Between adaptive and innate immunity: TLR4-mediated perforin production by CD28nullT-helper cells in ankylosing spondylitis
- Bernd Raffeiner†1,
- Christian Dejaco†1,
- Christina Duftner1,
- Werner Kullich2,
- Christian Goldberger1,
- Sandra C Vega3,
- Michael Keller3,
- Beatrix Grubeck-Loebenstein3 and
- Michael Schirmer1Email author
© Raffeiner et al.; licensee BioMed Central Ltd. 2005
Received: 21 April 2005
Accepted: 27 September 2005
Published: 18 October 2005
CD3+CD4+CD28null and CD3+CD8+CD28null T cells are enriched in patients with immune-mediated diseases compared with healthy controls. This study shows that CD4+CD28null T cells express Toll-like receptors recognizing bacterial lipopolysaccharides in ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis. In ankylosing spondylitis, TLR4 (23.1 ± 21.9%) and, to a smaller extent, TLR2 (4.1 ± 5.8%) were expressed on CD4+CD28null T cells, whereas expression was negligible on CD4+CD28+ and CD8+ T cells. CD4+CD28null T cells produced perforin upon stimulation with lipopolysaccharide, and this effect was enhanced by autologous serum or recombinant soluble CD14. Perforin production could be prevented with blocking antibodies directed against CD14 or TLR4. Incubation of peripheral blood mononuclear cells with tumour necrosis factor alpha led to an upregulation of TLR4 and TLR2 on CD4+CD28null T cells in vitro, and treatment of patients with antibodies specifically directed against tumour necrosis factor alpha resulted in decreased expression of TLR4 and TLR2 on CD4+CD28null T cells in vivo. We describe here a new pathway for direct activation of cytotoxic CD4+ T cells by components of infectious pathogens. This finding supports the hypothesis that CD4+CD28null T cells represent an immunological link between the innate immune system and the adaptive immune system.
Pattern recognition receptors (PRRs) are a family of receptors of the innate immune system binding to conserved pathogen-associated molecular patterns . The most important PRRs are the Toll-like receptors (TLRs), which allow monocytes, neutrophils, dendritic cells, natural killer (NK) cells and B cells to recognize bacterial components, viruses, fungi and host material such as heat shock proteins [2–5]. Receptor engagement leads to the translocation of NF-κB and to gene transcription of proinflammatory cytokines. Lipopolysaccharide (LPS) from Gram-negative bacteria is the main ligand for TLR4. LPS binding to TLR4 is promoted by CD14, which can be present either in a membrane-bound form (mCD14) or a soluble form (sCD14) [6, 7]. Whether LPS is a low-affinity ligand for TLR2 is still controversial .
The interactions between the innate and adaptive immune systems are crucial to promote proinflammatory reactions against pathogens and to ensure maintenance of vital self-tolerance. TLRs are expressed on both innate and adaptive immune cells and are critically involved in this interplay. TLR-stimulated dendritic cells induce specific T cells to differentiate into memory cells [9, 10], and microbial induction of the TLR pathway on dendritic cells also blocks the suppressive effects of regulatory T cells [11, 12]. In addition, TLRs are themselves expressed on T cells. In murine T cells, LPS signalling induces production of INF-γ and T-helper-1 accentuated immune responses [13, 14]. After in vitro activation, mouse CD4+ T cells express TLR3 and TLR9, and treatment of these cells with synthetic ligands for TLR3 and TLR9, viral dsRNA and bacterial unmethylated DNA enhances their survival . LPS can directly activate regulatory T cells, and can thereby increase their suppressive function . Activated human T cells express high levels of cell-surface TLR2 and produce elevated levels of cytokines in response to bacterial lipopeptide, a TLR2 ligand .
TLRs have also been shown to be important for the pathogenesis of immune-mediated diseases. In rheumatoid arthritis (RA), for example, TLR2 expression occurs in inflamed synovial tissue predominantly at sites of attachment and invasion into the cartilage and bone . The TLR2-mediated stimulation of synovial fibroblasts with bacterial components promotes the release of proinflammatory cytokines and leads to a higher expression of TLR2.
