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Alveolar type II epithelial cells present antigen to CD4+ T cells and are

capable to induce of Foxp3+ regulatory T cells

Marcus Gereke1, Steffen Jung2, Jan Buer3, Dunja Bruder1

1Immune Regulation Group, Helmholtz Centre for Infection Research, Braunschweig,

Germany. 2 Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.

3Department of Medical Microbiology, University Hospital Essen Essen, Germany

Correspondence and requests for reprints should be addressed to: Dunja Bruder, Immune

Regulation Group, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124

Braunschweig, Germany; dunja.bruder@helmholtz-hzi.de

Phone: ++49-531-6181-3051; Fax ++49-531-6181-3099

Grand support: This work was supported by a grant from the Deutsche

Forschungsgemeinschaft (SFB587) to Dunja Bruder and Jan Buer. FTY720 was provided by

Beata Zygmunt.

Running head: Treg cell induction in the lung

Descriptor number: 41

Word count: 6573

“At a Glance Commentary”: The contribution of alveolar type II epithelial cells (AECII) in

respiratory immune regulation has become increasingly appreciated. However, their precise

function in the induction and regulation of T cell reactivity to self-antigen remains poorly

understood. We show here that MHC class II expressing AECII present self-antigen to CD4+

T cells resulting in functional activation of lung-reactive T cells. Moreover, AECII support

the induction of Foxp3+ regulatory T cells thereby actively contribute to re-establishment of

peripheral T cell tolerance in the lung.

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This article has an online data supplement, which is accessible from this issue's table of

content online at www.atsjournals.org.

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Abstract

Background: Although the contribution of alveolar type II epithelial cells (AECII) in

respiratory immunity has become increasingly appreciated, their precise function in the

induction and regulation of T cell reactivity to self-antigen remains poorly understood.

Objectives: To investigate the role of AECII in the initiation of T cell reactivity to alveolar

self-antigen and to clarify their function in the peripheral induction of Foxp3+ regulatory

CD4+ T cells.

Methods: To dissect the complex cellular and molecular functions of AECII in lung

inflammation and immune regulation, we utilize a transgenic mouse model for CD4+ T cell

mediated pulmonary inflammation.

Measurements and Main Results: Here we report that AECII present endogenously

expressed antigen on MHC class II molecules to CD4+ T cells. Epithelial antigen display was

sufficient to induce primary T cell activation and pulmonary inflammation. Upon

inflammation AECII induce by a mechanism involving anti-proliferative soluble factors

including transforming growth factor-β (TGF-β) the differentiation of Foxp3+ regulatory T

cells (Tregs). Whereas in acute inflammation TGF-β appears to be the dominant factor to

induce Tregs, other AECII-derived factors can substitute for and/or synergize with TGF-β in

chronic pulmonary inflammations.

Conclusion: AECII are capably of priming naive CD4+ T cells demonstrating an active

participation of these cells in respiratory immunity. Moreover, AECII cells display so far

unrecognized functions in balancing inflammatory and regulatory T cell responses in the lung

by connecting innate and adaptive immune-mechanisms to establish peripheral T cell

tolerance to respiratory self-antigen.

Word count: 239

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Key words: peripheral tolerance, alveolar type II epithelial cells, immune regulation,

autoimmunity, transgenic mouse model

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INTRODUCTION

Immune reactions in the lung have to be tightly balanced since the mucosal immune system

has to discriminate between harmful invading pathogens against which an effective immune

response has to be generated and harmless inhaled and self antigens, against which tolerance

has to be established. Regulatory T cells (Tregs) fulfil a central task in the maintenance of

systemic self-tolerance. Tregs are capable to inhibit and modulate diverse immuno-pathologic

phenomena by controlling the proliferation of CD4+ and CD8+ T lymphocytes in vivo (1, 2).

There are at least two types of Tregs that perform similar functions. One class, defined by

expression of CD4 and CD25, develops in the thymus under the control of the transcription

factor Foxp3 and resides in all the secondary lymphoid organs (3, 4). In addition inducible

Tregs can be generated in the periphery as a consequence of alternative activation of naïve T

cells in the presence of specific cytokines. Inducible Tregs secrete IL-10 (5, 6) and/or TGF-β

(7, 8), both of which are potent regulators of inflammation capable of suppressing the

proliferation of effector cells.

