EXPERIMENTAL IMMUNOLOGY doi: 10.1111/sji.12221 ..................................................................................................................................................................
Inhibition of Human cd T Cell Proliferation and Effector Functions by Neutrophil Serine Proteases J. Fazio, S. Kalyan, D. Wesch & D. Kabelitz
Abstract Institute of Immunology, Christian-AlbrechtsUniversity of Kiel, Kiel, Germany
Received 18 July 2014; Accepted in revised form 16 August 2014 Correspondence to: D. Kabelitz, Institute of Immunology, Christian-Albrechts-University of Kiel, Arnold-Heller-Str. 3 Haus 17, D-24105 Kiel, Germany. E-mail: [email protected]
Human peripheral blood cd T cells expressing the Vc9Vd2 T cell receptor are activated by microbial or endogenous pyrophosphate antigens and indirectly by nitrogen-containing bisphosphonates. Apart from proliferation, such phosphoantigens induce proinflammatory cytokine production including TNF-a and IFN-c and trigger cytotoxic effector function. Neutrophil granulocytes are known to modulate T cell activation. The neutrophil serine proteases proteinase 3, elastase and cathepsin G have multiple potential targets and promote microbial killing. In this study, we investigated the effect of the three serine proteases on the in vitro proliferation and effector functions of cd T cells cultured in serum-free medium. All three proteases inhibited the proliferative activity, suppressed the cytokine production and decreased the cytotoxicity of cd T cells. Further studies indicated that proteolytic cleavage of IL-2 and modulation of butyrophilin 3A1 (CD277) expression might contribute to the overall inhibition.
Introduction cdT cells account for 1–10% of T lymphocytes in the peripheral blood of healthy adults. Subpopulations of cd T cells can be identified on the basis of T cell receptor (TCR) variable genes. The majority of blood cd T cells express Vc9 paired with Vd2 [1, 2]. Vc9Vd2 T cells recognize phosphorylated small molecules, so-called phosphoantigens (pAg), which are intermediates of the microbial and eukaryotic isoprenoid biosynthesis pathway. Many bacteria produce the pAg (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), which stimulates human cd T cells at pico- to nanomolar concentrations, whereas the eukaryotic pAg isopentenyl pyrophosphate (IPP) requires micromolar concentrations to activate cd T cells. The selective sensing of microbial pAg by Vc9Vd2 T cells suggests that these cells play a pivotal role in anti-infective immune responses [3]. The concentration of IPP generated in normal cells is insufficient to activate cd T cells, but higher concentrations accumulate upon cellular transformation which is associated with a dysregulated mevalonate pathway [4]. Vc9Vd2 T cells can be strongly and selectively activated in vitro by synthetic pAg such as bromohydrin pyrophosphate (BrHPP) or with nitrogencontaining bisphosphonates, which induce the intracellular accumulation of IPP [4–8]. Recently, a critical role of the butyrophilin family member BTN3A1 (CD277) in the activation of cd T cells by pAg has been discovered [9, 10]. Even though the precise role of BTN3A1 is still under
Ó 2014 John Wiley & Sons Ltd
investigation [11], BTN3A1 may serve as an extracellular [12] and/or intracellular receptor [13] for exogenous or endogenous pAg. Once activated, Vc9Vd2 T cells mediate diverse effector functions. They usually produce cytokines such as TNF-a and IFN-c, but can be also driven to secrete IL-4 or IL-17 [14, 15]. Vc9Vd2 T cells are unable to produce IL-2 in adequate amounts to sustain autocrine proliferation and therefore the addition of exogenous IL-2- or IL-2-producing CD4 T cells is required for proliferation [16]. Most cd T cells express the activating NK receptor NKG2D (Natural Killer Group 2 Member D), which recognizes stress-inducible MHC class-I-related molecules including MHC class I chain-related antigen A/B (MICA/B). Concomitantly, cd T cells can exert cytotoxicity against tumour cells through TCR- and/or NKG2D-dependent activation [17, 18]. In addition to cytokine production and cytotoxic activity, cd T cells can exert regulatory and antigen-presenting functions; thus, they display a surprisingly large functional plasticity [19–22]. Neutrophils are known to modulate T cell activation [23–25] and to inhibit cd T cell activation [26, 27]. Among the potent mediators produced by neutrophils are serine proteases that are stored in the azurophilic granules. Proteinase 3 (PR3), neutrophil elastase (NE) and cathepsin G (CG) are three serine proteases that are members of the chymotrypsin family. They are transcribed only in the early stages of myeloid differentiation of granulocytes and, to a lower extent, of monocytes. After maturation of
381
382 cd T Cells and Serine Proteases J. Fazio et al. .................................................................................................................................................................. granulocytes and monocytes, the transcription of mRNA for the three proteases is downregulated [28]. All three proteases are stored as zymogens in granulocytes and have to be cleaved by cathepsin C for activation [29]. Due to their multiple potential targets, the neutrophil proteases have manifold functions in inflammation and infection [28]. All three serine proteases are known to degrade components of the extracellular matrix and to promote microbial killing. In contrast to CG, PR3 and NE can cleave the IL-2 receptor a-chain from the cell surface, whereas CG is rather able to cleave the IL-6 receptor [30]. At high concentrations, CG has been reported to stimulate lymphocyte proliferation [31]. Neutrophil serine proteases are also associated with different diseases; the most prominent example is PR3, which is the target of autoantibodies in granulomatosis with polyangiitis (GPA), a rare autoimmune disease characterized by systemic vasculitis and granuloma formation [32]. Except for indirect evidence based on effects of the serine protease inhibitor a1-antitrypsin [26], there is no data available on possible effects of neutrophil serine proteases on cd T cell activation. We have previously reported that Vd2 T cells are significantly reduced in patients with GPA, which might be due to an effect of the serine proteases released by neutrophils at the site of inflammation [33]. Furthermore, serine proteases might also impact on the well-documented interplay between neutrophils and cd T cells during microbial infection [34]. In this study, we have investigated in detail the effect of the three serine proteases PR3, NE and CG on cdT cell functions in vitro.
Materials and methods Cell isolation. Blood of healthy adult donors was provided as leucocyte concentrate by the Department of Transfusion Medicine or from blood donors of the Institute of Immunology, both at UKSH Campus Kiel. The study was approved by the appropriate institutional clinical ethics board (code number: D 405/10), and informed consent was obtained from all donors. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll–Hypaque density gradient centrifugation (Biochrom AG, Berlin, Germany). For negative isolation of cd T cells from PBMC, the TCRc/d+ T Cell Isolation Kit was used following the manufacturer’s protocol. ab T cells were negatively isolated with the Pan T Cell Isolation Kit followed by depletion of cd T cells with the Anti-TCR c/d MicroBead Kit from this population. Positive isolation of cd T cells was performed with the antiTCR c/d MicroBead Kit. All kits were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). The purity of the cells was 99% for ab T cells and >92% for cd T cells. Analysis of T cell proliferation. PBMC containing 3–6% cd T cells were cultured at 1 9 105 cells per well in 96-well round-bottom microtiter plates in X-Vivo15
medium (Lonza, K€oln, Germany). Serum-free medium was selected to avoid interference of protease inhibitors present in serum. To selectively activate and expand cd T cells within PBMC, the cells were cultured in medium or in the presence of 300 nM BrHPP (Innate Pharma, Marseille, France) or 5 lM zoledronate and 50 IU IL-2/ml (both from Novartis, Basel, Switzerland) for up to 9 days. Isolated cd or ab T cells (1 9 104 per well) were stimulated with 2 9 104 MACSiBead particles per well loaded with anti-CD2, anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec). Purified natural proteases PR3, NE and CG (all from Athens Research & Technology, Athens GA, USA) were added at final concentrations indicated under Results. The proliferation of T cells was measured by the uptake of tritiated thymidine (3H-TdR). To this end, each well received 1 lCi 3H-TdR during the last 8 h of culture, in case of cd T cells on days 5, 7 and 9, and in the case of ab T cells on days 2, 4 and 6. Uptake of 3H-TdR was measured using a Wallac 1450 Microbeta Trilux counter (Perkin Elmer, Rodgau, Germany). Flow cytometry. For surface marker expression, the following monoclonal antibodies (mAb) were used: APCanti-pan-cd TCR (clone 11F2), PE-anti-CD25 (clone 2A3), FITC-anti-HLA-DR (clone L243) and APC-anti-CD14 (clone MoP9), all from BD Biosciences (Heidelberg, Germany); FITC-anti-pan-ab TCR (clone BMA031, from Life Technologies, Darmstadt, Germany) and PE-antiCD277 (clone BT3.1, from Biolegend, Fell, Germany). To detect intracellular cytokines, 3 lM monensin was added for the last 4 h of cell culture. Permeabilization and fixation of the cells for intracellular staining was performed with Cytofix/Cytoperm (BD Biosciences). PE-anti-IFN-c (clone 4S.B3) and PE-anti-TNF-a (clone MAb11, both from BD Biosciences) antibodies were used in combination with the anti-TCR cd mAb. Cell death was analysed by staining with 0.2 lg/ml propidium iodide (PI; Serva, Heidelberg, Germany). All samples were measured on a FACSCalibur (BD Biosciences) using the CellQuestPro Software. Both the percentage of positive cells and the mean fluorescence intensity (MFI) were measured. ELISA. Cytokines levels in cell culture supernatants were determined after 48 h of stimulation. IFN-c and TNF-a DuoSet ELISA kits were obtained from R&D Systems GmbH (Wiesbaden, Germany), and ELISAs were performed following the manufacturer’s protocol. Real-time cell analyzer. To determine the influence of serine proteases on the cytotoxicity of cd T cells against tumour cells, 5000 adherent pancreatic adenocarcinoma cell line Panc89 cells (kindly provided by Prof. Kalthoff, Section of Molecular Oncology, Kiel) per well were plated in X-Vivo15 medium in a 96-well micro-E-plate. During the first 24 h of tumour cell culture, positively isolated cd T cells were preincubated in parallel in X-Vivo15 medium with 300 nM BrHPP and 50 IU IL-2/ml in the absence or presence of 1 lg/ml PR3, NE or CG. Thereafter, cd T cells
Scandinavian Journal of Immunology, 2014, 80, 381–389
J. Fazio et al. cd T Cells and Serine Proteases 383 ..................................................................................................................................................................