In ankylosing spondylitis (AS), as in other immune-mediated diseases, an unusual proinflammatory and cytotoxic CD4+ T-cell subgroup has been described, which lacks the co-stimulatory molecule CD28. These CD4+CD28null T cells depend on alternative pathways for coactivation and can indeed obtain such signals by NK receptors recognizing ubiquitous major histocompatibility complex class I molecules [19–23]. Anomalous expression of NKG2D on these T cells together with upregulated MIC ligands in the inflamed synovial tissue of RA have also been shown to provide co-stimulatory signals . CD4+CD28null T cells have therefore been considered an immunological link between the adaptive and the innate defence system .
As CD4+CD28null T cells have NK cell features, and as NK cells express TLRs on their surface , we investigated the expression of TLRs as an alternative stimulatory pathway on CD4+CD28null T cells.
Patients and methods
A total of 90 consecutive patients with spondyloarthropathies, 72 patients with RA and 64 age-matched healthy controls were enrolled into the study. Spondyloarthropathy was defined according to the European Spondyloarthropathy Study Group criteria . Out of the spondyloarthropathy-defined patients, 65 patients had a diagnosis of AS according to the modified New York criteria  and 25 patients had psoriatic arthritis (PsA) as defined by the diagnostic criteria of Moll and Wright . RA was diagnosed according to the criteria of the American College of Rheumatology . Probands did not show any history, clinical or laboratory sign for infections nor malignant diseases. Healthy controls had no history of an immune-mediated disease. Heparinized blood samples were drawn from peripheral veins after informed and written consent according to the local ethics committee.
Controls (n = 64)
Ankylosing spondylitis (n = 65)
Psoriatic arthritis (n = 25)
Rheumatoid arthritis (n = 72)
49.2 ± 12.4
43.1 ± 10.9
52.2 ± 12
57.9 ± 11
Gender (% female)
Rheumatoid factor positivity (%)
CCP-Ab positivity (%)
HLA-B27 positivity (%)
Axial involvement (% positive)
Erythrocyte sedimentation rate >15 mm/hour (% positive)
C-reactive protein >0.7 μg/dl (% positive)
Cell preparation and surface staining
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. For surface staining, PBMCs were incubated, as appropriate, with fluorescein isothiocyanate-conjugated anti-CD4, anti-CD8 or anti-CD28, with phycoerythrin-conjugated anti-CD28 and with peridinin chlorophyll protein-conjugated anti-CD3, anti-CD4 and anti-CD8 monoclonal antibodies (Becton Dickinson, San Diego, CA, USA). Specific mAbs directed against CD14 (fluorescein isothiocyanate), TLR4 and TLR2 (phycoerythrin; eBioscience, San Diego, CA, USA) were used to analyse expression of PRRs. Cells were incubated for 30 minutes at 4°C with the antibodies. After washing with PBS, cells were fixed in 4% paraformaldehyde (cellfix; Becton Dickinson). Stained cells were analysed on a FACS-Calibur analyser (Becton Dickinson). At least 100,000 events were counted for each acquisition. Data were analysed using WinMDI software (version 2.5, Joseph Trotter; Scripps Research Institute, La Jolla, CA, USA). Cells were considered positively stained when fluorescence levels were higher than those of the corresponding isotype controls.
For the isolation of CD4+CD28+ and CD4+CD28null T cells, the MACS® CD4+ T-cell multisort kit and magnetic bead labelled anti-phycoerythrin antibodies were applied according to the manufacturer's instructions (Miltenyi Biotech, Amsterdam, The Netherlands). To further increase the purity of T cells, sorted fractions were incubated in 24-well plates for 2 hours at 37°C to allow adherence of contaminating monocytes. Purity of isolated fractions was determined by flow cytometry as already described.
Reverse transcription-polymerase chain reaction
Total RNA was extracted from isolated cell fractions using 1 ml Tri-Reagent (Sigma-Aldrich, St Louis, MO, USA), 200 μl chloroform (Sigma-Aldrich), 0.5 ml isopropanol (Sigma-Aldrich) and 1 μl glycogen (Roche, Basel, Switzerland) as previously described . RNA was reverse transcribed to cDNA applying the Reverse Transcription System (Promega, Madison, WI, USA). PCR amplification was performed on 1 μg total RNA and 10 pmol primers (TLR2: forward, 5'-GGCCAGCAAATTACCTGTGTG-3'; reverse, 5'-CTGAGCCTCGTCCATGGGCCACTCC-3'; TLR4: forward, 5'-TGCAATGGATCAAGGACCAGAGGC-3'; reverse, 5'-GTGCTGGGACACCACAACAATCACC-3'; and β2-microglobulin: forward, 5'-CTCCGTGGCCTTAGCTGTG-3'; reverse, 5'-TTTGGAGTACGCTGGATAGCC-3') using the HotStar Master Mix Kit (Qiagen Valencia, CA, USA). The PCR reaction was performed according to a modified protocol  on the PTC-100 Thermal Cycler (MJ Research, Waltham, MA, USA) at 95°C for 3 minutes, at 95°C for 1 minute, 58°C for 2 minute and 72°C for 1 minute (35 cycles), and at 72°C for 2 minutes. Negative control samples were prepared by amplification reactions in the absence of cDNA. PCR products were separated on 1% agarose gel containing ethidium bromide and were visualized by UV illumination.
