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Adaptive Immune ResponsesExtracellular Traps Links Innate and T Lymphocyte Priming by Neutrophil

SospedraKati Tillack, Petra Breiden, Roland Martin and Mireia

ol.1103414http://www.jimmunol.org/content/early/2012/02/20/jimmun

published online 20 February 2012J Immunol 

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The Journal of Immunology

T Lymphocyte Priming by Neutrophil Extracellular TrapsLinks Innate and Adaptive Immune Responses

Kati Tillack,* Petra Breiden,* Roland Martin,*,† and Mireia Sospedra*,†

Polymorphonuclear neutrophils constitute the first line of defense against infections. Among their strategies to eliminate pathogens

they release neutrophil extracellular traps (NETs), being chromatin fibers decoratedwith antimicrobial proteins. NETs trap and kill

pathogens very efficiently, thereby minimizing tissue damage. Furthermore, NETs modulate inflammatory responses by activating

plasmacytoid dendritic cells. In this study, we show that NETs released by human neutrophils can directly prime T cells by reducing

their activation threshold. NETs-mediated priming increases T cell responses to specific Ags and even to suboptimal stimuli, which

would not induce a response in resting T cells. T cell priming mediated by NETs requires NETs/cell contact and TCR signaling, but

unexpectedly we could not demonstrate a role of TLR9 in this mechanism. NETs-mediated T cell activation adds to the list of

neutrophil functions and demonstrates a novel link between innate and adaptive immune responses. The Journal of Immunology,

2012, 188: 000–000.

When fighting bacterial infections, cells of the innateimmune system recognize pathogen-associated mo-lecular patterns that are not displayed by host tissue.

Innate immune cells then secrete cytokines and chemokines thatsignal danger to other cells and induce inflammation at the siteof infection. Polymorphonuclear neutrophils (PMNs) are the firstimmune cells to be recruited to inflamed tissue (1), to contain andclear infectious organisms, and to direct the extravasation of adap-tive immune cells and their activation (2–4). Adaptive immunecells subsequently participate in the elimination of the pathogenand set up memory for the case of reinfection.The participation of PMNs in adaptive immune responses has not

been considered relevant until recently, when it has been recog-nized that PMNs and T cells may engage in multiple interactionsand mutual activation (5). PMNs release chemokines that attractT cells to the site of inflammation (5–7) and also cytokines thatinfluence T differentiation (8). Several cytokines and granuleproteins secreted by PMNs such as IFN-g, TNF-a, cathepsin G,and neutrophil elastase are able to increase T cell proliferation andcytokine production (9, 10), which together enhance adaptiveimmune responses. Paradoxically, PMNs also secrete mediatorssuch as oxygen species, arginase (11), IL-10, and TGF-b that may

suppress T cell activation and proliferation. Despite this evolvingevidence, a complete picture about PMN/T cell interactions re-quires further investigation.PMNs are endowed with a variety of weapons that enable them to

efficiently contain and clear infectious organisms. These includethe engulfment and intracellular degradation of microbes (12, 13),production of reactive oxygen species and granule proteins (14),and the recently described release of extracellular chromatin fibersdecorated with antimicrobial proteins called neutrophil extracel-lular traps (NETs) (15). NETs are the most efficient means tocontain and eliminate pathogens (16), since they not only trap andkill microbes, but also prevent collateral tissue damage by local-izing toxic proteases and reducing their proteolytic activity (17).Furthermore, NETs can modulate immune responses by activatingplasmacytoid dendritic cells (pDCs), an APC population special-ized in sensing infections. NETs activate pDCs through TLR9,an intracellular receptor that recognizes viral/bacterial DNA (18).Under physiological conditions, pDCs do not respond to self-DNA, most likely because self-DNA lacks CpG motifs found inviral/bacterial DNA and also because self-DNA is rapidly de-graded in the extracellular environment and therefore fails toaccess intracellular TLR9. However, damaged cells can releasefactors such as the neutrophil antimicrobial peptide LL37 and thehigh-mobility group box protein 1, which can protect self-DNAfrom DNase degradation and deliver it to the intracellular com-partment containing TLR9 in pDCs with the consequence ofTLR9-mediated activation (19–21). LL37 and high-mobilitygroup box protein 1 are also contained in NETs and hence con-fer to these structures the potential to activate pDCs via TLR9 (22,23). NETs activation of pDCs seems to play an important role inthe pathogenesis of some autoimmune diseases such as psoriasis(19) and systemic lupus erythematosus (22–25), for which it hasbeen suggested that the large amounts of IFN-g produced byNETs-activated pDCs can lead to the maturation of myeloid DCs(mDCs) and exert an effect on T cell function. If such an indirectinteraction between NETs and T cells were confirmed, it wouldrepresent a new NETs-mediated mechanism of communicationbetween PMNs and T cells. Furthermore, TLR9 is expressed inT lymphocytes, and CpG-containing oligodeoxynucleotides(CpG-ODN) can modulate T cell activation (26, 27). NETs maytherefore also be able to directly activate T cells through their

*Institute for Neuroimmunology and Clinical Multiple Sclerosis Research, Center forMolecular Neurobiology, University Medical Center Hamburg–Eppendorf, 20251Hamburg, Germany; and †Department of Clinical Neuroimmunology and MultipleSclerosis Research, Neurology Clinic, University Hospital Zurich, 8091 Zurich, Swit-zerland

Received for publication November 28, 2011. Accepted for publication January 21,2012.

This work was supported by Deutsche Forschungsgemeinschaft Grant SO 1029/1-1.The Institute for Neuroimmunology and Clinical Multiple Sclerosis Research wassupported by the Gemeinnutzige Hertie Stiftung.