Figure 1 Inhibition of T cell proliferation by neutrophil serine proteases. (A) Stimulation of cd T cells with zoledronate and IL-2 within PBMC. Proliferation was analysed on days 5, 7 and 9 by 3H-TdR uptake for the last 8 h as shown for one representative donor (mean cpm of triplicate cultures). (B) Mean standard deviation (SD) of proliferation of cd T cells within PBMC of four donors after stimulation with zoledronate and IL-2 for 7 days. (C) Negatively isolated cd T cells or (E) ab T cells were stimulated for 5–9 days (C) or 2–6 days (E) with anti-CD2-, anti-CD3- and antiCD28-coated beads and IL-2. Proliferation was analysed as mentioned above. Mean cpm of triplicate cultures of one representative experiment (C, E). Mean SD of three experiments analysing cd T cell proliferation on day 7 (D) or ab T cell proliferation on day 4 (F). Statistical analysis was performed with paired t-tests. *P < 0.05.
A
B
C
D
E
F
were washed twice and then added to the tumour cells at an effector/target ratio of 25:1 without any additional stimulus. The cell impedance (corresponding to cell death) was measured with the Real-Time Cell Analyzer (RTCA)Single plate (SP) assay (Roche, Mannheim, Germany) [35] . The plates were recorded every minute for the first 6 h and every 15 min for the residual course of the experiment. Proteolytic cleavage of IL-2. One microgram IL-2 (Proleukin; Novartis) was incubated with 0.1 lg/ml of each protease for 24 h at 37 °C. After denaturation of the samples for 4 min at 100 °C, samples were run on a 4–20% Mini-PROTEAN gradient gel (BioRad, Munich, Germany). After transferring the proteins from the gel to a nitrocellulose membrane, the membrane was blocked and then incubated overnight at 4 °C with anti-IL-2 mAb (Acris, Herford, Germany). After a washing step, the membrane was incubated with peroxidase-coupled goat anti-mouse antibody (Dianova, Hamburg, Germany) at room temperature for 1 h. For detection, we used the ECL Blotting Reagent (GE Healthcare, Munich, Germany). Statistical analysis. For processing of data and statistical analysis, Microsoft Office Excel and Prism GraphPad 6.0 (GraphPad Inc., La Jolla, CA) were used. Significances were
Ó 2014 John Wiley & Sons Ltd
analysed with paired t-tests, one-way ANOVA and Dunnett’s multiple comparison. Significance was set at P = 0.05 and is depicted as * for P ≤ 0.05 and ** for P ≤ 0.01.
Results Inhibition of cd T cell proliferation by neutrophil serine proteases
Selective proliferation of cd T cells within PBMC can be induced by stimulation with the nitrogen-containing bisphosphonate zoledronate or the pAg BrHPP in the presence of exogenous IL-2. As shown in Fig. 1, cd T cell proliferation was strongly inhibited in the presence of all three serine proteases. Dose titration experiments over a range of 0.01 to 10 lg/ml indicated that inhibition occurred at concentrations of 0.1 lg/ml and higher (data not shown). Therefore, we selected a concentration of 0.1 lg/ml for each of the proteases. As exemplified for one representative donor in Fig. 1A, the proliferation of zoledronate-stimulated cd T cells within PBMC was inhibited over the whole time period of analysis from day 5 through day 9. A summary of experiments with four
384 cd T Cells and Serine Proteases J. Fazio et al. .................................................................................................................................................................. A
B
C
D
E
F
Figure 2 Inhibition of cytokine production by serine proteases. PBMC were stimulated for 48 h with zoledronate and IL-2. (A) The amounts of TNF-a or (D) IFN-c in cell culture supernatants were analysed by ELISA (n = 9). Intracellular cytokine expression was analysed after addition of 3 lM monensin for the last 4 h (B,C,E,F). The final concentration of the proteases was 1 lg/ml. A gate was set on cd T cells. (B) Overlay of TNF-a-positive cd T cells of one representative donor (control = black, PR3 = red, NE = green, CG = blue) and (C) mean fluorescence intensity (MFI) of TNF-a-positive cd T cells of three donors. (E) Overlay of IFN-c-positive cd T cells as described for (A). (F) MFI of IFN-c-positive cd T cells of three donors. Statistical analysis was performed using paired t-tests. *P < 0.05, **P < 0.01.
donors analysed on day 7 is presented in Fig. 1B. Similar results were obtained with BrHPP stimulation (data not shown). While the addition of proteases at the initiation of cell culture was most efficient, delayed addition on day 3 of a 7-day culture period still resulted in significant inhibition of zoledronate- or BrHPP-stimulated cellular proliferation (not shown). All three proteases also inhibited the proliferation of purified cd T cells stimulated with anti-CD2/CD3/ CD28-coated beads in the presence of IL-2 proliferated (Fig. 1C). A summary of four experiments analysed on day 7 is shown in Fig. 1D. Even though we used 3H-TdR uptake as a read-out for cell proliferation in our studies, pilot experiments indicated that this was in complete agreement with enumerating the actual number of proliferating cd T cells by a previously established flow cytometric method [36]. While the proliferation of cd T cells was significantly inhibited, the proliferation of negatively isolated and antiCD2/CD3/CD28 bead-stimulated ab T cells was not influenced by PR3 and CG, and was only slightly reduced by NE, each used at 0.1 lg/ml (Fig. 1E,F). Serine proteases inhibit the cytokine production in T cells
Apart from proliferation, we also analysed the effect of the serine proteases on effector functions of cd T cells. As
illustrated in Fig. 2, the production of the inflammatory cytokines TNF-a and IFN-c was again inhibited in the presence of neutrophil serine proteases. The analysis of culture supernatants after 48 h of zoledronate-stimulated PBMC revealed that there was significantly less TNF-a (Fig. 2A) and IFN-c (Fig. 2D) released after stimulation in the presence of serine proteases compared to the medium control. As cytokines measured in supernatants of activated PBMC might have been produced by cells other than cd T cells, we next analysed intracellular cytokines by flow cytometry in cd T cells after 48 h of zoledronate stimulation. As shown in Fig. 2B and E for individual donors, all three proteases inhibited the induction of TNFa (Fig. 2B) and IFNc (Fig. 2E). A summary of experiments performed with three donors is shown for TNFa in Fig. 2C and for IFNc in Fig. 2F. Again, comparable results were obtained after stimulation with BrHPP (data not shown). Serine proteases reduce the cytotoxic activity of cd T cells
To investigate the influence of the three serine proteases on cd T cell cytotoxicity, we used the RTCA system that measures the impedance of the adherent tumour cells. When the tumour cells are killed by cd T cells, the impedance is reduced, thereby decreasing the cell index
Scandinavian Journal of Immunology, 2014, 80, 381–389
J. Fazio et al. cd T Cells and Serine Proteases 385 .................................................................................................................................................................. A
B
C
D
Figure 3 Cytotoxicity of cd T cells against Panc89 tumour cells. Freshly isolated cd T cells were preincubated for 24 h with BrHPP and IL-2 in the presence or absence of proteases. After washing twice, cd T cells were added to the tumour cells at an effector/target ratio of 25:1 without any further stimulus. The cell index was normalized to 1 at the time of addition of cd T cells as indicated by the vertical black thin line. Every line presents the average of duplicate or triplicate values; cyan = tumour cells alone, magenta = Triton X-100 (maximal lysis), black = cd T cells/medium, red = cd T cells/PR3, green = cd T cells/NE, blue = cd T cells/CG. Results of four individual experiments (A–D) with cd T cells from different donors are shown.
