treg cell–mediated suppression ([15]). Previously, Akt hyperactivation in Teff cells has been shown to cause resistance to suppression in patients with juvenile idiopathic arthritis ([18]). In this study, we showed that Akt is hyperactivated in CD4+CD45RA−FoxP3low and FoxP3− T cells from patients with lupus nephritis. Therefore, we asked whether these T cell subpopulations display a heightened proliferative response upon stimulation in the presence of autologous Treg cells. It is important in this context to note that in some studies true Treg cells from patients with SLE have been shown to display intact suppressive activities ([15, 41]). PBMCs from children with lupus nephritis or controls were labeled with CFSE and activated with anti-CD3/CD28 for 4 or 7 days. Proliferation was assessed by measuring the extent of CFSE dilution of CD4+CD45RA−FoxP3low and FoxP3− T cells. As shown in Figure 6A, CD4+CD45RA−FoxP3low and FoxP3− T cells from children with lupus nephritis had a significantly higher proliferation index than those from controls. Since autologous Treg cells were present in the PBMC cultures, these results suggest that CD4+CD45RA−FoxP3low and FoxP3− T cells might be insensitive to Treg cell–mediated suppression in children with lupus nephritis.

Figure 6.

Inhibition of Akt activity reduces the enhanced proliferative response of Teff cells from pediatric patients with lupus nephritis. A, Proliferation index for the indicated cell populations from patients with lupus nephritis (n = 17) and healthy controls (n = 15). Peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3/CD28 for 7 days. Cells were gated on CD4+CD45RA−FoxP3− and CD4+CD45RA−FoxP3low T cells, and proliferation was analyzed by quantifying 5,6-carboxyfluorescein succinimidyl ester (CFSE) dilution using flow cytometry. Bars show the mean ± SD. P values were determined by Mann-Whitney U test. B, Effect of Akt inhibition on cells from patients with lupus nephritis and healthy controls. CFSE-labeled PBMCs were activated with anti-CD3/CD28 in the presence or absence of the Akt inhibitor VIII (Akt Inh; 0.2 μM). Top, Representative fluorescence-activated cell sorting dot plots of gated CD4+CD45RA− FoxP3− T cells from a patient with lupus nephritis and a healthy control, analyzed by flow cytometry on day 4. Numbers indicate the percentage of nonproliferating cells. Bottom, Percentage of proliferating cells in the absence or presence of the Akt inhibitor. Values are the mean ± SD change in CD4+CD45RA−FoxP3− T cell proliferation (n = 3 subjects per group). See Figure 2 for other definitions.

To test whether hyperactivation of Akt in Teff cells from lupus patients contributes to enhanced proliferation, a specific Akt inhibitor was added to the cultures. Inhibition of Akt activity significantly reduced the proliferative response of CD4+CD45RA−FoxP3− Teff cells from lupus patients (Figure 6B).

DISCUSSION

Treg cells from patients with SLE have been shown in some studies to exhibit intact suppressive activity, but Teff cells are resistant to suppression ([15, 41]). The molecular mechanisms underlying the resistance of Teff cells to Treg cell–mediated suppression in patients with SLE remain unclear.

In this study, we have shown that the serine/threonine kinase Akt is hyperactivated in CD4+CD45RA−FoxP3low and FoxP3− Teff cells from pediatric patients with lupus nephritis, resulting in the down-regulation of the proapoptotic protein Bim. In contrast, in resting and activated Treg cells, the levels of activated Akt and Bim were comparable in children with lupus nephritis and controls. Moreover, the proliferative response upon stimulation of CD4+CD45RA−FoxP3low and FoxP3− T cells was higher in pediatric patients with lupus nephritis than controls, and inhibition of Akt activity markedly reduced the proliferation of these cells. Furthermore, the activity of Akt inversely correlated with the impaired induction of TRAF6, whereas OX40 was up-regulated in Teff cells from lupus patients. However, stimulation of OX40 failed to significantly induce TRAF6 expression in Teff cells from lupus patients in contrast to healthy controls, but led to further increased Akt activation that was reversed by the inhibition of OX40 signaling.

These results suggest that the deficient induction of TRAF6 and up-regulation of OX40 might contribute to the resistance of Teff cells to Treg cell–mediated suppression by up-regulating the activity of Akt in children with lupus nephritis. This conclusion is supported by several previous findings. In murine lupus models, CD4+ T cells show high levels of activated Akt ([26]) and are resistant to Treg cell–mediated suppression ([13]). In addition, in patients with rheumatoid arthritis, high levels of activated Akt in inflammatory Teff cells cause resistance to suppression, and the inhibition of Akt restores responsiveness to suppression ([18]).