Address correspondence and reprint requests to Dr. Mireia Sospedra, Department ofNeuroimmunology and Multiple Sclerosis Research, Neurology Clinic, UniversityHospital Zurich, Frauenklinikstrasse 26, 8091 Zurich, Switzerland. E-mail address:Mireia.SospedraRamos@usz.ch

The online version of this article contains supplemental material.

Abbreviations used in this article: DC, dendritic cell; DPI, diphenyleneiodonium;mDC, myeloid dendritic cell; NET, neutrophil extracellular trap; ODN, oligodeox-ynucleotide; pDC, plasmacytoid dendritic cell; PFA, paraformaldehyde; PMN, poly-morphonuclear neutrophil.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

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TLR9, and this would represent a second and, in this case, directmechanism of a NETs-mediated communication between PMNsand T cells.As outlined above, there are at least two possibilities of how

PMNs could interact with T cells via NETs production. In this studywe have examined whether NETs released by human PMNs areable to exert direct or indirect effects on T cell activation. We foundthat NETs were able to directly prime T cells by reducing theiractivation threshold, which increased T cell responses to specificAgs and even to suboptimal stimuli. Priming by NETs requiredNETs/cell contact and TCR signaling, but unexpectedly we failedto show a role of TLR9 in this mechanism. Our results demonstratea novel strategy how PMNs are capable of activating adaptiveimmune responses via NETs.

Materials and MethodsCell purification and TCC36

Cells were isolated from healthy donors at the Department of TransfusionMedicine, University Medical Center Hamburg–Eppendorf, after informedconsent was obtained. PMNs were isolated from blood or buffy coats usingdextran-Ficoll, as described previously (28). The purity and viability ofPMNs was $97 and $95%, respectively, as assessed by expression of theneutrophil-specific marker CD16b, annexin V expression, and trypan blueexclusion (Supplemental Fig. 1). PBMCs were isolated by density gradi-ent. CD4+ T cells were enriched using the BD IMag human CD4T lymphocyte enrichment set–DM (BD Biosciences, Franklin Lakes, NJ),naive CD4+ T cells using the BD IMag human naive CD4 T lymphocyteenrichment set–DM (BD Biosciences), memory CD4+ T cells using the BDIMag human memory CD4 T lymphocyte enrichment set–DM (BD Bio-sciences), CD8+ T cells using the BD IMag anti-human CD8 magneticparticles–DM (BD Biosciences), DCs using the Blood Dendritic CellIsolation Kit II (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany).Cells were separated according to the manufacturers’ instructions.

TCC36 was established from cerebrospinal fluid of an untreated multiplesclerosis patient by limiting dilution as previously described (29). Stimu-latory peptides were identified using positional scanning combinatorialpeptide libraries (29).

Cell stimulation

Purified PMNs were resuspended in HBSS+ medium (Invitrogen, Carlsbad,CA) supplemented with 10 mM HEPES (Invitrogen). Then, 5 3 105–106

PMNs/ml were seeded into tissue culture plates on glass coverslips(Menzel-Glaser, Braunschweig, Germany) pretreated with 0.001% poly-L-lysine (Sigma-Aldrich, Steinheim, Germany). PMNs were stimulated with25 nM PMA (Sigma-Aldrich) (15 min or 3 h) and with 100 nM fMLP(Sigma-Aldrich) (3 h). When indicated, PMNs were preincubated with 100mM diphenyleneiodonium (DPI; Sigma-Aldrich) 30 min before stimulationwith PMA (3 h) (30). Also when indicated, PMNs were fixed with 4%paraformaldehyde (PFA) (Roth, Karlsruhe, Germany). To induce apoptosisand secondary necrosis, PMNs were exposed to UV light (60 min) andsubsequently incubated (16 h). Purified CD4+ T cells were stimulated with25 nM PMA.

Isolation and quantification of NETs

NETs released by activated PMNs were digested with 10 U/ml micrococcalnuclease (Worthington Biochemical, Lakewood, NJ) as previously de-scribed (31). NETs (myeloperoxidase/DNA complexes) in supernatantswere quantified using a capture ELISA as previously described (32). Ab-sorbance was measured at 405 nm using a mQuant microplate reader (Bio-Tek, Winooski, VT).

Cell cocultures

PMNs stimulated or unstimulated were seeded on coverslips and placed intotissue culture plates. Autologous PBMCs (33 106), purified DCs (43 105),purified CD4+ (2 3 106), purified naive CD4+ (2 3 106), purified memoryCD4+ (2 3 106), or purified CD8+ (2 3 106) T cells were added to thewells. When indicated cells were incubated with PMN-free supernatantcontaining NETs or were physically separated by a transwell poly-carbonate permeable membrane (0.4 mm pore size, Costar; Corning,Acton, MA). Also when indicated 2.5 mM chloroquine (Sigma-Aldrich), 1mM TLR9 antagonist (ODN TTAGGG) (CAYLA-InvivoGen, Toulouse,France), 3 mM herbimycin A (Sigma-Aldrich), 60 mg/ml anti–HLA-DR

blocking Ab (provided by Dr. H.G. Rammensee, Department of Immu-nology, University of Tubingen, Tubingen, Germany) or 60 mg/ml corre-sponding isotype control (BioLegend, San Diego, CA) was added into theculture. As positive control, 1 mg/ml soluble anti-CD3 Ab (OKT3; OrthoBiotech Products, Raritan, NJ) or anti-hCD2/anti-hCD3/anti-hCD28 beads(cell/bead ratio of 2:1) (T cell activation/expansion kit; Miltenyi Biotec)were used (data not shown).