[35]. As shown in Fig. 3, freshly isolated cd T cells from four different donors were able to lyse Panc89 tumour cells after preincubation with BrHPP and IL-2 (black line), compared to tumour cells cultured in medium alone (cyan line). Overnight preincubation of cd T cells with PR3, NE or CG in the presence of BrHPP and IL-2 resulted in reduced killing activity. While the preincubation of cd T cells from some donors with the proteases led to a complete abrogation of their cytotoxicity against Panc89 tumour cells (Fig. 3A,B), the cytotoxic activity was diminished only to some degree in other donors (Fig. 3C,D). However, the three serine proteases negatively influenced the cytotoxic activity of cd T cells against Panc89 tumour cells in all tested donors. Importantly, this effect was not due to a direct activity of the proteases on the tumour cells as the cd T cells had been washed twice following preincubation with the proteases. Effect of serine proteases on IL-2 receptor expression and IL-2
The following experiments were designed to get more insight into the mechanisms of protease-dependent inhibition of cd T cell activation. As revealed by propidium iodide staining and flow cytometry, the serine proteases did
Ó 2014 John Wiley & Sons Ltd
not induce cell death in cd T cells (not shown). Next we investigated the influence of the proteases on IL-2 and its receptor. To this end, we analysed the possible modulation of CD25 expression on the cell surface of cd T cells after incubation with the serine proteases in the presence of zoledronate and IL-2. In these experiments, the proteases were used at 1 lg/ml, and analysis was performed after 48 h. As shown in Fig. 4A, PR3, NE and CG did not modulate the relative proportion of CD25 positive cd T cells upon zoledronate plus IL-2 stimulation. Some reduction of CD25 expression was noted, however, when the MFI rather than the percentage of positive cells was analysed (Fig. 4B). Here, particularly, NE reduced the MFI of CD25 expression, as also published for PHA-activated T cells by Bank et al. [30]. Therefore, the reduced intensity of CD25 expression on the cell surface of cd T cells after protease treatment might contribute to the inhibition of proliferation. We then analysed the influence of the three proteases on the growth factor IL-2 itself. As illustrated in Fig. 4C, incubation of IL-2 with PR3, NE or CG led to proteolytic cleavage. In the case of PR3 and CG, a smaller cleavage fragment with less than the 17 kDa of IL-2 appeared. The incubation with NE resulted in a strong reduction of the overall amount of IL-2 protein.
386 cd T Cells and Serine Proteases J. Fazio et al. .................................................................................................................................................................. A
A
B B
C
C
Figure 4 Influence of serine proteases on CD25 expression and IL-2. (A) Flow cytometric analysis of CD25 expression on cd T cells after 48 h of stimulation with zoledronate and IL-2 is presented as the percentages of CD25-positive cd T cells and (B) the MFI of CD25 on cd T cells (n = 3). Statistical analysis was performed using Dunnett’s multiple comparison. (C) Western blot analysis of IL-2 preincubated for 24 h with serine proteases or medium (one representative experiment out of three).
PR3 and NE but not CG modulate cell surface expression of CD277
BTN3A1 (CD277) was recently shown to be absolutely required for the activation of cd T cells with phosphoantigens and with nitrogen-containing bisphosphonates [9–13]. Therefore, we analysed possible effects of the serine proteases on the cell surface expression of CD277 on PBMC stimulated for 48 h with zoledronate and IL-2 in the absence of presence of proteases. The expression of CD277 in the presence of the proteases (PR3 = red, NE = green, CG = blue) is shown in comparison with the medium control (black) for one representative donor in Fig. 5A. Both PR3 and NE reduced the CD277 expression in comparison with medium or CG. A summary of experiments with nine donors is presented in Fig. 5B. Isotype controls were not affected by any of the proteases compared to the medium control (not shown). In all cases, the percentage of CD277positive cells remained unchanged (Fig. 5C). The expression of CD277 on cd T cells themselves was also significantly
Figure 5 Modulation of cell surface expression of CD277 by serine proteases. PBMC were cultured for 48 h in the presence of zoledronate/IL2 and absence or presence of serine proteases. (A) Flow cytometric analysis of one representative donor with an overlay of the CD277 expression under the four different conditions as follows: medium = black, PR3 = red, NE = green, CG = blue. (B) MFI SD of CD277 on PBMC and (C) percentages of CD277 positive cells. A gate was set on all living cells using the forward and side scatter. The cells were stained with mAb anti-CD277 (n = 9). Statistical analysis was performed with paired t-tests. * P < 0.05.
reduced after incubation with PR3, and somewhat reduced in the presence of NE, but not affected by CG (Fig. S1).
Discussion In this study, we have identified a strong inhibitory effect of the three neutrophil serine proteases PR3, NE and CG on the proliferation, cytokine release and cytotoxic activity of cd T cells. Inhibitory concentrations of the serine proteases on cd T cells had only a minor impact on ab T
Scandinavian Journal of Immunology, 2014, 80, 381–389
J. Fazio et al. cd T Cells and Serine Proteases 387 ..................................................................................................................................................................
cells. Proliferation of T cells depends on the availability of IL-2 and the activation-induced expression of the IL-2 receptor complex [37]. The two proteases PR3 and NE, but not CG, slightly modulated the surface expression of the IL-2 receptor a-chain (CD25), in line with published reports [30]. In our studies, all three proteases strongly inhibited cd T cell proliferation and also restrained their production of TNF-a and IFN-c. Given the differential effect of PR3 and NE versus CG, reduction of cell surface CD25 expression alone cannot explain the inhibitory effect on cd T cell proliferation. In addition, however, the proteases proteolytically cleave IL-2, which might contribute to the overall inhibition of cd T cell proliferation, as cd T cells are unable to produce substantial amounts of IL-2 [38]. While similar inhibition of ab T cell proliferation might be expected if cleavage of IL-2 was the major mechanism, it is conceivable that ab T cell proliferation is much less affected due to massive autocrine IL-2 production by activated CD4 T cells. Repetitive addition of exogenous IL-2 at different time points after initial culture initiation in the presence of PR3 only partially abrogated the inhibition of cd T cell proliferation, possibly due to the remaining protease activity (results not shown). Nevertheless, degradation of the essential growth factor IL-2 might be a major contributing factor in the inhibition of cd T cell proliferation and subsequent production of TNF-a and IFN-c. The activation of cd T cells by pAg and nitrogencontaining bisphosphonates critically depends on BTN3A1/CD277 [9–13]. When PBMC were incubated for 48 h with the proteases, we observed that PR3 and to a lower extent NE reduced the intensity of CD277 expression on the cell surface of PBMC (Fig. 5B), while the percentages of CD277-positive cells remained unchanged in the presence of all three proteases (Fig. 5C). Again, modulation of CD277 expression is not the only underlying mechanism of the inhibitory effect of the serine proteases, because CG did not modulate CD277 expression despite its strong suppression of cd T cell proliferation, cytokine production and cytotoxic activity. Serine proteases do not always exert inhibitory effects on immune cells. For instance, PR3 induces dendritic cell (DC) maturation by interaction with the cell surface receptor PAR-2 (protease-activated receptor 2) and thus exerts positive effects [39]. In a study published in the mid-1990s, CG was reported to actually stimulate lymphocyte proliferation [31]. In this study, however, much higher concentrations of CG were used, and the experiments were performed in serum-containing medium. Of note, all experiments in our study were performed in serum-free medium. Due to the presence of protease inhibitors in serum (notably a1-antitrypsin [AAT]) [40, 41], the inhibitory effect of serine proteases on cd T cell activation was strongly diminished in serum-containing medium. However, there was also a slight but inconsistent decrease of cd T cell proliferation in the presence of Ó 2014 John Wiley & Sons Ltd
PR3 when medium was supplemented with human serum (data not shown). The inhibitory activity of the serine proteases requires enzymatically active proteins. In our study, we used proteolytically active proteases purified from human neutrophils. The inhibitory activity of PR3 was abrogated by heat inactivation (not shown). Furthermore, recombinant PR3 expressed in E. coli did not have any inhibitory activity (not shown). While our in vitro studies in serum-free medium have clearly identified a preferential and previously unrecognized susceptibility of cd as opposed to ab T cells to inhibitory effects of serine proteases, the in vivo relevance of these findings remains to be clarified. Reciprocal interactions between neutrophils and cd T cells have been investigated. Neutrophils harbouring phagocytosed bacteria actually activate Vc9Vd2 T cells through the release of microbial pAg [34]. However, neutrophils can also inhibit cd T cell activation, and reactive oxygen species production by neutrophils has been identified as a major mechanism [26, 27]. In our previous study, we also noted that inhibition of cd T cell expansion in whole leucocyte cultures stimulated with zoledronate and IL-2 could be partially reverted by AAT, suggesting that neutrophil proteases contributed to the overall inhibition of cd T cell expansion [26]. However, all studies identifying positive and negative reciprocal interactions between human neutrophils and cd T cells were carried out in serum-supplemented culture medium, thereby precluding the detection of a major effect of neutrophil serine proteases [26, 27, 34]. Despite the limitations of our in vitro observations made under serumfree culture conditions, it can be envisioned that there are situations where the reported effect of serine proteases on cd T cells could play a role in vivo. At the sites of inflammation in tissue, where neutrophils release their effector molecules including serine proteases, protease inhibitors are not necessarily present in equally high concentrations as in serum. Furthermore, patients with AAT deficiency have strongly reduced AAT serum levels [42]. Here, low levels of AAT might less efficiently counteract the suppressive capacity of neutrophil serine proteases on cd T cells in these patients. In conclusion, we found that all three neutrophil serine proteases inhibit cd T cell activation. We were unable, however, to identify a single underlying mechanism. It rather appears that several mechanisms including modulation of cell surface antigen expression (CD25, CD277) but also proteolytic cleavage of IL-2 contribute to the overall inhibition.