The ability to induce Tregs from the naïve T cell pool may be of particular benefit at mucosal

surfaces. Due to their physiological function these tissues interact with profuse microbial

organisms and other potentially noxious factors, which are potent immune stimulants (9-12).

Therefore, T cell tolerance in mucosal tissues must be actively enforced to avoid

inappropriate inflammation (13). Alveolar type II epithelial cells (AECII) are strategically

located in alveoli were they can contribute to the innate immune response against airborne

pathogens by the production of various antimicrobial and proinflammatory effector molecules

(14-17). It has been described previously that AECII show constitutive MHC II surface

expression (18, 19). More recently Debbai and colleagues have shown that upon microbial

stimulation, AECII become activated and up-regulate expression of MHC II. This was

accompanied with MHC II presentation of pathogen-derived antigen and activation of anti-

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microbial T cell response (20). Therefore, not only AECII can contribute to pulmonary

immunity by secreting chemokines that recruit inflammatory cells to the lung, but they can

also serve as antigen-presenting cells, suggesting a novel role for AECII in the immunological

response to respiratory pathogens. In contrast a recent study defined that AECII naïve or

INF-γ treated pulsed with exogenous antigen were unable to efficiently activate CD4+ T cells

in vitro (21). The authors proposed that antigen recognition on AECII cells in the absence of

costimulation tolerizes T cells in the lung. Whereas these data may suggest a possible

contribution of AECII in the establishment of T cell tolerance to exogenous antigen, so far no

data exist regarding the functional involvement of AECII in the maintenance of peripheral T

cell tolerance against self-antigens expressed in the lung epithelium. In light of recent findings

indicating that common destructive lung diseases, such as chronic obstructive pulmonary

diseases (COPD) and emphysema can be considered as autoimmune disorders (22, 23),

further studies are required focussing on maintenance and breakdown of T cell tolerance in

the lung and the possible implication of AECII in these processes.

In order to study the complex mechanisms that underlie AECII mediated T cell

priming and T cell tolerance induction in the lung in more detail, we made use of surfactant

protein C (SPC)-hemagglutinin (HA) transgenic mice expressing influenza strain A/PR8/34

HA under control of the SPC promoter specifically in type II alveolar epithelial cells (AECII).

To establish an autoimmune environment, these mice were crossed to mice expressing a

transgenic T cell receptor specific for a MHC II restricted HA-derived peptide (TCR-HA).

Concomitant presence of a lung-specific self-antigen and self-reactive CD4+ T cells in

transgenic mice was sufficient to initiate mucosal inflammation progressing to severe

autoimmune pneumonia with emphysema (24). We demonstrate here a previously unknown

immunological attribute of lung epithelial cells in the MHC class II dependent initiation of

CD4+ T cell reactivity to self-antigen. Moreover, we elucidate their active contribution in the

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establishment of peripheral T cell tolerance in the lung. We show that AECII secrete soluble

factors including transforming growth factor-β (TGF-β), which suppress T cell proliferation

and induce Foxp3 expression in CD4+ T cells. As a part of the innate immune system AECII

thus exhibit so far underestimated immune regulatory function and synergize with adaptive

immune mechanisms to re-establish tolerance to self-antigen in the lung.

Some of the results of these studies have been previously reported in the form of abstracts

(25-27).

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METHODS

Mice. BALB/c mice were obtained from Harlan (Borchen, Germany). TCR-HA transgenic

mice expressing a TCR αβ specific for the HA-peptide110–120 from A/PR8/34 HA have been

described previously (28). SPC-HA mice expressing the influenza A/PR8/34 HA under the

transcriptional control of the human surfactant protein C (SP-C) promoter specifically in

AECII have been described elsewhere (24). The generation and screening of CD11c-DTR Tg

mice has been reported previously (29). Mice were bred in the animal facility at the Helmholtz

Centre for Infection Research and were kept under SPF conditions and experiments were all

performed according to national and institutional guidelines.