To study the threshold for activation, NETs-primed CD4+ T cells werestimulated with 0.025 mg/ml soluble OKT3 (Ortho Biotech Products)(48 h). NETs-primed TCC36 was seeded in quadruplicate in 96-well plates(25,000 cells/well) together with autologous irradiated PBMCs (1 3 105

cells/well) (3000 rad) with or without different concentrations of thespecific stimulatory peptide (72 h).

Proliferation assays

Proliferation in the cocultures was measured after 48 h using a Click-iT EdUAlexa Fluor 647 flow cytometry assay kit (Molecular Probes/Invitrogen)following the manufacturer’s instructions. Cells were stained with anti-CD3 (PE-Cy7; eBioscience, San Diego, CA), anti-CD4 (allophycocyanin;eBioscience), and anti-CD8 (Pacific Blue; Dako) and analyzed by flowcytometry. Sample acquisition was done with a LSRII (BD Biosciences)flow cytometer and data were analyzed with FACSDiva (BD Biosciences)and FlowJo (Tree Star) softwares.

Proliferation of TCC36 was measured by [3H]thymidine (HartmannAnalytic, Braunschweig, Germany) incorporation using a scintillation betacounter (Wallac 1450; PerkinElmer, Rodgau-Jurgesheim, Germany).

Cytokine production

After 24 h coculture, supernatants were collected and cytokine levels weremeasured by ELISA using the following kits: human IFN-g (BioLegend);IL-4, IL-10, and IL-2 CytoSet (BioSource International, Camarillo, CA);and IL-17A (eBioscience). Reactions were performed according to themanufacturers’ instructions.

Flow cytometry analysis of surface markers

T cell activation was assessed using the following Abs: anti-CD3 (PE-Cy7;eBioscience), anti-CD4 (allophycocyanin; eBioscience), anti-CD8 (PacificBlue; Dako), anti-CD25 (PE; eBioscience), and anti-CD69 (FITC; BDBiosciences). DC activation was assessed using the following Abs: anti-CD3 (Pacific Blue; eBioscience), anti-CD14 (Pacific Blue; BD Biosci-ences), anti-CD19 (Pacific Blue; BD Biosciences), anti-CD56 (Pacific Blue;BDBiosciences), anti-CD11c (PerCP-Cy5.5; BioLegend), anti-CD123 (PE-Cy7; BioLegend), anti-CD40 (PE; Miltenyi Biotec), anti-CD83 (allophy-cocyanin; BD Biosciences), anti-CD80 (PE; eBioscience), anti-CD86(FITC; Dako), and anti-HLA class II (FITC; BD Biosciences). FITC(BD Biosciences), PE (BD Biosciences), allophycocyanin (eBioscience),Pacific Blue (Dako), PerCP-Cy5.5 (BD Biosciences), and PE-Cy7 (eBio-science) isotype controls were also used.

Microscopy assays

NETs formation was visualized using fluorescence microscopy in PMNsfixed with 4% PFA and blocked overnight with PBS containing 5% donkeyserum (The Jackson Laboratory, Bar Harbor, ME) and 0.6% Triton X-100(Roth), by staining with a primary anti-humanmyeloperoxidase-specific Ab(AbD Serotec) followed by the secondary goat anti-mouse Cy3 Ab (TheJackson Laboratory) and the DNA dye Hoechst 33258 (Sigma-Aldrich).T cell cluster formation was analyzed in transmission light. Specimenswere analyzed with a confocal microscope F1000 (Olympus, Hicksville,NY) and Axio Imager M1 (Zeiss, Gottingen, Germany), respectively.

Analysis of T cell signaling

Purified CD4+ T cells cocultured with NETs-supernatant (10 min) wereharvested and TCR signal transduction was analyzed by intracellularstaining using an Alexa Fluor 488 mouse anti-ZAP70 phosphorylated ontyrosine 319 (pY319) (BD Phosflow) according to the manufacturer’sinstructions.

Statistical analysis

Statistical analyses were performed with Prism 5.02 (GraphPad Software,San Diego, CA). Descriptive statistics are reported as means 6 SEM.Parametric tests were applied for two-group comparisons using unpaired ttests with two-tailed p values. Comparisons of three groups and more wereassessed by one-way ANOVA with Bonferroni’s correction for multiplecomparisons. A p value ,0.05 was considered statistically significant.

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ResultsNETs induce cluster formation, upregulation of the activationmarkers CD25 and CD69, and phosphorylation of theTCR-associated signaling kinase ZAP70 but not proliferationin CD4+ T cells

To examine the NETs-mediated effect on T cells, purified humanCD4+ T cells were cocultured with PMNs releasing NETs

(NETting-PMNs) and unstimulated PMNs. We stimulated NET

formation by incubating highly pure PMNs with 25 nM PMA for

3 h (Fig. 1A). To assure that we examine the role of NETs and not

of other PMA-induced mediators, we also incubated CD4+ T cells

with PMNs pretreated with diphenyleneiodonium (DPI) (DPI-

PMNs), a NADPH inhibitor of NET formation, before stimula-

tion with PMA. Furthermore, we digested NETs from stimulated

PMNs with micrococcal nuclease and incubated purified CD4+

T cells with these cell-free supernatants containing NETs (NETs-

supernatant). CD4+ T cells cocultured with NETting-PMNs (Fig.