Acknowledgment This work was supported by the Deutsche Forschungsgemeinschaft (KA 502/16-1 and KFO170, project 3) and a grant from the Medical Faculty of Kiel University to SK. We thank Ina Martens for technical assistance and
388 cd T Cells and Serine Proteases J. Fazio et al. .................................................................................................................................................................. all members of the Wesch and Kabelitz groups for support.
Conflict of interest All authors state that they have no conflict of interests.
References 1 Bonneville M, O’Brien RL, Born WK. cd T cell effector functions: a blend of innate programming and acquired plasticity. Nat Rev Immunol 2010;10:467–78. 2 Kabelitz D, Wesch D, Hinz T. cd T cells, their T cell receptor usage and role in human diseases. Springer Semin Immunpathol 1999;21:55–75. 3 Kalyan S, Kabelitz D. Defining the nature of human cd T cells: a biographical sketch of the highly empathetic. Cell Mol Immunol 2013;10:21–9. 4 Grober H, Kistowska M, Angman L, Jen€o P, Mori L, De Libero G. Human T cell receptor cd cells recognize endogenous mevalonate metabolites in tumor cells. J Exp Med 2003;197:163–8. 5 Tanaka Y, Sano S, Nieves E et al. Nonpeptide ligands for human cd T cells. Proc Natl Acad Sci USA 1994;91:8175–9. 6 Kunzmann V, Bauer E, Feurle J, Weissinger F, Tony HP, Wilhelm M. Stimulation of cd T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 2000;96:384– 92. 7 Espinosa E, Belmants C, Pont F, Luciani B, Poutpot R, Romagne F. Chemical synthesis and biological activity of bromohydrin pyrophosphate, a potent stimulator of human cd T cells. J Biol Chem 2001;276:18337–44. 8 Kabelitz D. Small molecules for the activation of human cd T cell responses against infection. Recent Pat Antiinfect Drug Discov 2008;3:1–9. 9 Harly C, Guillaume Y, Nedellec S et al. Key implication of CD277/ butyrophilin-3 (BTN3A) in cellular stress sensing by a major human cd T cell subset. Blood 2012;120:2269–79. 10 Wang H, Henry O, Distefano M et al. Butyrophilin 3A1 plays an essential role in prenyl pyrophosphate stimulation of human Vc2Vd2 T cells. J Immunol 2013;191:1029–42. 11 Kabelitz D. Critical role of butyrophilin 3A1 in presenting prenyl pyrophosphate antigens to human cd T cells. Cell Mol Immunol 2014;11:117–9. 12 Vavassori S, Kumar A, Wan G et al. Butyrophilin 3A1 binds phosphorylated antigens and stimulates human cd T cells. Nat Immunol 2013;14:908–16. 13 Sandstrom A, Peigne CM, Leger A et al. The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate activation of human Vc9Vd2 T cells. Immunity 2014;40:490–500. 14 Wesch D, Glatzel A, Kabelitz D. Differentiation of resting human peripheral blood cd T cells toward Th1- or Th2-phenotype. Cell Immunol 2001;212:110–7. 15 Ness-Schwickerath KJ, Jin C, Morita CT. Cytokine requirements for the differentiation and expansion of IL-17A- and IL-22-producing human Vc2Vd2 T cells. J Immunol 2010;184:7268–80. 16 Pechhold K, Wesch D, Schondelmaier S, Kabelitz D. Primary activation of Vc9-expressing cdT cells by Mycobacterium tuberculosis. Requirement for Th1-type CD4 T cell help and inhibition by IL-10. J Immunol 1994;152:4984–92. 17 Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced MHC molecules by intestinal cd T cells. Science 1998;279:1737–40. 18 Wrobel P, Shojaei H, Schittek B et al. Lysis of a broad range of epithelial tumour cells by human cd T cells: involvement of NKG2D ligands and T-cell receptor- versus NKG2D-dependent recognition. Scand J Immunol 2007;66:320–8.
19 Peters C, Oberg H, Kabelitz D, Wesch D. Phenotype and regulation of immunosuppressive V d2-expressing cd T cells. Cell Mol Life Sci 2014;71:1943–60. 20 K€uhl A, Pawlowski N, Grollich K et al. Human peripheral blood cd T cells possess regulatory potential. Immunology 2009;128:580–8. 21 Brandes M, Willimann K, Moser B. Professional antigen-presentation function by human cd T cells. Science 2005;309:264–8. 22 Kabelitz D, He W. The multifunctionality of human Vc9Vd2 cd T cells: clonal plasticity or distinct subsets? Scand J Immunol 2012;76:213–22. 23 Thewissen M, Damoiseaux J, van de Gaar J, Tervaert JW. Neutrophils and T cells: bidirectional effects and functional interferences. Mol Immunol 2011;48:2094–101. 24 Hock BD, Taylor KG, Cross NB, Kettle AJ, Hampton MB, McKenzie JL. Effect of activated human polymorphonuclear leucocytes on T lymphocyte proliferation and viability. Immunology 2012;137:249–58. 25 Kalyan S, Kabelitz D. When neutrophils meet T cells: beginnings of a tumultuous relationship with under-appreciated potential. Eur J Immunol 2014;44:627–33. 26 Kalyan S, Chandrasekaran V, Quabius S, Lindhorst T, Kabelitz D. Neutrophil uptake of nitrogen-bisphosphonates leads to the suppression of human peripheral blood cd T cells. Cell Mol Life Sci 2014;71:2335–46. 27 Sabbione F, Gabelloni ML, Ernst G et al. Neutrophils suppress cd Tcell function. Eur J Immunol 2014;44:819–30. 28 Korkmaz B, Thierry M, Gauthier F. Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions. Biochem 2008;90:227–42. 29 Methot N, Rubin J, Guay D et al. Inhibition of activation of multiple serine proteases with a cathepsin C inhibitor requires sustained exposure to prevent pro-enzyme processing. J Biol Chem 2007;282:20836–46. 30 Bank U, Reinhold D, Schneemilch C, Kunz D, Synowitz H, Ansorge S. Selective proteolytic cleavage of IL-2 receptor and IL-6 receptor ligand binding chains by neutrophil-derived serine proteases at foci of inflammation. J Interferon Cytokine Res 1999;19:1277–87. 31 Hase-Yamazaki T, Aoki Y. Stimulation of human lymphocytes by cathepsin G. Cell Immunol 1995;160:24–32. 32 Niles J, McCluskey R, Ahmad M, Arnaout M. Wegener’s granulomatosis autoantigen is a novel neutrophil serine proteinase. Blood 1989;74:1888–93. 33 Fazio J, Quabius ES, M€uller A et al. Vd2 T cell deficiency in granulomatosis with polyangiitis (Wegener’s granulomatosis). Clin Immunol 2013;149:65–72. 34 Davey MS, Lin CY, Roberts GW et al. Human neutrophil clearance of bacterial pathogens triggers anti-microbial cd T cell responses in early infection. PLoS Pathog 2011;7:e1002040. 35 Oberg HH, Peipp M, Kellner C et al. Novel bispecific antibodies increase cd T-cell cytotoxicity against pancreatic cancer cells. Cancer Res 2014;74:1349–60. 36 Pechhold K, Pohl T, Kabelitz D. Rapid quantification of lymphocyte subsets in heterogeneous cell populations by flow cytometry. Cytometry 1994;16:152–9. 37 Wesch D, Marx S, Kabelitz D. Comparative analysis of ab and cd T cell activation by Mycobacterium tuberculosis and isopentenyl pyrophosphate. Eur J Immunol 1997;27:952–6. 38 Wesch D, Kabelitz D, Friese K, Pechhold K. Mycobacteria-reactive cd T cells in HIV-infected individuals: lack of Vc9 cell responsiveness is due to deficiency of antigen-specific CD4 T helper type 1 cells. Eur J Immunol 1996;26:557–62. 39 Csernok E, Ai M, Gross WL, Wicklein D et al. Wegener autoantigen induces maturation of dendritic cells and licenses them for Th1 priming via the protease-activated receptor-2 pathway. Blood 2006;107:4440–8.
Scandinavian Journal of Immunology, 2014, 80, 381–389
J. Fazio et al. cd T Cells and Serine Proteases 389 .................................................................................................................................................................. 40 Carell R. The molecular structure and pathology of alpha1-antitrypsin. Lung 1990;168 (Suppl):530–4. 41 Wood A, Stockley R. Alpha 1 antitrypsin deficiency: from gene to treatment. Respiration 2007;74:481–92. 42 Kueppers F, Briscoe W, Bearn A. Hereditary deficiency of serum alpha1-antitrypsin. Science 1964;146:1678–9.
Ó 2014 John Wiley & Sons Ltd
Supporting Information Additional supporting information may be found in the online version of this article: Figure S1. Modulation of cell surface expression of CD277 on cd T cells.