Isolation of cells. CD4+ T cells were isolated from spleen and lymph nodes by negative

selection by AutoMACS using the CD4+ T cell isolation Kit (Miltenyi Biotec). CD4+CD25- T

cells were isolated by adding biotinylated anti-CD25 mAB (7D4) to the CD4+ T cell isolation

Kit for adoptive transfer experiments. For in vitro proliferation assays splenocytes were

stained with anti-CD4 (RM4-5) and anti-CD25 (PC61) and subjected to one-step cell sorting

using a MoFlow cell sorter (Cytomation, Fort Collins, CO) to obtain highly pure CD4+CD25-

T cell population and to exclude contaminations with professional APCs in T cell

preparations (see Figure E1 in the online data supplement). T cells from the lung were

isolated as described before (24). Primary AECII were as described before (30). Briefly, mice

were killed and exsanguinated by serving the inferior vena cava and left renal artery. The

tracheae was exposed and cannulated, and lungs were perfused with 10 to 20ml sterile

phosphate buffered saline via the pulmonary artery until visually free of blood. 2ml dispase

(BD Biosciences, Heidelberg, Germany) was instilled into lungs via the tracheal catheter

followed by instillation of 500μl 1% low-melt agarose prior warmed to 45°C. Instilled lungs

were immediately covered with ice and incubated for 2min to gel the agarose. Lungs were

then dissected, placed in a culture tube containing an additional 1ml of dispase, and incubated

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for 45min at room temperature. Lungs were then transferred to a culture dish and 7ml serum

free DMEM + 25mM HEPES (GIBCO, Eggenstein, Germany) containing 100U/ml DNAse I

(Sigma, Hannover, Germany) was added. The tissue was gently teased away from the airways

using forceps and lungs were carefully dissociated before agitating the tissue for 10min on a

shaker. Crude cell suspension was sequentially filtered through nylon gauze (100μm, 45μm,

30μm) followed by centrifugation (12min, 130xg) to pellet the cells. For fluorescence

activated cell sorting of alveolar type II epithelial cells, cells were washed with serum free

DMEM + 25mM HEPES and subsequently labeled with anti-CD45, anti-CD32/CD16, anti-

CD11b, anti-CD11c, anti-CD19 and anti-F4/80 antibodies and PE-conjugated goat anti rat-

IgG as secondary antibody. After staining the cells were washed with PBS containing 2%

fetal calf serum and 2mM EDTA and subjected to one-step cell sorting using a MoFlow cell

sorter (Cytomation, Fort Collins, CO). Granular alveolar type II epithelial cells were

identified as SSChigh population. PE (CD45/CD32/CD16/CD11b/F4/80/CD11c/CD19)-positive

cells were excited by an argon ion laser emitted at the wavelength of 488 nm and the

fluorescence was collected after a 580/±30nm band-pass filter. A two parameter sorting

window (side light scattering and PE fluorescent intensity) was used to identify the PE-

negative, side scatter high AEC II population. Cells were sorted through a flow chamber with

a 100μm nozzle tip under 25 psi sheath fluid pressure. Using this protocol a purity of 97-99%

(see Figure E1 in the online data supplement) and viability of 90% was obtained. DCs were

cultured and purified as described (31). Immature bone marrow-derived DCs were collected

on day 5 and purified further by positive selection with AutoMACS using the α-CD11c

Isolation Kit (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated cells were

used for co-culture experiments.

Antibodies and flow cytometry. The monoclonal antibodies 6.5 (α-TCR-HA) and α-MHC II

(M5/114) were purified from hybridoma supernatant by affinity chromatography. The

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monoclonal antibodies α-CD45 (30-F11), α-CD16/CD32 (2.4G2), α-CD11b (M1/70), α-

F4/80, α-CD4 (RM4-5), α-CD25 (PC61 and 7D4), α-Foxp3 (FJK-16s), α-PD-1 (J43), α-

GITR/ TNFRSF18 (108619), α-CD103/Integrin α-IEL chain (2E7), α-CD152/CTLA-4 (UC

10-4F10-11), α-CD19 (1D3), and CD11c (HL3) were obtained from BD Biosciences

Heidelberg, Germany. Foxp3 antibody was obtained by eBioscience, San Diego, CA, USA.

Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labelling and adoptive transfer

of T cells. MACS-isolated CD4+ T cells from the spleens of TCR-HA mice were washed in

RPMI without FCS, re-suspended at a number of 107 lymphocytes/ml and incubated with 2.5

μM CFSE (Molecular Probes, Göttingen, Germany) according to the manufactures

instructions followed by i.v. injection of 1 x 106 antigen-specific CD4+ T cells into mice.

Proliferation assay and co-culture experiments. For co-culture experiments 1 x 105 freshly

isolated AECII were plated in 96-well flat-bottom plates in a final volume of 200μl IMDM

containing 10% FCS. Where indicated, AECII were incubated with a purified blocking MHC

class II monoclonal antibody (14-4-4S) at 35μg/ml for 1 h at 4°C. The cells were then washed

twice with PBS, re-suspended in medium and used as APCs for co-culture experiments. 2.5-3

x 105 CD4+ T cells isolated from TCR-HA transgenic mice were added as responder cells. For

standard proliferation assays cells were cultured for 48h at 37°C and proliferation was

determined by 3[H]-thymidine incorporation. In case AECII and professional APC were used

for co-culture experiment 1 x 105 AECII were plated together with 2.5 x 104 immature DCs.

After 48h the supernatants were discarded and 2.5 x 105 CD4+ T cells isolated from TCR-HA

transgenic mice were added together with fresh medium to a final volume of 200 μl and

cultured at 37 °C for additional 48 h. For measurement of proliferation of CD4+ T cells in

AECII conditioned media 1 x 105 CD4+ T cells were cultured in 200μl AECII conditioned

medium in the presence of 1 μg/ml plate-bound α-CD3ε (145-2C11) (BD Bioscience, San

Jose, CA) at 37 °C for 48 h. 3[H]-thymidine was added for the last 15h of culture and

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incorporation was measured by scintillation counting to asses proliferation. AECII

conditioned media were generated by culturing freshly isolated AECII in IMDM with 10 %

FCS for 48h.

DCs depletion in CD11c-DTR/SPC-HA mice. One day before adoptive transfer of 1x106

antigen-specific CFSE labeled CD4+ T cells CD11c-DTR/SPC-HA mice received an i.t.

injection of 100 ng Diphteria toxin (Sigma-Aldrich) or equal volume of saline to deplete

CD11c+ cells. At day 4 after transfer injected mice were sacrificed and lymphocytes were

isolated from lung and BLN and the proliferation pattern was determined by the loss of CFSE

dye.

FTY720 treatment. Mice were injected daily intra peritoneally with 0,5 mg FTY720/kg body

weight (Novartis) or with an equivalent volume of saline starting two days before adoptive

transfer of 1 x 106 CFSE labelled HA-specific CD4+ T cells. FTY720 treatment was continued

for the following four days and proliferation of transferred T cells was analyzed on day five

after adoptive transfer.

Real-time RT-PCR. Total RNA was prepared from sorted AECII using the RNeasy kit

(Qiagen, Hilden, Germany) and cDNA synthesis was done using Superscript II Reverse

Transcriptase, Oligo dT and random hexamer primers (Invitrogen). Quantitative Real-time

RT-PCR was performed on an ABI PRISM cycler (Applied Biosystems) as described before

(23).

In vitro induction of Foxp3 expression in T cells. 2 x 105 freshly isolated CD4+CD25- cells

and 5 x 105 irradiated syngeneic APC were added to 12-well plates in 1 ml IMDM complete

or AECII conditioned media. Cells were stimulated with soluble anti-CD3 mAb (1 μg/ml) and

cultured for 5 days. At day 3, 50 U/ml recombinant human IL-2 were added to the culture.

Phenotype of the cells was analyzed by flow cytometry. Recombinant human TGF-β1 (R&D

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Systems) and/ or α-TGF-β (1D11) (R&D Systems) were added to the culture in some cases as

indicated in the figure legends.