1B) and NETs-supernatant (data not shown) formed clusters in-

dicative of cell activation or proliferation that were absent when

cells were cocultured with unstimulated PMNs (Fig. 1B) or DPI-

PMNs (data not shown). As a further sign of activation, CD25

and CD69 expression was upregulated on purified CD4+ T cells

cocultured during 24 h with NETting-PMNs and NETs-super-

natant, but not on T cells cocultured with unstimulated PMNs or

DPI-PMNs (Fig. 1C). To examine whether these effects of NETs

on CD4+ T cells involved TLR9 and TCR signaling, CD4+ T cells

were cocultured with NETs-supernatant in the presence or absenceof two TLR9 inhibitors: chloroquine, which blocks TLR9/DNA

interactions in endosomes (33) and the TLR9 antagonist (ODN

TTAGGG), or the antibiotic herbimycin A, which inhibits TCR-

mediated signaling (34). The upregulation of CD25 and CD69 on

CD4+ T cells cocultured with NETs-supernatant remained un-

changed in the presence of chloroquine and TLR9 antagonist

(ODN TTAGGG), whereas it was reduced by herbimycin A (Fig.

1D), suggesting that the effect of NETs is independent of TLR9,

but involves TCR signaling.To investigate further the TCR-mediated effect of NETs but also

to exclude the possibility that this effect was mediated by PMA thatwas taken up by neutrophils during stimulation and then releasedbound to NETs, we analyzed whether NETs induced recruitment ofthe TCR-proximal tyrosine kinase ZAP70. ZAP70 plays a criticalrole during early steps of TCR-mediated signaling, but is bypassedby PMA that directly activates protein kinase C without involve-ment of ZAP70 (35). Purified CD4+ T cells were cocultured for10 min with unstimulated PMNs, NETting-PMNs, or NETs-supernatant, and ZAP70 (pY319) phosphorylation was measuredby flow cytometry. ZAP70 phosphorylation was upregulated inCD4+ T cells cocultured with NETting-PMNs or NETs-super-natant compared with unstimulated PMNs (Fig. 1E), showing aneffect of NETs on purified CD4+ T cells that involves TCR sig-naling and is not mediated by PMA. Furthermore, a putative roleof PMA bound to NETs was also excluded by the observation thatpurified CD4+ T cells stimulated with PMA for 48 h proliferated

FIGURE 1. CD4+ T cell priming induced by NETs. (A) Representative images of unstimulated neutrophils and NETting-PMNs visualized using fluores-

cence microscopy. DNA is shown in blue (Hoechst 33258) and myeloperoxidase in red. (B) Transmission light images of cluster formation of purified CD4+

T cells cocultured with unstimulated PMNs and NETting-PMNs (NETs). Original magnification (A, B) 340. (C and D) CD25 and CD69 surface expression

on purified CD4+ T cells cocultured 24 h with unstimulated PMNs, NETting-PMNs, DPI-PMNs, and NETs-supernatant with or without chloroquine,

TLR9 antagonist (ODN TTAGGG), or herbimycin A. Graphs show mean fluorescence intensity (MFI)6 SEM from five independent experiments. *p, 0.05,

**p, 0.01. (E) Flow cytometry analysis of ZAP70 (pY319) phosphorylation in CD4+ T cells cocultured for 10 min with unstimulated PMNs, NETting-PMNs,

and NETs-supernatant. Graph represents MFI 6 SEM from three independent experiments. *p , 0.05. (F) CD4+ T cell proliferation assessed by EdU in-

corporation after culture during 48 h with NETting-PMNs, NETs-supernatant, and PMA. Histograms represent the proliferation from a representative ex-

periment. Graph represents the percentage of EdU-positive cells (mean values 6 SEM) from three or more independent experiments. ***p , 0.001.

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whereas purified CD4+ T cells cocultured with NETting-PMNs orNETs-supernatant did not (Fig. 1F). The lack of proliferation ofpurified CD4+ T cells cocultured with NETs indicated that theincrease in ZAP70 phosphorylation induced by NETs was ap-parently insufficient to fully activate T cells and support theirproliferation.

T cell priming by NETs lowers the activation threshold

The changes that NETs exerted on CD4+ T cells suggested thatthey led to a state of preactivation or priming. We therefore ex-amined whether interaction with NETs reduces the activationthreshold of CD4+ T cells. First, we studied the effect of NETs onAg-specific responses using a previously well-characterized CD4+

T cell clone (TCC36) (29). TCC36 was precultured for 24 h withunstimulated PMNs or NETs-supernatant and then seeded withautologous irradiated PBMCs pulsed or not with its specific targetpeptide. TCC36 primed by NETs proliferated significantly morevigorously in response to PBMCs loaded with 10 and 1 mg/mlspecific peptide when compared with the unprimed TCC36, thatis, precultured with unstimulated PMNs (Fig. 2A).Then, we addressed whether priming by NETs reduces the acti-

vation threshold of CD4+ T cells and renders them capable to beactivated by suboptimal stimuli. Purified CD4+ T cells were pre-cultured with unstimulated PMNs, NETting-PMNs, DPI-PMNs, orNETs-supernatant for 24 h. Next, T cells were carefully washed andcultured for another 48 h with low concentrations of soluble anti-CD3 Ab (OKT3) (signal 1) in the absence of APC (signal 2), asuboptimal stimulus, which alone is not able to induce proliferationof resting T cells. Purified CD4+ T cells precultured with unstimu-lated PMNs or with DPI-PMNs failed to proliferate (Fig. 2B). Incontrast, 27.2 6 6.7% of CD4+ T cells precultured with NETting-PMNs and 13.27 6 3.5% precultured with NETs-supernatant pro-liferated (Fig. 2B).