Inhibition of Human cd T Cell Proliferation and Effector Functions by Neutrophil Serine Proteases J. Fazio, S. Kalyan, D. Wesch & D. Kabelitz
Abstract Institute of Immunology, Christian-AlbrechtsUniversity of Kiel, Kiel, Germany
Received 18 July 2014; Accepted in revised form 16 August 2014 Correspondence to: D. Kabelitz, Institute of Immunology, Christian-Albrechts-University of Kiel, Arnold-Heller-Str. 3 Haus 17, D-24105 Kiel, Germany. E-mail: [email protected]
Human peripheral blood cd T cells expressing the Vc9Vd2 T cell receptor are activated by microbial or endogenous pyrophosphate antigens and indirectly by nitrogen-containing bisphosphonates. Apart from proliferation, such phosphoantigens induce proinflammatory cytokine production including TNF-a and IFN-c and trigger cytotoxic effector function. Neutrophil granulocytes are known to modulate T cell activation. The neutrophil serine proteases proteinase 3, elastase and cathepsin G have multiple potential targets and promote microbial killing. In this study, we investigated the effect of the three serine proteases on the in vitro proliferation and effector functions of cd T cells cultured in serum-free medium. All three proteases inhibited the proliferative activity, suppressed the cytokine production and decreased the cytotoxicity of cd T cells. Further studies indicated that proteolytic cleavage of IL-2 and modulation of butyrophilin 3A1 (CD277) expression might contribute to the overall inhibition.
Introduction cdT cells account for 1–10% of T lymphocytes in the peripheral blood of healthy adults. Subpopulations of cd T cells can be identified on the basis of T cell receptor (TCR) variable genes. The majority of blood cd T cells express Vc9 paired with Vd2 [1, 2]. Vc9Vd2 T cells recognize phosphorylated small molecules, so-called phosphoantigens (pAg), which are intermediates of the microbial and eukaryotic isoprenoid biosynthesis pathway. Many bacteria produce the pAg (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), which stimulates human cd T cells at pico- to nanomolar concentrations, whereas the eukaryotic pAg isopentenyl pyrophosphate (IPP) requires micromolar concentrations to activate cd T cells. The selective sensing of microbial pAg by Vc9Vd2 T cells suggests that these cells play a pivotal role in anti-infective immune responses [3]. The concentration of IPP generated in normal cells is insufficient to activate cd T cells, but higher concentrations accumulate upon cellular transformation which is associated with a dysregulated mevalonate pathway [4]. Vc9Vd2 T cells can be strongly and selectively activated in vitro by synthetic pAg such as bromohydrin pyrophosphate (BrHPP) or with nitrogencontaining bisphosphonates, which induce the intracellular accumulation of IPP [4–8]. Recently, a critical role of the butyrophilin family member BTN3A1 (CD277) in the activation of cd T cells by pAg has been discovered [9, 10]. Even though the precise role of BTN3A1 is still under
Ó 2014 John Wiley & Sons Ltd
investigation [11], BTN3A1 may serve as an extracellular [12] and/or intracellular receptor [13] for exogenous or endogenous pAg. Once activated, Vc9Vd2 T cells mediate diverse effector functions. They usually produce cytokines such as TNF-a and IFN-c, but can be also driven to secrete IL-4 or IL-17 [14, 15]. Vc9Vd2 T cells are unable to produce IL-2 in adequate amounts to sustain autocrine proliferation and therefore the addition of exogenous IL-2- or IL-2-producing CD4 T cells is required for proliferation [16]. Most cd T cells express the activating NK receptor NKG2D (Natural Killer Group 2 Member D), which recognizes stress-inducible MHC class-I-related molecules including MHC class I chain-related antigen A/B (MICA/B). Concomitantly, cd T cells can exert cytotoxicity against tumour cells through TCR- and/or NKG2D-dependent activation [17, 18]. In addition to cytokine production and cytotoxic activity, cd T cells can exert regulatory and antigen-presenting functions; thus, they display a surprisingly large functional plasticity [19–22]. Neutrophils are known to modulate T cell activation [23–25] and to inhibit cd T cell activation [26, 27]. Among the potent mediators produced by neutrophils are serine proteases that are stored in the azurophilic granules. Proteinase 3 (PR3), neutrophil elastase (NE) and cathepsin G (CG) are three serine proteases that are members of the chymotrypsin family. They are transcribed only in the early stages of myeloid differentiation of granulocytes and, to a lower extent, of monocytes. After maturation of
381
382 cd T Cells and Serine Proteases J. Fazio et al. .................................................................................................................................................................. granulocytes and monocytes, the transcription of mRNA for the three proteases is downregulated [28]. All three proteases are stored as zymogens in granulocytes and have to be cleaved by cathepsin C for activation [29]. Due to their multiple potential targets, the neutrophil proteases have manifold functions in inflammation and infection [28]. All three serine proteases are known to degrade components of the extracellular matrix and to promote microbial killing. In contrast to CG, PR3 and NE can cleave the IL-2 receptor a-chain from the cell surface, whereas CG is rather able to cleave the IL-6 receptor [30]. At high concentrations, CG has been reported to stimulate lymphocyte proliferation [31]. Neutrophil serine proteases are also associated with different diseases; the most prominent example is PR3, which is the target of autoantibodies in granulomatosis with polyangiitis (GPA), a rare autoimmune disease characterized by systemic vasculitis and granuloma formation [32]. Except for indirect evidence based on effects of the serine protease inhibitor a1-antitrypsin [26], there is no data available on possible effects of neutrophil serine proteases on cd T cell activation. We have previously reported that Vd2 T cells are significantly reduced in patients with GPA, which might be due to an effect of the serine proteases released by neutrophils at the site of inflammation [33]. Furthermore, serine proteases might also impact on the well-documented interplay between neutrophils and cd T cells during microbial infection [34]. In this study, we have investigated in detail the effect of the three serine proteases PR3, NE and CG on cdT cell functions in vitro.
Materials and methods Cell isolation. Blood of healthy adult donors was provided as leucocyte concentrate by the Department of Transfusion Medicine or from blood donors of the Institute of Immunology, both at UKSH Campus Kiel. The study was approved by the appropriate institutional clinical ethics board (code number: D 405/10), and informed consent was obtained from all donors. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll–Hypaque density gradient centrifugation (Biochrom AG, Berlin, Germany). For negative isolation of cd T cells from PBMC, the TCRc/d+ T Cell Isolation Kit was used following the manufacturer’s protocol. ab T cells were negatively isolated with the Pan T Cell Isolation Kit followed by depletion of cd T cells with the Anti-TCR c/d MicroBead Kit from this population. Positive isolation of cd T cells was performed with the antiTCR c/d MicroBead Kit. All kits were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). The purity of the cells was 99% for ab T cells and >92% for cd T cells. Analysis of T cell proliferation. PBMC containing 3–6% cd T cells were cultured at 1 9 105 cells per well in 96-well round-bottom microtiter plates in X-Vivo15
medium (Lonza, K€oln, Germany). Serum-free medium was selected to avoid interference of protease inhibitors present in serum. To selectively activate and expand cd T cells within PBMC, the cells were cultured in medium or in the presence of 300 nM BrHPP (Innate Pharma, Marseille, France) or 5 lM zoledronate and 50 IU IL-2/ml (both from Novartis, Basel, Switzerland) for up to 9 days. Isolated cd or ab T cells (1 9 104 per well) were stimulated with 2 9 104 MACSiBead particles per well loaded with anti-CD2, anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec). Purified natural proteases PR3, NE and CG (all from Athens Research & Technology, Athens GA, USA) were added at final concentrations indicated under Results. The proliferation of T cells was measured by the uptake of tritiated thymidine (3H-TdR). To this end, each well received 1 lCi 3H-TdR during the last 8 h of culture, in case of cd T cells on days 5, 7 and 9, and in the case of ab T cells on days 2, 4 and 6. Uptake of 3H-TdR was measured using a Wallac 1450 Microbeta Trilux counter (Perkin Elmer, Rodgau, Germany). Flow cytometry. For surface marker expression, the following monoclonal antibodies (mAb) were used: APCanti-pan-cd TCR (clone 11F2), PE-anti-CD25 (clone 2A3), FITC-anti-HLA-DR (clone L243) and APC-anti-CD14 (clone MoP9), all from BD Biosciences (Heidelberg, Germany); FITC-anti-pan-ab TCR (clone BMA031, from Life Technologies, Darmstadt, Germany) and PE-antiCD277 (clone BT3.1, from Biolegend, Fell, Germany). To detect intracellular cytokines, 3 lM monensin was added for the last 4 h of cell culture. Permeabilization and fixation of the cells for intracellular staining was performed with Cytofix/Cytoperm (BD Biosciences). PE-anti-IFN-c (clone 4S.B3) and PE-anti-TNF-a (clone MAb11, both from BD Biosciences) antibodies were used in combination with the anti-TCR cd mAb. Cell death was analysed by staining with 0.2 lg/ml propidium iodide (PI; Serva, Heidelberg, Germany). All samples were measured on a FACSCalibur (BD Biosciences) using the CellQuestPro Software. Both the percentage of positive cells and the mean fluorescence intensity (MFI) were measured. ELISA. Cytokines levels in cell culture supernatants were determined after 48 h of stimulation. IFN-c and TNF-a DuoSet ELISA kits were obtained from R&D Systems GmbH (Wiesbaden, Germany), and ELISAs were performed following the manufacturer’s protocol. Real-time cell analyzer. To determine the influence of serine proteases on the cytotoxicity of cd T cells against tumour cells, 5000 adherent pancreatic adenocarcinoma cell line Panc89 cells (kindly provided by Prof. Kalthoff, Section of Molecular Oncology, Kiel) per well were plated in X-Vivo15 medium in a 96-well micro-E-plate. During the first 24 h of tumour cell culture, positively isolated cd T cells were preincubated in parallel in X-Vivo15 medium with 300 nM BrHPP and 50 IU IL-2/ml in the absence or presence of 1 lg/ml PR3, NE or CG. Thereafter, cd T cells
Scandinavian Journal of Immunology, 2014, 80, 381–389
J. Fazio et al. cd T Cells and Serine Proteases 383 ..................................................................................................................................................................