LPS studies and intracellular cytokine staining
Isolated PBMCs were resuspended in RPMI 1640 containing 2 mM L-glutamine and were distributed on 24-well plates at a density of 1 × 106 cells per well. LPS of Escherichia coli serotype 026:B6 (Sigma) was added at a concentration of 10 μg/ml [16, 17], sCD14 (R&D systems, Minneapolis, MN, USA) at 25 μg/ml and autologous serum at a concentration of 5% as indicated.
To test inhibitory effects of blocking anti-CD14 or anti-TLR4 antibodies, cells were resuspended in RPMI 1640 with 5% autologous serum and were incubated with 10 μg/ml blocking anti-CD14 antibody (R&D Systems), 10 μg/ml anti-TLR4 antibody (Torrey Pines Biolabs, TX, USA) and 10 μg/ml isotype control antibody (Becton Dickinson) for 1 hour before adding LPS at a concentration of 10 μg/ml. Brefeldin A (Sigma) was added to functional experiments at a concentration of 10 μg/ml to avoid release of produced cytokines. After incubation for 16 hours at 37°C the cells were washed, stained with CD4-peridinin chlorophyll protein mAbs and CD28-phycoerythrin mAbs and were permeabilized with 0.05% Tween 20 to stain intracellularly with fluorescein isothiocyanate-conjugated anti-perforin mAbs or control immunoglobulin (Becton Dickinson).
Stimulation of short-term cell lines
Short-term cell lines were established after incubation of fresh PBMCs on stimulating immobilized anti-CD3 mAbs (OKT3; eBioscience) for 18 hours. Cells were then cultured in densities of 0.5 × 106 – 2 × 106 in RPMI 1640 containing 10% FCS, 2 mM L-glutamine and 20 U/ml recombinant human IL-2 (Sigma). The medium was changed every 2–3 days. Cell lines were used after 7 days for stimulation assays over 24 hours with 20 ng/ml recombinant human TNF-α (Prepotech, London, UK).
Enzyme-linked immunosorbent assay
Soluble levels of CD14 in blinded sera were tested in duplicates with an ELISA kit (R&D systems) according to the manufacturer's instructions.
Statistical analysis was performed using the SPSS program (version 11.5; SPSS Inc., Chicago, IL, USA). The Kolmogorov–Smirnov test was used to test for normal distribution, and the Mann–Whitney U test and the Wilcoxon test were used as appropriate. At least six assays were performed for each experiment. P < 0.05 was considered significant and P < 0.01 was highly significant. Data are shown as box plots with the lines within the boxes representing the median, the boxes representing the 25th–75th percentiles and the lines outside the boxes including all values except mavericks.
Comparison between the prevalences of circulating CD4+CD28null and CD8+CD28nullT cells in AS, PsA, RA and healthy controls
Increased expression of pattern recognition receptors on CD4+CD28nullT cells
Overall, all analysed PRRs (CD14, TLR4 and TLR2) were detected on the surface of CD4+CD28null T cells but not on CD28+ T cells irrespective of the underlying diseases tested. CD14 was expressed on 13.3 ± 20.4% of CD4+CD28null T cells versus 0.7 ± 1% of CD4+CD28+ T cells in AS, on 8.8 ± 15.7% of CD4+CD28null T cells versus 1.0 ± 2.3% of CD4+CD28+ T cells in PsA, and on 11.3 ± 17.2% of CD4+CD28null T cells versus 0.6 ± 0.6% of CD4+CD28+ T cells in RA (each with P < 0.001). TLR-4, the main receptor for LPS recognition, was significantly expressed on CD4+CD28null T cells in AS (23.1 ± 21.9% versus 0.9 ± 1.2%), in PsA (12.4 ± 18.1% versus 0.4 ± 0.5%) and in RA (23.1 ± 24.7% versus 0.6 ± 0.9%; each with P < 0.001). TLR2 was more frequently expressed on CD4+CD28null T cells than on CD28+ cells (4.1 ± 5.8% versus 1.0 ± 1.1%, P < 0.001) in AS, but not in PsA or in RA (Figure 3b). Negligible surface expression of PRRs without any difference between CD28null and CD28+ T cells were seen on CD8+ T cells from patients (data not shown). CD4+CD28+, CD8+CD28+ and CD8+CD28null T cells from healthy controls showed no significant expression of PRRs. CD4+CD28null T cells from healthy controls expressed PRR to some extent but, as the prevalence of this subpopulation is low, no significant data could be acquired (data not shown).