Resting DCs are able to activate NETs-primed CD4+ T cells inthe absence of specific Ag

Next, we examined whether a more physiologic, but suboptimalstimulus such as the interaction of T cells with resting DCs in theabsence of specific Ag, which alone does not induce proliferation

of resting T cells, stimulates a response of NETs-primed CD4+

T cells. CD4+ T cells were precultured for 24 h with unstimulatedPMNs (unprimed CD4) or with NETs-supernatant (NETs-primedCD4) for 24 h. Simultaneously, purified DCs were preculturedwith unstimulated PMNs (resting DCs). After this period, both celltypes were carefully washed and cocultured for an additional 48 h.NETs-primed purified CD4+ T cells (8.7 6 1.3%) proliferated andreleased 2092 6 459 pg/ml IFN-g, whereas no activation wasobserved in unprimed CD4+ T cells (Fig. 3A). To confirm thatNETs priming is TLR9 independent, but involves TCR signaling,CD4+ T cells were primed with NETs-supernatant in the presenceor absence of the TLR9 inhibitor chloroquine, TLR9 antagonist(ODN TTAGGG), or herbimycin A. Proliferation was not affectedin CD4+ T cells primed in the presence of chloroquine or TLR9antagonist (ODN TTAGGG); however, herbimycin A clearly re-duced proliferation (Fig. 3B), confirming that T cell priming byNETs is independent of TLR9 but involves TCR signaling.To better understand the interaction between NETs-primed

CD4+ T cells and resting DCs, NETs-primed purified CD4+

T cells and resting DCs were cocultured in the presence or ab-sence of a blocking anti–HLA-DR Ab, the corresponding isotypecontrol, or herbimycin A. The presence of an anti–HLA-DR Abdid not induce a significant reduction of primed CD4+ T cellproliferation (Fig. 3C). However, because class II moleculesother than DR are expressed on DCs, that is, HLA-DQ and HLA-DP molecules, we cannot rule out that HLA class II molecules areinvolved. The presence of herbimycin A completely abrogated theproliferation of primed CD4+ T cells, indicating that activation ofprimed T cells by DCs requires TCR signaling (Fig. 3C).

NETs-activated pDCs are not able to activate unprimed T cells

Next, we also examined whether DCs, particularly pDCs, pre-cultured with NETs-supernatant (NETs-activated DCs) were ableto activate CD4+ T cells. Following purification, ∼40% of DCswere pDCs and 60% were mDCs. After 24 h coculture with NETs,all mDCs died, whereas pDCs survived and upregulated somematuration and activation markers such as CD40, CD80, CD83and CD86, but not HLA class II (Fig. 4A). Unprimed CD4+ T cellscocultured with NETs-activated pDCs did not proliferate nor did

FIGURE 2. T cell priming by NETs lowers the activation threshold. (A) T cell proliferation assessed by thymidine incorporation in TCC36 precultured with

unstimulated PMNs orNETs-supernatant and stimulatedwith autologous irradiated PBMCs pulsedwith a specific peptide. Graph represents cpms (meanvalues

6 SEM) from three or more independent experiments. (B) T cell proliferation assessed by EdU incorporation in purified CD4+ T cells precultured with

unstimulated PMNs, NETting-PMNs, DPI-PMNs, or NETs-supernatant and stimulated or not with low concentrations of anti-CD3 Ab (OKT3). Graph rep-

resents the percentage of EdU-positive cells (mean values 6 SEM) from three or more independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

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they produce IFN-g (Fig. 4B). NETs-activated pDCs were able toinduce proliferation and IFN-g release only in NETs-primedCD4+ T cells (Fig. 4B), suggesting that the NETs-mediated acti-vation of pDCs in our in vitro setting does not exert an effect on

T cell activation. NETs activation of pDCs seems to play a role insome autoimmune diseases (19, 22–25), and it has been suggestedthat the large amounts of IFN-a produced by NETs-activatedpDCs can activate mDCs and increase Ag presentation to

0 2 4 6 8 10 12 14 16

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Pre-culture

CD4 +PMNs

DC +PMNs

Unstimulated Unstimulated

UnstimulatedNETsSupernatant

Co-culture

CD4 DC

Resting

Resting

Unprimed

NETs-primed

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Co-culture

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+ Anti-HLA-DR

+ Isotype control

+ Herbimycin A

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RestingNETs-primed

RestingNETs-primed(TLR9 Antagonist)

RestingNETs-primed(Chloroquine)

RestingNETs-primed(Herbimycin A)

FIGURE 3. Resting DCs are able to activate NETs-primed CD4+ T cells in the absence of specific Ag. (A–C) CD4+ T cell proliferation assessed by EdU

incorporation. Histograms represent the proliferation from a representative experiment. Graph represents the percentage of EdU-positive cells and pg/ml

IFN-g (mean values 6 SEM) from three or more independent experiments. (A) Purified CD4+ T cells precultured with unstimulated PMNs (unprimed) or

with NETs-supernatant (NETs-primed) were cocultured with DCs previously precultured with unstimulated PMNs (resting). *p , 0.014, **p , 0.003. (B)

CD4+ T cells precultured with NETs-supernatant in presence or absence of TLR9 inhibitors chloroquine and TLR9 antagonist (ODN TTAGGG) or TCR

signaling inhibitor herbimycin A, and then cocultured with resting DCs. *p, 0.05. (C) NETs-primed CD4+ T cells cocultured with resting DCs in presence

or absence of a HLA-DR blocking Ab, the corresponding isotype control, or herbimycin A. **p , 0.01.

FIGURE 4. NETs-activated pDCs do not exert an effect on unprimed CD4+ T cells. (A) HLA class II, CD40, CD80, CD83, and CD86 expression on gated

NETs-activated pDCs. Values showmean fluorescence intensity (MFI)6 SEM. *p = 0.0263, **p = 0.0041, ***p = 0.0004. (B) PurifiedCD4+ T cells precultured

with unstimulated PMNs (unprimed) or with NETs-supernatant (NETs-primed) were cocultured with DCs previously precultured with NETs-supernatant

(NETs-activated). CD4+ T cell proliferationwas assessed by EdU incorporation. Histograms represent the proliferation from a representative experiment. Graph

represents the percentage of EdU-positive cells and pg/ml IFN-g (mean values 6 SEM) from three or more independent experiments. *p , 0.04.