Figure 1 Inhibition of T cell proliferation by neutrophil serine proteases. (A) Stimulation of cd T cells with zoledronate and IL-2 within PBMC. Proliferation was analysed on days 5, 7 and 9 by 3H-TdR uptake for the last 8 h as shown for one representative donor (mean cpm of triplicate cultures). (B) Mean standard deviation (SD) of proliferation of cd T cells within PBMC of four donors after stimulation with zoledronate and IL-2 for 7 days. (C) Negatively isolated cd T cells or (E) ab T cells were stimulated for 5–9 days (C) or 2–6 days (E) with anti-CD2-, anti-CD3- and antiCD28-coated beads and IL-2. Proliferation was analysed as mentioned above. Mean cpm of triplicate cultures of one representative experiment (C, E). Mean SD of three experiments analysing cd T cell proliferation on day 7 (D) or ab T cell proliferation on day 4 (F). Statistical analysis was performed with paired t-tests. *P < 0.05.
A
B
C
D
E
F
were washed twice and then added to the tumour cells at an effector/target ratio of 25:1 without any additional stimulus. The cell impedance (corresponding to cell death) was measured with the Real-Time Cell Analyzer (RTCA)Single plate (SP) assay (Roche, Mannheim, Germany) [35] . The plates were recorded every minute for the first 6 h and every 15 min for the residual course of the experiment. Proteolytic cleavage of IL-2. One microgram IL-2 (Proleukin; Novartis) was incubated with 0.1 lg/ml of each protease for 24 h at 37 °C. After denaturation of the samples for 4 min at 100 °C, samples were run on a 4–20% Mini-PROTEAN gradient gel (BioRad, Munich, Germany). After transferring the proteins from the gel to a nitrocellulose membrane, the membrane was blocked and then incubated overnight at 4 °C with anti-IL-2 mAb (Acris, Herford, Germany). After a washing step, the membrane was incubated with peroxidase-coupled goat anti-mouse antibody (Dianova, Hamburg, Germany) at room temperature for 1 h. For detection, we used the ECL Blotting Reagent (GE Healthcare, Munich, Germany). Statistical analysis. For processing of data and statistical analysis, Microsoft Office Excel and Prism GraphPad 6.0 (GraphPad Inc., La Jolla, CA) were used. Significances were
Ó 2014 John Wiley & Sons Ltd
analysed with paired t-tests, one-way ANOVA and Dunnett’s multiple comparison. Significance was set at P = 0.05 and is depicted as * for P ≤ 0.05 and ** for P ≤ 0.01.
Results Inhibition of cd T cell proliferation by neutrophil serine proteases
Selective proliferation of cd T cells within PBMC can be induced by stimulation with the nitrogen-containing bisphosphonate zoledronate or the pAg BrHPP in the presence of exogenous IL-2. As shown in Fig. 1, cd T cell proliferation was strongly inhibited in the presence of all three serine proteases. Dose titration experiments over a range of 0.01 to 10 lg/ml indicated that inhibition occurred at concentrations of 0.1 lg/ml and higher (data not shown). Therefore, we selected a concentration of 0.1 lg/ml for each of the proteases. As exemplified for one representative donor in Fig. 1A, the proliferation of zoledronate-stimulated cd T cells within PBMC was inhibited over the whole time period of analysis from day 5 through day 9. A summary of experiments with four
384 cd T Cells and Serine Proteases J. Fazio et al. .................................................................................................................................................................. A
B
C
D
E
F
Figure 2 Inhibition of cytokine production by serine proteases. PBMC were stimulated for 48 h with zoledronate and IL-2. (A) The amounts of TNF-a or (D) IFN-c in cell culture supernatants were analysed by ELISA (n = 9). Intracellular cytokine expression was analysed after addition of 3 lM monensin for the last 4 h (B,C,E,F). The final concentration of the proteases was 1 lg/ml. A gate was set on cd T cells. (B) Overlay of TNF-a-positive cd T cells of one representative donor (control = black, PR3 = red, NE = green, CG = blue) and (C) mean fluorescence intensity (MFI) of TNF-a-positive cd T cells of three donors. (E) Overlay of IFN-c-positive cd T cells as described for (A). (F) MFI of IFN-c-positive cd T cells of three donors. Statistical analysis was performed using paired t-tests. *P < 0.05, **P < 0.01.
donors analysed on day 7 is presented in Fig. 1B. Similar results were obtained with BrHPP stimulation (data not shown). While the addition of proteases at the initiation of cell culture was most efficient, delayed addition on day 3 of a 7-day culture period still resulted in significant inhibition of zoledronate- or BrHPP-stimulated cellular proliferation (not shown). All three proteases also inhibited the proliferation of purified cd T cells stimulated with anti-CD2/CD3/ CD28-coated beads in the presence of IL-2 proliferated (Fig. 1C). A summary of four experiments analysed on day 7 is shown in Fig. 1D. Even though we used 3H-TdR uptake as a read-out for cell proliferation in our studies, pilot experiments indicated that this was in complete agreement with enumerating the actual number of proliferating cd T cells by a previously established flow cytometric method [36]. While the proliferation of cd T cells was significantly inhibited, the proliferation of negatively isolated and antiCD2/CD3/CD28 bead-stimulated ab T cells was not influenced by PR3 and CG, and was only slightly reduced by NE, each used at 0.1 lg/ml (Fig. 1E,F). Serine proteases inhibit the cytokine production in T cells
Apart from proliferation, we also analysed the effect of the serine proteases on effector functions of cd T cells. As
illustrated in Fig. 2, the production of the inflammatory cytokines TNF-a and IFN-c was again inhibited in the presence of neutrophil serine proteases. The analysis of culture supernatants after 48 h of zoledronate-stimulated PBMC revealed that there was significantly less TNF-a (Fig. 2A) and IFN-c (Fig. 2D) released after stimulation in the presence of serine proteases compared to the medium control. As cytokines measured in supernatants of activated PBMC might have been produced by cells other than cd T cells, we next analysed intracellular cytokines by flow cytometry in cd T cells after 48 h of zoledronate stimulation. As shown in Fig. 2B and E for individual donors, all three proteases inhibited the induction of TNFa (Fig. 2B) and IFNc (Fig. 2E). A summary of experiments performed with three donors is shown for TNFa in Fig. 2C and for IFNc in Fig. 2F. Again, comparable results were obtained after stimulation with BrHPP (data not shown). Serine proteases reduce the cytotoxic activity of cd T cells
To investigate the influence of the three serine proteases on cd T cell cytotoxicity, we used the RTCA system that measures the impedance of the adherent tumour cells. When the tumour cells are killed by cd T cells, the impedance is reduced, thereby decreasing the cell index
Scandinavian Journal of Immunology, 2014, 80, 381–389
J. Fazio et al. cd T Cells and Serine Proteases 385 .................................................................................................................................................................. A
B
C
D
Figure 3 Cytotoxicity of cd T cells against Panc89 tumour cells. Freshly isolated cd T cells were preincubated for 24 h with BrHPP and IL-2 in the presence or absence of proteases. After washing twice, cd T cells were added to the tumour cells at an effector/target ratio of 25:1 without any further stimulus. The cell index was normalized to 1 at the time of addition of cd T cells as indicated by the vertical black thin line. Every line presents the average of duplicate or triplicate values; cyan = tumour cells alone, magenta = Triton X-100 (maximal lysis), black = cd T cells/medium, red = cd T cells/PR3, green = cd T cells/NE, blue = cd T cells/CG. Results of four individual experiments (A–D) with cd T cells from different donors are shown.