LPS-mediated perforin production of CD4+CD28nullT cells depends on CD14 and TLR4
Blocking assays with antibodies directed against TLR4 were then performed to specifically address the role of TLR4 in LPS-mediated perforin production of CD4+CD28null T cells. As shown in Figure 5c, preincubation with anti-TLR4 antibody inhibited activation of CD4+CD28null T cells by LPS (3.4 ± 2.3% with anti-TLR4 antibody versus 23.8 ± 14.7% perforin+ cells with isotype control antibody, P = 0.002).
Effects of TNF-α in vitro and therapeutic blockade of TNF-α in vivo on PRR expression of CD4+CD28nullT cells
To examine the effects of TNF-α blocking treatment on the expression of PRRs on CD4+CD28null T cells in vivo, peripheral CD4+CD28null T cells from AS patients were tested before and after successful treatment with TNF-α-specific chimeric antibodies. As shown in Figure 6b, CD4+CD28null T cells from patients during active AS disease showed higher levels of PRRs than after successful treatment with the TNF-α blocking agent. CD14 was reduced from 10.6 ± 16.6% before treatment to 2.5 ± 2% after treatment (P = 0.011), TLR4 was reduced from 46.9 ± 32.7% to 11.7 ± 12.5% (P = 0.008) and TLR2 was reduced from 11.7 ± 19.4% to 1.8 ± 2.9% (P = 0.012).
The present study shows an increased expression of PRRs on human circulating CD4+ T cells lacking the CD28 co-stimulatory molecule. TLR4 can be considered an alternative signalling pathway for cytotoxic CD4+CD28null T cells, but neither for their CD28+ counterparts nor for CD8+ T cells. The concomitant expression of T-cell receptor (TCR) and PRRs on the cell surface further supports the role of CD4+CD28null T cells as an immunological link between the adaptive and the innate defence system, and is in accordance with earlier descriptions of co-existing NK receptors and TCR on these cells . In all chronic immune diseases tested (AS, PsA and RA) more CD4+CD28null T cells expressed TLR4 than TLR2, thus stressing the superior role of TLR4 over TLR2. Indeed, a significant surface expression of TLR2 on CD4+CD28null T cells has only been found in patients with AS, but not in patients with PsA and RA (Figure 1b), which is consistent with an earlier histological study in RA that did not find TLR2 on CD3+ T cells in the synovial tissue .
To assure a high purity of T-cell populations was a critical part in this study. T cells were therefore not only purified by MACS® technology for the RT-PCR assays, but were also monocyte depleted. A high purity of CD3+CD4+CD28null cells ranging from 94.2% up to 99.1% was thus obtained, as shown in Figure 2a. In a separate approach, surface expression of TLRs was studied by fluorescence-activated cell sorting analysis with gates carefully set to focus on the lymphocyte population on the forward scatter and the side scatter. An additional gate was then set on the population expressing high levels of CD4, ensuring monocytes that express lower levels of CD4 were excluded . Detection of mRNA and surface expression of TLRs were therefore used as two independent techniques to ensure the presence of TLRs in the examined cell populations.