Moreover, TRAF6-deficient T cells show high levels of activated Akt and are resistant to suppression ([27]). In this study, TRAF6 expression was slightly higher in fresh CD4+CD45RA−FoxP3− T cells from children with active lupus nephritis who display activated T cells than in those from patients with inactive lupus nephritis or healthy controls, whereas its expression was significantly reduced in Teff cells from lupus patients upon activation in vitro. Since TRAF6 is rapidly induced following T cell activation ([27]), these results indicate that the induction of TRAF6 upon activation is impaired in Teff cells from lupus patients. TRAF6 also plays a crucial role in the induction of T cell anergy ([39]). This is important because T cells were found to resist anergy induction in an experimental murine model of SLE ([12]), and anergy avoidance has been proposed to contribute to the inappropriate responses of T cells to autoantigens ([12]). Thus, the reduced induction of TRAF6 and its inverse correlation with Akt activity in CD4+CD45RA−FoxP3low and FoxP3− T cells, as demonstrated in this study, could be involved in two pathogenic mechanisms of SLE, resistance to suppression and anergy avoidance. However, a direct causal link between the impaired induction of TRAF6 and hyperactivation of Akt in Teff cells from lupus patients remains to be elucidated.

In activated conventional CD4+ T cells, TRAF6 is increasingly induced after ligation of OX40 ([40]). While our findings show that OX40 expression is up-regulated in CD4+CD45RA−FoxP3low and FoxP3− T cells from children with lupus nephritis, we found that OX40 stimulation failed to significantly increase TRAF6 expression in lupus CD4+CD45RA−FoxP3low and FoxP3− T cells in contrast to healthy controls. This also seems to be consistent with previous studies showing that after ligation of OX40, TRAF6 also plays a pivotal role in the activation of the transcription factor NF-κB ([40]), the activity of which is decreased in T cells from lupus patients ([42, 43]).

In addition, stimulation of OX40 further increases Akt activation in lupus CD4+CD45RA−FoxP3low and FoxP3− T cells, whereas the blockade of OX40 signaling results in the down-regulation of Akt activity. This is consistent with previous findings showing that OX40 promotes Akt activation ([28]) and inhibits the generation of new Treg cells ([29]). Interestingly, Teff cells are insensitive to Treg cell–mediated suppression in the presence of an agonistic OX40 antibody ([31]), further supporting the hypothesis that OX40 up-regulation in CD4+CD45RA−FoxP3low and FoxP3− T cells from lupus patients, among other mechanisms, underlies their resistance to suppression. However, inhibition of OX40 signaling did not normalize the levels of activated Akt in Teff cells from lupus patients to those observed in Teff cells from controls, and activation of Akt was enhanced in the absence of OX40 stimulation. Thus, Akt hyperactivation also occurs by OX40-independent mechanisms.

Further, high levels of activated Akt may favor Teff cell expansion in SLE, as we found significantly increased proliferation of CD4+CD45RA−FoxP3low and FoxP3− T cells from children with lupus nephritis compared to controls, and inhibition of Akt activity significantly reduced proliferation.

Akt enhances cell survival by inhibiting the function of proapoptotic proteins and processes. For instance, Akt blocks the FOXO-mediated transcription of the BH3-only domain protein Bim, a key trigger of apoptosis that plays a critical role in the physiologic regulation of immune homeostasis, terminating the normal immune response, and preventing autoimmunity ([44, 45]). Down-regulation of Bim in CD4+CD45RA−FoxP3low and FoxP3− T cells from pediatric patients with lupus nephritis as a consequence of Akt overactivation, as observed in this study, may contribute to the perturbed T cell homeostasis in SLE. This is consistent with previous findings in Bim-deficient mice that show an altered lymphocyte homeostasis and fatal systemic autoimmune disease with renal failure that has similarities to human SLE ([46]).

Importantly, reduced activity of Akt in Treg cells is required for their suppressive function ([47]). We observed that the levels of activated Akt in resting and activated Treg cells were comparable in children with lupus nephritis and controls, suggesting that Treg cells in children with lupus nephritis may exert regular functions. This is supported by the findings of previous studies showing that the suppressive capacity of Treg cells from patients with SLE is indeed intact ([15]).

To date, therapeutic agents that target specific pathogenic mechanisms of childhood lupus nephritis are lacking. Therapeutic down-regulation of Akt activity by modulating the expression of OX40 or TRAF6 may render Teff cells susceptible to Treg cell–mediated suppression without affecting Treg cell function and thus effectively control autoimmune T cell responses in children with lupus nephritis.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Edelbauer had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Kshirsagar, Wechselberger, Steichen, Edelbauer.

Acquisition of data. Kshirsagar, Binder, Riedl, Edelbauer.

Analysis and interpretation of data. Kshirsagar, Binder, Riedl, Wechselberger, Steichen, Edelbauer.

Acknowledgments

We are very grateful to the staff at Zentrum für Kinder- und Jugendmedizin Heidelberg (Heidelberg, Germany) Universitäts-Kinder- und Jugendklink Rostock (Rostock, Germany), and Klinikum Links der Weser, Bremen (Bremen, Germany) for collecting blood samples and to all of the patients and healthy subjects for their participation.

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