[35]. As shown in Fig. 3, freshly isolated cd T cells from four different donors were able to lyse Panc89 tumour cells after preincubation with BrHPP and IL-2 (black line), compared to tumour cells cultured in medium alone (cyan line). Overnight preincubation of cd T cells with PR3, NE or CG in the presence of BrHPP and IL-2 resulted in reduced killing activity. While the preincubation of cd T cells from some donors with the proteases led to a complete abrogation of their cytotoxicity against Panc89 tumour cells (Fig. 3A,B), the cytotoxic activity was diminished only to some degree in other donors (Fig. 3C,D). However, the three serine proteases negatively influenced the cytotoxic activity of cd T cells against Panc89 tumour cells in all tested donors. Importantly, this effect was not due to a direct activity of the proteases on the tumour cells as the cd T cells had been washed twice following preincubation with the proteases. Effect of serine proteases on IL-2 receptor expression and IL-2
The following experiments were designed to get more insight into the mechanisms of protease-dependent inhibition of cd T cell activation. As revealed by propidium iodide staining and flow cytometry, the serine proteases did
Ó 2014 John Wiley & Sons Ltd
not induce cell death in cd T cells (not shown). Next we investigated the influence of the proteases on IL-2 and its receptor. To this end, we analysed the possible modulation of CD25 expression on the cell surface of cd T cells after incubation with the serine proteases in the presence of zoledronate and IL-2. In these experiments, the proteases were used at 1 lg/ml, and analysis was performed after 48 h. As shown in Fig. 4A, PR3, NE and CG did not modulate the relative proportion of CD25 positive cd T cells upon zoledronate plus IL-2 stimulation. Some reduction of CD25 expression was noted, however, when the MFI rather than the percentage of positive cells was analysed (Fig. 4B). Here, particularly, NE reduced the MFI of CD25 expression, as also published for PHA-activated T cells by Bank et al. [30]. Therefore, the reduced intensity of CD25 expression on the cell surface of cd T cells after protease treatment might contribute to the inhibition of proliferation. We then analysed the influence of the three proteases on the growth factor IL-2 itself. As illustrated in Fig. 4C, incubation of IL-2 with PR3, NE or CG led to proteolytic cleavage. In the case of PR3 and CG, a smaller cleavage fragment with less than the 17 kDa of IL-2 appeared. The incubation with NE resulted in a strong reduction of the overall amount of IL-2 protein.
386 cd T Cells and Serine Proteases J. Fazio et al. .................................................................................................................................................................. A
A
B B
C
C
Figure 4 Influence of serine proteases on CD25 expression and IL-2. (A) Flow cytometric analysis of CD25 expression on cd T cells after 48 h of stimulation with zoledronate and IL-2 is presented as the percentages of CD25-positive cd T cells and (B) the MFI of CD25 on cd T cells (n = 3). Statistical analysis was performed using Dunnett’s multiple comparison. (C) Western blot analysis of IL-2 preincubated for 24 h with serine proteases or medium (one representative experiment out of three).
PR3 and NE but not CG modulate cell surface expression of CD277
BTN3A1 (CD277) was recently shown to be absolutely required for the activation of cd T cells with phosphoantigens and with nitrogen-containing bisphosphonates [9–13]. Therefore, we analysed possible effects of the serine proteases on the cell surface expression of CD277 on PBMC stimulated for 48 h with zoledronate and IL-2 in the absence of presence of proteases. The expression of CD277 in the presence of the proteases (PR3 = red, NE = green, CG = blue) is shown in comparison with the medium control (black) for one representative donor in Fig. 5A. Both PR3 and NE reduced the CD277 expression in comparison with medium or CG. A summary of experiments with nine donors is presented in Fig. 5B. Isotype controls were not affected by any of the proteases compared to the medium control (not shown). In all cases, the percentage of CD277positive cells remained unchanged (Fig. 5C). The expression of CD277 on cd T cells themselves was also significantly
Figure 5 Modulation of cell surface expression of CD277 by serine proteases. PBMC were cultured for 48 h in the presence of zoledronate/IL2 and absence or presence of serine proteases. (A) Flow cytometric analysis of one representative donor with an overlay of the CD277 expression under the four different conditions as follows: medium = black, PR3 = red, NE = green, CG = blue. (B) MFI SD of CD277 on PBMC and (C) percentages of CD277 positive cells. A gate was set on all living cells using the forward and side scatter. The cells were stained with mAb anti-CD277 (n = 9). Statistical analysis was performed with paired t-tests. * P < 0.05.
reduced after incubation with PR3, and somewhat reduced in the presence of NE, but not affected by CG (Fig. S1).
Discussion In this study, we have identified a strong inhibitory effect of the three neutrophil serine proteases PR3, NE and CG on the proliferation, cytokine release and cytotoxic activity of cd T cells. Inhibitory concentrations of the serine proteases on cd T cells had only a minor impact on ab T
Scandinavian Journal of Immunology, 2014, 80, 381–389
J. Fazio et al. cd T Cells and Serine Proteases 387 ..................................................................................................................................................................
cells. Proliferation of T cells depends on the availability of IL-2 and the activation-induced expression of the IL-2 receptor complex [37]. The two proteases PR3 and NE, but not CG, slightly modulated the surface expression of the IL-2 receptor a-chain (CD25), in line with published reports [30]. In our studies, all three proteases strongly inhibited cd T cell proliferation and also restrained their production of TNF-a and IFN-c. Given the differential effect of PR3 and NE versus CG, reduction of cell surface CD25 expression alone cannot explain the inhibitory effect on cd T cell proliferation. In addition, however, the proteases proteolytically cleave IL-2, which might contribute to the overall inhibition of cd T cell proliferation, as cd T cells are unable to produce substantial amounts of IL-2 [38]. While similar inhibition of ab T cell proliferation might be expected if cleavage of IL-2 was the major mechanism, it is conceivable that ab T cell proliferation is much less affected due to massive autocrine IL-2 production by activated CD4 T cells. Repetitive addition of exogenous IL-2 at different time points after initial culture initiation in the presence of PR3 only partially abrogated the inhibition of cd T cell proliferation, possibly due to the remaining protease activity (results not shown). Nevertheless, degradation of the essential growth factor IL-2 might be a major contributing factor in the inhibition of cd T cell proliferation and subsequent production of TNF-a and IFN-c. The activation of cd T cells by pAg and nitrogencontaining bisphosphonates critically depends on BTN3A1/CD277 [9–13]. When PBMC were incubated for 48 h with the proteases, we observed that PR3 and to a lower extent NE reduced the intensity of CD277 expression on the cell surface of PBMC (Fig. 5B), while the percentages of CD277-positive cells remained unchanged in the presence of all three proteases (Fig. 5C). Again, modulation of CD277 expression is not the only underlying mechanism of the inhibitory effect of the serine proteases, because CG did not modulate CD277 expression despite its strong suppression of cd T cell proliferation, cytokine production and cytotoxic activity. Serine proteases do not always exert inhibitory effects on immune cells. For instance, PR3 induces dendritic cell (DC) maturation by interaction with the cell surface receptor PAR-2 (protease-activated receptor 2) and thus exerts positive effects [39]. In a study published in the mid-1990s, CG was reported to actually stimulate lymphocyte proliferation [31]. In this study, however, much higher concentrations of CG were used, and the experiments were performed in serum-containing medium. Of note, all experiments in our study were performed in serum-free medium. Due to the presence of protease inhibitors in serum (notably a1-antitrypsin [AAT]) [40, 41], the inhibitory effect of serine proteases on cd T cell activation was strongly diminished in serum-containing medium. However, there was also a slight but inconsistent decrease of cd T cell proliferation in the presence of Ó 2014 John Wiley & Sons Ltd
PR3 when medium was supplemented with human serum (data not shown). The inhibitory activity of the serine proteases requires enzymatically active proteins. In our study, we used proteolytically active proteases purified from human neutrophils. The inhibitory activity of PR3 was abrogated by heat inactivation (not shown). Furthermore, recombinant PR3 expressed in E. coli did not have any inhibitory activity (not shown). While our in vitro studies in serum-free medium have clearly identified a preferential and previously unrecognized susceptibility of cd as opposed to ab T cells to inhibitory effects of serine proteases, the in vivo relevance of these findings remains to be clarified. Reciprocal interactions between neutrophils and cd T cells have been investigated. Neutrophils harbouring phagocytosed bacteria actually activate Vc9Vd2 T cells through the release of microbial pAg [34]. However, neutrophils can also inhibit cd T cell activation, and reactive oxygen species production by neutrophils has been identified as a major mechanism [26, 27]. In our previous study, we also noted that inhibition of cd T cell expansion in whole leucocyte cultures stimulated with zoledronate and IL-2 could be partially reverted by AAT, suggesting that neutrophil proteases contributed to the overall inhibition of cd T cell expansion [26]. However, all studies identifying positive and negative reciprocal interactions between human neutrophils and cd T cells were carried out in serum-supplemented culture medium, thereby precluding the detection of a major effect of neutrophil serine proteases [26, 27, 34]. Despite the limitations of our in vitro observations made under serumfree culture conditions, it can be envisioned that there are situations where the reported effect of serine proteases on cd T cells could play a role in vivo. At the sites of inflammation in tissue, where neutrophils release their effector molecules including serine proteases, protease inhibitors are not necessarily present in equally high concentrations as in serum. Furthermore, patients with AAT deficiency have strongly reduced AAT serum levels [42]. Here, low levels of AAT might less efficiently counteract the suppressive capacity of neutrophil serine proteases on cd T cells in these patients. In conclusion, we found that all three neutrophil serine proteases inhibit cd T cell activation. We were unable, however, to identify a single underlying mechanism. It rather appears that several mechanisms including modulation of cell surface antigen expression (CD25, CD277) but also proteolytic cleavage of IL-2 contribute to the overall inhibition.