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T cells. The loss of mDCs after culture with NETs most likelyprevented this indirect effect of NETs-activated pDCs on T cells inour in vitro system.

Coculture of T cells, NETting-PMNs, and DCs results in T cellactivation

We then asked whether coculture of T cells, DCs, and NETting-PMNs results in T cell activation and whether NETs exert thesame effect on CD4+ and CD8+ T cells. We used NETting-PMNsinstead of NETs-supernatant as the more physiologic condition.Purified CD4+ or CD8+ T cells were cocultured with unstimulatedor NETting-PMNs in the absence or presence of purified DCs.Proliferation and secretion of IFN-g, IL-17A, IL-4, IL-10, andIL-2 were measured as indicators of T cell activation (Fig. 5).Purified CD4+ and CD8+ T cells cocultured with unstimulatedPMNs alone or in the presence of purified DCs did not proliferateor produce cytokines (Fig. 5A, 5B). Similarly, no proliferation orcytokine release was observed upon coculture with NETting-PMNs in the absence of DCs (Fig. 5A, 5B). In contrast, we ob-served both proliferation and cytokine release when purified DCswere added to the cocultures. We found that 16.26 2.8% of CD4+

T cells and 11.6 6 1.3% of CD8+ T cells proliferated (Fig. 5A,5B). Regarding cytokine production, purified CD4+ T cellscocultured with NETting-PMNs and DCs secreted 901 6 233pg/ml IFN-g and 289 6 99 pg/ml IL-2. Purified CD8+ T cellssecreted only IFN-g (981.66 293 pg/ml) (Fig. 5B). Direct contactof T cells with DCs and NETting-PMNs was required for T cellactivation. When CD4+ or CD8+ T cells were physically separatedfrom DCs and NETting-PMNs by a transwell, no activation wasdetected (Fig. 5A, 5B). The proliferation of CD4+ T cells cocul-tured with NETting-PMNs and DCs remained unchanged whenCD8+ T cells were added (18.5 6 6.5%); conversely, the prolif-eration of purified CD8+ T cells cocultured with NETting-PMNsand DCs increased when purified CD4+ cells were added to thecoculture (27 6 6.7%) (Fig. 5A).We also addressed whether the ability of NETs to induce T cell

proliferation was comparable between naive and memory CD4+

T cells. Purified naive or memory CD4+ T cells were coculturedwith unstimulated PMNs or NETting-PMNs in the presence of pu-rified DCs. We found that 14.7 6 6.8% of naive T cells and 11.1 65% of memory CD4+ T cells cocultured with NETting-PMNsand purified DCs proliferated, whereas neither naive nor memory

FIGURE 5. Coculture of T cells with NETting-PMNs and DCs results in T cell activation. (A) T cell proliferation assessed by EdU incorporation in

purified CD4+ (red) and purified CD8+ (blue) T cells cocultured with unstimulated PMNs in the presence or absence of DCs, NETting-PMNs (NETs) in the

presence or absence of DCs and separated or not by a transwell, and in the presence or absence of additional purified CD8+ or CD4+ T cells, respectively.

Histograms represent the proliferation from a representative experiment. Graphs represent the percentage of EdU-positive cells (mean values 6 SEM) from

three or more independent experiments. **p, 0.01, ***p, 0.001. (B) IFN-g, IL-17A, IL-4, IL-10, and IL-2 produced by purified CD4+ (in red), CD8+ (in

blue), or both T cells together (in gray) cocultured as indicated in (A). Values show mean pg/ml 6 SEM. *p , 0.05, ***p , 0.001. (C) T cell proliferation

assessed by EdU incorporation in purified naive (open bars) and memory (filled bars) CD4+ T cells cocultured with unstimulated PMNs or NETting-PMNs

in presence of DCs. Graph represents the percentage of EdU-positive cells (mean values 6 SEM) from three independent experiments.

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CD4+ T cells proliferated upon coculture with unstimulated PMNsand DCs (Fig. 5C).

Characterization of NETs-mediated T cell activation inPBMCs

Finally, we characterized the activation of CD4+ and CD8+ T cellsmediated by NETs in bulk PBMCs, a condition that reflects betterthe circumstances under which T cells will encounter NETting-PMNs in vivo. PBMCs include, in addition to T cells and DCs,monocytes and B cells that could both influence T cell activationmediated by NETs. PBMCs cocultured with NETting-PMNsformed large clusters that were absent when cells were cocul-tured with unstimulated PMNs (Fig. 6A) and strongly upregulatedCD25 and CD69 expression on both CD4+ and CD8+ T cells (Fig.6B). Additionally, within the PBMCs, 10.5 6 1.8% of CD4+

T cells and 32.3 6 3.1% of CD8+ proliferated after coculture withNETting-PMNs, whereas no proliferation was observed whenPBMCs were cocultured with unstimulated PMNs (Fig. 6C). Theability of NETting-PMNs to mediate proliferation in CD4+ andCD8+ T cells showed great interindividual variability (Fig. 6D).PBMCs cocultured with NETting-PMNs also secreted 17776 272pg/ml IFN-g and 93.5 6 25 pg/ml IL-17A, but IL-4 and IL-10 didnot (Fig. 6D). No cytokine secretion was detected when PBMCswere cocultured with unstimulated PMNs.