From the functional perspective, TLR4 is the key receptor for Gram-negative bacteria. In our in vitro model the effect of bacterial exposure on the cytotoxic function of CD4+CD28null T cells was simulated by addition of LPS from an E. coli strain. TLR4 binds LPS and thus provides activating signals to the CD4+CD28null T cells, which can be reversed with TLR4 blocking antibodies (Figure 5c). This mechanism clearly depends on CD14, which allows signal transmission . CD14 in AS may either occur on cell membranes of CD4+CD28null T cells (Figure 3b) or as a soluble molecule in the serum (Figure 5a). As the percentages of CD4+CD28null T cells expressing mCD14 were much lower than the percentages of cells expressing TLR4, we added either recombinant CD14 or autologous sera for stimulation assays (Figure 4a,b). Indeed, addition of CD14 nearly doubled the percentage of perforin+CD4+CD28null T cells upon LPS stimulation, whereas the addition of anti-CD14 antibody completely abolished the effect of LPS (Figure 5b). LPS binding protein, another TLR-related molecule, is also known to support binding of LPS to TLR4. Serum levels of LPS binding protein correlate with inflammation in RA and reactive arthritis , but have not so far been studied in AS. In our experiments both the addition of recombinant sCD14 and autologous serum had a comparable additional effect on LPS-induced perforin production of CD4+CD28null T cells, which indicates serum LPS binding protein not to be indispensable in AS. Taking these facts together, activated CD4+CD28null T cells produce perforin upon LPS-mediated activation in a CD14-dependent and TLR4-dependent manner.
As we used PBMCs for functional assays, we cannot exclude that LPS also activated antigen-presenting cells within the PBMCs. However, direct LPS-mediated effects on TLR4+ T cells appear more relevant: TLR- T cells were not activated in the presence of LPS, and the percentage of perforin+CD4+CD28null T cells correlated well with the prevalence of TLR4-expressing CD4+CD28null T cells (data not shown). Antigen-presenting cells would not need addition of CD14 for activation by LPS anyway, as CD14 is widely expressed on antigen-presenting cells.
Direct TLR-mediated activation of human T cells has been previously shown for activated CD8+ T cells and CD4+CD45RO+ memory T cells from healthy individuals with high surface levels of TLR2, but not TLR4 . Although a number of CD4+CD28+ T cells express the memory marker CD45RO (data not shown), we did not detect TLR2 on these cells. A possible explanation for the discrepancy with our results may be that the mAbs used recognize different epitopes or variants of TLRs. We showed that expression of mRNA for TLR2 was present in both CD4+CD28+ and CD4+CD28null T cells to a varying extent (Figure 2b). In contrast, we found TLR4 exclusively in CD4+CD28null T cells on both the mRNA and the protein level. The activation of TLR4 on CD4+CD28null T cells was independent of TCR-mediated stimulation for perforin production, and TLR4 signalling did not lead to an additive effect on concomitant cross-linking of TCR (data not shown). The high affinity of TLR4 to LPS without the obligatory need of the TCR signal may therefore have an influence on the susceptibility of CD4+CD28null T cells from AS patients to Gram-negative bacterial components.
As TNF-α directly influences CD28 gene transcription and may facilitate the emergence of CD4+CD28null T cells in chronic inflammatory syndromes , we also studied the effects of TNF-α on PRRs in vitro. In line with its effects on monocytic TLRs on the mRNA level , TNF-α also resulted in an increased protein expression of TLR4 and TLR2 on CD4+CD28null T cells (Figure 6a). Accordingly, expression of TLRs on CD4+CD28null T cells from patients with active AS disease before treatment (Figure 6b) were higher than those of unselected AS patients (examined for Figure 3b). The expression of CD14, TLR4 and TLR2 was then reduced on fresh CD4+CD28null T cells from AS patients treated with TNF-α blocking agents, further indicating the important role of TNF-α for the upregulation of surface expression of these PRRs also on CD4+CD28null T cells (Figure 6b).
The finding of PRRs on cytotoxic CD4+CD28null T cells of patients with AS, PsA or RA represents a new pathophysiological link between the innate and the adaptive immune system. In vitro activation of CD4+CD28null T cells by LPS is mediated by TLR4 and depends on CD14. Additional work has to be carried out to explain the downstream mechanisms of action and the clinical implications of these findings.
enzyme-linked immunosorbent assay
foetal calf serum
peripheral blood mononuclear cell
polymerase chain reaction
pattern recognition receptor
tumour necrosis factor alpha.
This work was supported by the Innsbruck Medical University, the 'Verein zur Förderung der Hämatologie, Onkologie und Immunologie' (Innsbruck, Austria) and by the 'Verein zur Förderung der Ausbildung und wissenschaftlichen Tätigkeit von Südtirolern an der Universität Innsbruck' (Innsbruck, Austria) (to C Dejaco).
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