Acknowledgment This work was supported by the Deutsche Forschungsgemeinschaft (KA 502/16-1 and KFO170, project 3) and a grant from the Medical Faculty of Kiel University to SK. We thank Ina Martens for technical assistance and
388 cd T Cells and Serine Proteases J. Fazio et al. .................................................................................................................................................................. all members of the Wesch and Kabelitz groups for support.
Conflict of interest All authors state that they have no conflict of interests.
References 1 Bonneville M, O’Brien RL, Born WK. cd T cell effector functions: a blend of innate programming and acquired plasticity. Nat Rev Immunol 2010;10:467–78. 2 Kabelitz D, Wesch D, Hinz T. cd T cells, their T cell receptor usage and role in human diseases. Springer Semin Immunpathol 1999;21:55–75. 3 Kalyan S, Kabelitz D. Defining the nature of human cd T cells: a biographical sketch of the highly empathetic. Cell Mol Immunol 2013;10:21–9. 4 Grober H, Kistowska M, Angman L, Jen€o P, Mori L, De Libero G. Human T cell receptor cd cells recognize endogenous mevalonate metabolites in tumor cells. J Exp Med 2003;197:163–8. 5 Tanaka Y, Sano S, Nieves E et al. Nonpeptide ligands for human cd T cells. Proc Natl Acad Sci USA 1994;91:8175–9. 6 Kunzmann V, Bauer E, Feurle J, Weissinger F, Tony HP, Wilhelm M. Stimulation of cd T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 2000;96:384– 92. 7 Espinosa E, Belmants C, Pont F, Luciani B, Poutpot R, Romagne F. Chemical synthesis and biological activity of bromohydrin pyrophosphate, a potent stimulator of human cd T cells. J Biol Chem 2001;276:18337–44. 8 Kabelitz D. Small molecules for the activation of human cd T cell responses against infection. Recent Pat Antiinfect Drug Discov 2008;3:1–9. 9 Harly C, Guillaume Y, Nedellec S et al. Key implication of CD277/ butyrophilin-3 (BTN3A) in cellular stress sensing by a major human cd T cell subset. Blood 2012;120:2269–79. 10 Wang H, Henry O, Distefano M et al. Butyrophilin 3A1 plays an essential role in prenyl pyrophosphate stimulation of human Vc2Vd2 T cells. J Immunol 2013;191:1029–42. 11 Kabelitz D. Critical role of butyrophilin 3A1 in presenting prenyl pyrophosphate antigens to human cd T cells. Cell Mol Immunol 2014;11:117–9. 12 Vavassori S, Kumar A, Wan G et al. Butyrophilin 3A1 binds phosphorylated antigens and stimulates human cd T cells. Nat Immunol 2013;14:908–16. 13 Sandstrom A, Peigne CM, Leger A et al. The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate activation of human Vc9Vd2 T cells. Immunity 2014;40:490–500. 14 Wesch D, Glatzel A, Kabelitz D. Differentiation of resting human peripheral blood cd T cells toward Th1- or Th2-phenotype. Cell Immunol 2001;212:110–7. 15 Ness-Schwickerath KJ, Jin C, Morita CT. Cytokine requirements for the differentiation and expansion of IL-17A- and IL-22-producing human Vc2Vd2 T cells. J Immunol 2010;184:7268–80. 16 Pechhold K, Wesch D, Schondelmaier S, Kabelitz D. Primary activation of Vc9-expressing cdT cells by Mycobacterium tuberculosis. Requirement for Th1-type CD4 T cell help and inhibition by IL-10. J Immunol 1994;152:4984–92. 17 Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced MHC molecules by intestinal cd T cells. Science 1998;279:1737–40. 18 Wrobel P, Shojaei H, Schittek B et al. Lysis of a broad range of epithelial tumour cells by human cd T cells: involvement of NKG2D ligands and T-cell receptor- versus NKG2D-dependent recognition. Scand J Immunol 2007;66:320–8.
19 Peters C, Oberg H, Kabelitz D, Wesch D. Phenotype and regulation of immunosuppressive V d2-expressing cd T cells. Cell Mol Life Sci 2014;71:1943–60. 20 K€uhl A, Pawlowski N, Grollich K et al. Human peripheral blood cd T cells possess regulatory potential. Immunology 2009;128:580–8. 21 Brandes M, Willimann K, Moser B. Professional antigen-presentation function by human cd T cells. Science 2005;309:264–8. 22 Kabelitz D, He W. The multifunctionality of human Vc9Vd2 cd T cells: clonal plasticity or distinct subsets? Scand J Immunol 2012;76:213–22. 23 Thewissen M, Damoiseaux J, van de Gaar J, Tervaert JW. Neutrophils and T cells: bidirectional effects and functional interferences. Mol Immunol 2011;48:2094–101. 24 Hock BD, Taylor KG, Cross NB, Kettle AJ, Hampton MB, McKenzie JL. Effect of activated human polymorphonuclear leucocytes on T lymphocyte proliferation and viability. Immunology 2012;137:249–58. 25 Kalyan S, Kabelitz D. When neutrophils meet T cells: beginnings of a tumultuous relationship with under-appreciated potential. Eur J Immunol 2014;44:627–33. 26 Kalyan S, Chandrasekaran V, Quabius S, Lindhorst T, Kabelitz D. Neutrophil uptake of nitrogen-bisphosphonates leads to the suppression of human peripheral blood cd T cells. Cell Mol Life Sci 2014;71:2335–46. 27 Sabbione F, Gabelloni ML, Ernst G et al. Neutrophils suppress cd Tcell function. Eur J Immunol 2014;44:819–30. 28 Korkmaz B, Thierry M, Gauthier F. Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions. Biochem 2008;90:227–42. 29 Methot N, Rubin J, Guay D et al. Inhibition of activation of multiple serine proteases with a cathepsin C inhibitor requires sustained exposure to prevent pro-enzyme processing. J Biol Chem 2007;282:20836–46. 30 Bank U, Reinhold D, Schneemilch C, Kunz D, Synowitz H, Ansorge S. Selective proteolytic cleavage of IL-2 receptor and IL-6 receptor ligand binding chains by neutrophil-derived serine proteases at foci of inflammation. J Interferon Cytokine Res 1999;19:1277–87. 31 Hase-Yamazaki T, Aoki Y. Stimulation of human lymphocytes by cathepsin G. Cell Immunol 1995;160:24–32. 32 Niles J, McCluskey R, Ahmad M, Arnaout M. Wegener’s granulomatosis autoantigen is a novel neutrophil serine proteinase. Blood 1989;74:1888–93. 33 Fazio J, Quabius ES, M€uller A et al. Vd2 T cell deficiency in granulomatosis with polyangiitis (Wegener’s granulomatosis). Clin Immunol 2013;149:65–72. 34 Davey MS, Lin CY, Roberts GW et al. Human neutrophil clearance of bacterial pathogens triggers anti-microbial cd T cell responses in early infection. PLoS Pathog 2011;7:e1002040. 35 Oberg HH, Peipp M, Kellner C et al. Novel bispecific antibodies increase cd T-cell cytotoxicity against pancreatic cancer cells. Cancer Res 2014;74:1349–60. 36 Pechhold K, Pohl T, Kabelitz D. Rapid quantification of lymphocyte subsets in heterogeneous cell populations by flow cytometry. Cytometry 1994;16:152–9. 37 Wesch D, Marx S, Kabelitz D. Comparative analysis of ab and cd T cell activation by Mycobacterium tuberculosis and isopentenyl pyrophosphate. Eur J Immunol 1997;27:952–6. 38 Wesch D, Kabelitz D, Friese K, Pechhold K. Mycobacteria-reactive cd T cells in HIV-infected individuals: lack of Vc9 cell responsiveness is due to deficiency of antigen-specific CD4 T helper type 1 cells. Eur J Immunol 1996;26:557–62. 39 Csernok E, Ai M, Gross WL, Wicklein D et al. Wegener autoantigen induces maturation of dendritic cells and licenses them for Th1 priming via the protease-activated receptor-2 pathway. Blood 2006;107:4440–8.
Scandinavian Journal of Immunology, 2014, 80, 381–389
J. Fazio et al. cd T Cells and Serine Proteases 389 .................................................................................................................................................................. 40 Carell R. The molecular structure and pathology of alpha1-antitrypsin. Lung 1990;168 (Suppl):530–4. 41 Wood A, Stockley R. Alpha 1 antitrypsin deficiency: from gene to treatment. Respiration 2007;74:481–92. 42 Kueppers F, Briscoe W, Bearn A. Hereditary deficiency of serum alpha1-antitrypsin. Science 1964;146:1678–9.
Ó 2014 John Wiley & Sons Ltd
Supporting Information Additional supporting information may be found in the online version of this article: Figure S1. Modulation of cell surface expression of CD277 on cd T cells.