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RESULTS

Epithelial self-antigen expression leads to the induction of regulatory T cells in the lung

We have reported previously the SPC-HA/TCR-HA mouse as a model for chronic T cell-

mediated lung inflammation (24). In this study we demonstrated that epithelial cell-specific

CD4+ T cells derived from the lung of diseased SPC-HA/TCR-HA mice exhibit an activated

phenotype but at the same time express numerous T cell markers, including CD25, cytotoxic

T lymphocyte antigen-4 (CTLA-4), αEβ7/CD103, glucocorticoid-induced TNF receptor

(GITR) or programmed death ligand (PD-1) which have been discussed in the context of

Tregs (32-37). To substantiate the gene profiling data, we performed a flow cytometry

analysis of the pulmonary CD4+ T cells of SPC-HA/TCR-HA mice and confirmed the

“regulatory signature” of self-reactive CD4+ T cells (Fig. 1A). Since surface expression of the

above mentioned molecules is also induced upon T cell activation, it is difficult to distinguish

whether self-reactive CD4+ T cells isolated from the heavily inflamed lung are activated or

Tregs. Therefore, we also included in our analysis staining for the transcription factor Foxp3

(Fig. 1B) that is considered the most specific marker for Tregs in the mouse (3, 4, 38). For all

molecules analyzed we could detect increased expression on HA-specific and thereby

autoreactive CD4+ T cells when comparing T cells derived from diseased SPC-HA/TCR-HA

with healthy TCR-HA control mice. Elevated expression of Tregs cell-specific molecules was

most prominent in the lung suggesting that chronic epithelial self-antigen exposure in the lung

leads to the induction of Foxp3+ Tregs at the site of inflammation.

Next we performed phenotypic analysis of HA-specific CD4+ T cells that were

adoptively transferred into SPC-HA mice. In addition to providing information on the kinetics

of Tregs induction in the lung, this approach avoids the analysis of mixture of naïve thymic

emigrants, recently activated CD4+ T cells and already converted Tregs that are present in the

lung of SPC-HA/TCR-HA double transgenic mice. We have previously shown that adoptive

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transfer of naïve CD4+ T cells recognizing epithelial neo-self-antigen HA results in

autoimmune interstitial pneumonia characterized by a perivascular and peribronchiolar

infiltration with mature lymphocytes in the lung of SPC-HA mice (30). Consistent with the

observed immuno-pathology, transferred T cells show an activated phenotype as

demonstrated by the production of proinflammotory cytokines like IL-2 and INF-γ (30).

Accordingly, adoptive transfer of carboxyfluorescein succinimidyl diester (CFSE) labelled

HA-specific CD4+ T cells results in an intense antigen-specific proliferation in lung and

bronchial lymph nodes (BLN) of SPC-HA mice 7 days after transfer (Fig. 2A). As expected,

due to the lack of HA expression, no T cell activation/proliferation was observed in peripheral

lymphatic organs of SPC-HA mice, nor in lungs of wildtype (wt) BALB/c mice.

Transferred CD4+ HA-specific T cells recovered from lung and BLN of SPC-HA mice

were further analyzed for the expression of Foxp3, CTLA-4, GITR, αEβ7/CD103 and PD-1

(Fig. 2B). For that purpose we defined the CFSElow population as self-reactive T cells that

underwent several rounds of divisions and the CFSEhigh population as undivided T cells being

naïve or recently activated. Interestingly, the elevated expression of GITR, CTLA-4, PD-1

and CD103/αEβ7 was found to be restricted to the CFSElow CD4+ T cells. Furthermore,

proliferation was associated with a dramatic increase in Foxp3 expression. These data

collectively indicate the induction of Tregs in the lung after a short effector phase that is

associated with strong T cell proliferation and pulmonary immuno-pathology. These in situ

induced Tregs specific for epithelial self-antigen may be part of regulatory mechanisms that

counteract the progression of inflammatory processes thus preventing overwhelming immune

reactions and tissue destruction in the lung.

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Alveolar type II epithelial cells induce MHC class II dependent priming of self-reactive

CD4+ T cells

In order to further dissect the mechanisms that underlie the induction and regulation of T cell

reactivity to epithelial self-antigen in the lung, we performed studies regarding the priming of

self-reactive T cells. Dendritic cells (DCs) and macrophages are discussed to act as

professional antigen presenting cells (APCs) which are able to take up and cross-present self-

antigen from surrounding cells. To investigate whether AECII derived self-antigen is

accessible for DCs cross-presentation resulting in the priming of epithelial cell-specific CD4+

T cells, bone marrow-derived wt DCs and AECII isolated from SPC-HA mice were co-

cultured in vitro together with HA-specific CD4+ T cells. The results suggested that as

expected DCs had the ability to cross-present epithelial antigen and activate naïve HA-

specific CD4+ T cells in vitro (Fig. 3A). However, surprisingly, AECII albeit not as efficient

as professional APC were able to stimulate antigen-specific T cell proliferation as well (Fig.