NETs and not other factors mediate T cell activation in PBMCs

To confirm that NETs and not other factors mediated the substantialT cell activation in bulk PBMCs, we included a broad range ofadditional controls. First, to discard that residual contaminatingPMA activates PBMCs despite careful washing of NETting-PMNsafter PMA stimulation, PBMCswere cocultured with PMNs treatedwith PMA for only 15 min, a period too short to induce substantialNET production. No upregulation of CD25 and CD69 expression(Fig. 7A), T cell proliferation (Fig. 7B), or production of cytokines(Fig. 7C) was observed under this condition. PMNs activated withfMLP, a stimulus less efficient than PMA in inducing NETs, alsofailed to mediate T cell activation (Fig. 7). NETting-PMNs, that is,PMNs undergoing NETosis, are dying cells. To exclude an effectof dead cells on T cell activation, we cocultured PBMCs withPMNs dying by other mechanisms than NETosis. We inducedapoptosis in PMNs by exposure to UV light for 60 min. Cells weresubsequently incubated for additional 16 h to increase the numberof dying cells and then cocultured with PBMCs. No activation wasdetected under these coculture conditions (Fig. 7).To exclude that PMA-induced mediators other than NETs were

responsible for T cell activation, we stimulated PMNs with PMAfor 3 h and then fixed them with PFA prior to coculture withPBMCs. After fixation, PMNs are not able to produce any medi-ators, and only NETs remained on cells. PBMCs cocultured withNETting, PFA-fixed PMNs showed upregulation of CD25 andCD69 expression (Fig. 7A), T cell proliferation (Fig. 7B), andcytokine release (Fig. 7C) similar to those observed with unfixedcells. As an additional control, PBMCs were cocultured withNETting-PMNs, but physically separated by a transwell to avoidcell/cell or NETs/cell contact. No upregulation of CD25 expres-sion, T cell proliferation, or release of cytokines (Fig. 7) wasdetected under this condition, excluding that soluble mediatorsproduced by PMNs are responsible for T cell activation. Contactbetween NETs and PBMCs was required for T cell activation,as already observed with purified cells (Fig. 5). Interestingly,some upregulation of CD69 was observed in this condition, sug-gesting that the expression of this molecule is partially mediatedby PMA-induced soluble factors (Fig. 7A). Finally, as expected,the coculture of PBMCs with DPI-PMNs did not induce T cell

activation, whereas the coculture with NETs-supernatants inducedproliferation and upregulation of activation markers but not cy-tokine production (Fig. 7). Proteases released by neutrophils thatare eliminated during the careful washing of NETting-PMNs arenot eliminated, but concentrated in NETs-supernatants and mostlikely degradated cytokines (36).

DiscussionThe main function of the innate immune system during infection isto rapidly sense microbial pathogens, limit their spread, and elim-inate them with minimum collateral tissue damage. The com-position of NETs, being fibers of chromatin decorated with anti-microbial proteins, turns them into optimal structures to performthis task (16). The main advantages of NETs are the following: 1)NET fibers trap pathogens and act as physical barriers, preventingmicrobial spread; 2) NETs render antimicrobial proteins moreefficient by concentrating them on the DNA/protein fibers, whichkeeps them together and allows them to act synergistically; 3) thecollateral tissue damage is reduced since proteases do not diffuseinto the tissue, and, additionally, binding to NETs reduces thetoxic activity of some of these proteases (17); and 5) NETs alsoallow colocalization of adjuvants and danger signals that canmodulate inflammation, for example of self-DNA and LL-37,which are able to activate pDCs. In this study, we describe asa novel function of NETs their ability to directly prime T cells byreducing their activation threshold and in consequence mediateT cell activation. This previously unknown property of NETsdemonstrates that their role is not limited to innate immunemechanisms, but is also involved in activating the adaptive im-mune system.CD4+ T cell activation mediated by NETs unfolds as a two-step

process. In the first step, NETs prime CD4+ T cells by directcontact, which reduces their threshold of activation. CD4+ T cellsprimed by NETs showed increased Ag-specific responses and canbe activated by suboptimal stimuli such as soluble anti-CD3 Ab(signal 1) in the absence of APCs (signal 2) or resting DCs in theabsence of specific Ag, which are both not sufficient to inducea response of resting CD4+ T cells. We observed that NETs/T cellcontact induced the formation of cell clusters, upregulation of theactivation markers CD25 and CD69, as well as some phosphor-ylation of ZAP70 in CD4+ T cells. These changes were insuffi-cient to fully activate T cells and support T cell proliferation, butthey lowered their activation threshold. Unexpectedly, we couldnot demonstrate a role of TLR9 in this NETs-mediated T cellpriming because neither of the two TLR9 inhibitors, the TLR9inhibitor chloroquine and the TLR9 antagonist (ODN TTAGGG),had an effect. Whether T cell priming mediated by NETs is aDNA activation pathway that is TLR9 independent, such as thoseinduced by CpG in monocytes (37) or nucleosomes in neutrophils(38), requires further investigation. Although TLR9 does not seemto play a role, T cell priming by NETs apparently involves TCRengagement and signaling since they induced phosphorylation ofthe TCR-associated signaling kinase ZAP70, and herbimycin A,a well-known inhibitor of TCR-mediated signal transduction,strongly reduced T cell priming. Further studies need to determinewhich of the many components of NETs engage the TCR andinduce TCR signaling.The lower activation threshold of NETs-primed T cells has been

demonstrated only for CD4+ T cells. The survival of CD8+ T cellsprecultured with NETs and subsequently incubated with DCs wasvery low and prevented us from performing priming experimentswith CD8+ T cells. It has been reported that the antimicrobialpeptide LL-37, which is present in NETs, induces granzyme-mediated apoptosis of cytotoxic T lymphocytes (39), which

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could explain the lower survival of CD8+ T cells pre-exposed toNETs, but we did not address this possibility in this study. CD8+

T cells primed with NETs showed cluster formation and upregu-lation of CD25 and CD69 expression comparable to that observed

in NETs-primed CD4+ T cells (Supplemental Fig. 2), whichsuggests a similar behavior of both cell types after NETs priming.Furthermore, only minor differences were found in proliferationand release of IFN-g between CD8+ and CD4+ T cells cocultured

FIGURE 6. NETs-mediated T cell activation in PBMCs. (A) Transmission light images of cluster formation of PBMCs cocultured with unstimulated

PMNs and NETting-PMNs. Original magnification 340. (B) CD25 and CD69 surface expression after gating on CD4+ (red) and CD8+ (blue) T cells.