3B), strongly suggesting that AECII themselves have antigen-presenting capacity. AECII and

responder T cells used for these in vitro studies were highly pure (see Figure E1 in the online

data supplement) excluding the possibility that T cell activation resulted from contaminating

professional APCs. To confirm that AECII driven CD4+ T cell proliferation is mediated by

MHC class II dependent antigen recognition, sorted AECII were co-cultured with HA-specific

CD4+ T cells in the presence or absence of an MHC class II blocking antibody. Blocking of

MHC class II dependent antigen recognition resulted in complete abrogation of antigen-

specific CD4+ T cell proliferation (Fig. 3C), which became even more apparent when AECII

loaded in vitro with high doses of exogenous antigenic peptide were used as APC (Fig. 3D).

To scrutinize antigen presenting function of airway epithelial cells we extended our

investigation on the capacity of AECII to induce CD4+ T cell proliferation to a second,

exogenously applied antigen ovalbumin (OVA). Interestingly, both in vitro and in vivo

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application of antigen to AECII resulted in OVA-specific CD4+ T cell priming, supporting the

existence of the antigen uptake, processing and presentation machinery in these cells. As

observed before with AECII derived from SPC-HA mice, OVA-specific CD4+ T cell priming

was demonstrated to be MHC class II dependant (see Figure E2 in the online data

supplement).

Taken together we conclude that antigen presentation and T cell priming in the lung may not

exclusively be dependent on professional APCs like DCs, but that AECII themselves might

contribute to the priming and re-stimulation of antigen-specific T cells and thus may actively

contribute to the initiation and progression of inflammation in the lung tissue.

To further investigate the function of AECII in the priming of T cells reactive to self-

antigen in vivo, we made use of the CD11c-DTR transgenic mice in which the administration

of Diphtheria toxin (DTx) allows the conditional ablation of CD11c+ antigen presenting cells

in the lung (29, 39, 40). SPC-HA transgenic mice were mated with CD11c-DTR transgenic

BALB/c mice and double transgenic mice were treated intra-tracheally (i.t.) with the toxin.

FACS analysis revealed that a single dose of 100 μg DTx injected i.t. in SPC-HA/CD11c-

DTR mice strongly reduced the frequencies of local CD11c+MHCII+ DCs (39) in lung tissue

and draining lymph nodes (BLN) (Fig. 4A, B). DTx-induced depletion of CD11c+ cells

allowed us to study epithelial-specific T priming in the presence or absence of professional

APCs. To clarify the role of AECII in T cell priming in the lung, SPC-HA/CD11c-DTR mice

were treated with DTx on day 0. At day 1 HA-specific CD4+ T cells were adoptively

transferred and proliferation of adoptively transferred T cells in lung and BLN of these mice

was determined at day 4 (summarized in Fig. 4A). As shown in Figure 4c we observed a

strong and comparable T cell proliferation in the lung of both CD11c+ depleted SPC-

HA/CD11c-DTR mice as well as in mice that harbour normal numbers of CD11c+ cells in

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lung and BLN (PBS treated control, Fig. 4B). As expected, we also observed massive T cell

proliferation in the BLN of PBS-treated mice. However, T cells failed to proliferate in BLN

after conditional depletion of CD11c+ cells indicating that in the absence of professional,

mobile APCs no transfer of epithelial antigen from the lung to the BLN does occur.

Importantly, histological analysis of the lungs from CD11c+ cell-depleted versus non-depleted

SPC-HA/CD11c-DTR mice did not reveal any significant differences in the degree of

inflammation (data not shown). Collectively, these data suggest that CD11c+ professional

APCs are not necessary to trigger T cell responses to epithelial self-antigen in the lung. Thus,

in line with our in vitro data (Fig. 3), AECII mediated self-antigen presentation in vivo may

contribute to unconventional lymph node-independent T cell priming in the respiratory tract.