Histograms represent the expression from a representative experiment. Dotted line indicates PBMCs cocultured with unstimulated PMNs; solid line

indicates PBMCs cocultured with NETting-PMNs (NETs). Graphs represent mean fluorescence intensity (MFI)6 SEM from five independent experiments.

***p , 0.001. (C) T cell proliferation in PBMCs cocultured with unstimulated PMNs and NETting-PMNs. T cell proliferation was assessed by EdU

incorporation after gating on CD4+ (red) and CD8+ (blue) T cells. Graphs represent the percentage of EdU-positive cells (mean values 6 SEM) from 21

independent experiments. **p , 0.01, ***p , 0.001. Histograms represent the proliferation from a representative experiment. (D) Scatter plot showing T

cell proliferation, in which each dot represents one individual donor. (E) IFN-g, IL-17A, IL-4, and IL-10 produced by PBMCs cocultured with unstimulated

PMNs and NETting-PMNs (NETs). Values show mean pg/ml 6 SEM from five independent experiments. **p , 0.01, ***p , 0.001.

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with NETting-PMNs and DCs. However, higher proliferation andIFN-g release were found in CD8+ T cells cocultured with NETsand DCs in the presence of CD4+ T cells. These data suggest that,although CD8+ T cells were activated by NETs, CD4+ T cellsmost likely enhanced CD8+ T cell activation via IL-2 productionwhen both cell types were present. Unexpectedly, NETs mediatedthe activation of both CD4+ naive and memory T cells. It is wellknown that memory CD4+ T cells have less stringent activationrequirements than do naive CD4+ cells and are able to respondfaster and stronger to lower doses of Ag and to lower levels ofcostimulation than do naive CD4+ T cells (40–42). In this context,our results suggest that priming of naive CD4+ T cells by NETsmay induce changes in these cells similar to those that accompanymemory T cell generation.Despite the well-documented importance of NETs as effective

antimicrobial first-line defense mechanism, there is increasing evi-dence that NETs occur in various clinical settings in the absence ofmicrobial infections and that they are probably also associated withpathophysiological conditions (43). Abnormally high productionand/or low degradation of NETs can lead to tissue damage, andthe activation of pDCs and simultaneous exposure of the immunesystem to autoantigens may result in the generation of anti-selfAbs, which in addition can stimulate NET formation, creatinga vicious cycle. Aberrant NETs formation can therefore not onlyparticipate in the organ damage observed in chronic inflammatorydisorders (44–47), but furthermore contribute to the developmentand perpetuation of autoimmune diseases such as systemic lupuserythematosus (19, 22, 23, 48, 49), small-vessel vasculitis (32),

and psoriasis (19). Because of the central role of T cells in mostof these disorders, our finding that NETs are able to mediate Tcell proliferation and secretion of Th1 proinflammatory cytokinesrepresents a novel mechanism of how NETs may contribute toT cell-mediated autoimmune diseases or certain aspects of theirpathogenesis.In conclusion, we describe in this study for the first time, to our

knowledge, the ability of NETs to directly prime T cells by re-ducing their activation threshold and consequently to enhanceadaptive immune responses. This novel feature adds to the list ofinteresting properties of NETs and the functions of neutrophils ingeneral. NETs as a highly specialized structure of PMNs are,according to our observations, not only important for the first-lineinnate immune defense, but they also participate in renderingadaptive immune responses more efficient. Our data on the in-volvement of NETs in adaptive immunity still leaves many im-portant questions, and, among them, it will be very interesting toexamine the role of NETs in pathological immune responses andautoimmune diseases and explore whether a better understandingmay lead to new therapeutic strategies.

AcknowledgmentsWe thank all healthy donors for blood donations and the staff of the Depart-

ment for Transfusion Medicine, University Medical Centre Hamburg–

Eppendorf, for providing the samples. We also thank Nancy Martinez

(Max Planck Institute for Molecular Physiology, Dortmund, Germany)

for technical advice, and Raquel Planas and Benjamin Schattling for crit-

ical reading of the manuscript.

FIGURE 7. NETs and not other

factors mediate T cell activation. (A)

CD25 and CD69 surface expression

after gating on CD4+ (red) and CD8+

(blue) T cells in PBMCs cocultured

with unstimulated PMNs, NETting-

PMNs (NETs), PMNs stimulated with

PMA for 15 min, PMNs stimulated

with fMLP, dying PMNs exposed to UV

light, NETting-PMNs fixed with PFA,

NETting-PMNs physically separated by

a transwell, DPI-PMNs, and NETs-su-

pernatant. Graphs represent mean fluo-

rescence intensity (MFI) 6 SEM from

five independent experiments. (B) T cell

proliferation assessed by EdU incorpo-

ration after gating on CD4+ (red) and

CD8+ (blue) T cells in PBMCs cocul-

tured as indicated in (A). Graphs rep-

resent the percentage of EdU-positive

cells (mean values 6 SEM) from five

independent experiments. (C) IFN-g,

IL-17A, IL-4, and IL-10 produced by

PBMCs cocultured as indicated in (A).

Values show mean pg/ml 6 SEM from

five independent experiments. *p ,0.5, **p , 0.01, ***p , 0.001.

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DisclosuresThe authors have no financial conflicts of interest.

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