As a second approach to scrutinize the capacity of AECII in priming CD4+ T cell

responses the lung we utilized the sphingosine-1-phosphate receptor agonist FTY720 to block

the egress of T cells from lymphoid organs (41). Treatment of mice with FTY720 before

adoptive T cell transfer should allow the identification of the site where the priming of CD4+

T cells specific for epithelial self-antigen takes place. We reasoned that if HA-specific T cells

were migrating to the lung after antigen exposure in the lung BLN, than inhibition of

sphingosine 1-phosphate signalling should trap activated CD4+ T cells within the BLN. In

contrast, in case T cell priming would occur directly in the lung by resident AECII we would

observe T cell proliferation in the lung even when egress of T cells from lymphoid organs is

prevented. Following the experimental setting illustrated in Fig. 4D, SPC-HA transgenic mice

received daily either FTY720 or sterile PBS before adoptive transfer of CFSE labelled HA-

specific CD4+ T cells. As a consequence of FTY720 treatment all mice that received the drug

showed lymphopenia, a clear indicator for the success of FTY720 treatment (data not shown)

(42). Day 5 after adoptive transfer proliferation of T cells was analyzed in lung and BLN of

FTY720 treated and control mice (Fig. 4E). As expected, treatment with FTY720 did not

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reduce the proliferation of transferred CD4+ T cells in the BLN, indicating that HA-specific

CD4+ T cells encountered epithelium-derived HA in BLN, probably by DCs that acquired the

proteins from epithelial cells in the lung and transported them to the BLN. Importantly, we

observed a strong proliferation of HA-specific CD4+ T cells in lung tissue of FTY720 treated

mice. Since due to FTY720 treatment conventionally primed T cell are impeded to home from

the BLN to the lung and are captured within the lymph nodes T cell proliferation in the lung

of FTY720 treated mice indicates that CD4+ T cell priming can take place in the lung tissue

and does not require cross-presentation by professional APCs in the BLN. Collectively, our in

vitro and in vivo data indicate a contribution of AECII to the priming of self-reactive T cells

in the lung.

Alveolar type II epithelial cells interfere with T cell proliferation

We previously reported the phenotypic characterization and whole genome expression

profiling on AECII isolated from the lungs of healthy SPC-HA and diseased SPC-HA/TCR-

HA mice (30). This study revealed that AECII become activated and massively change their

phenotype during pulmonary inflammation, suggesting their active participation in

inflammatory, but also in immuno-regulatory processes. By transcriptional profiling we found

that AECII from SPC-HA/TCR-HA transgenic mice showed among others elevated

expression of genes involved in antigen processing and presentation (30). In light of this

finding, we expected AECII isolated from inflamed SPC-HA/TCR-HA lungs to be superior in

the priming CD4+ T cells when compared to AECII of healthy SPC-HA mice. However,

strikingly despite their activated phenotype and elevated MHC II surface expression (Fig.

5A), AECII isolated from diseased SPC-HA/TCR-HA mice failed to stimulate epithelial cell-

specific CD4+ T cell proliferation (Fig. 5B). To elucidate whether the lack of T cell

proliferation is the consequence of inappropriate T cell activation or indicative of active T cell

suppression, analysis for surface expression of co-stimulatory and inhibitory molecules on

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AECII was performed. Interestingly, we observed lack of CD80 and CD86 expression both in

the steady state as well as on activated AECII isolated from the inflamed lung. This was also

true for inhibitory molecules related with down-modulation of T cell responses such as

ICOSL or 41BBL which were absent at the surface of AECII, both in the healthy and in the

inflamed lung (see Figure E3 in the online data supplement).

Assuming that AECII upon inflammation might release mediators that directly affect

T cell proliferation AECII were isolated from SPC-HA, SPC-HA/TCR-HA and BALB/c mice

and cultured for 48 h in vitro. Supernatants were collected and AECII-conditioned media

were used as culture media for in vitro activated CD4+ T cells. As seen in Figure 5C, the

results of this experiment show that AECII of diseased SPC-HA/TCR-HA mice release

soluble mediators that efficiently suppress T cell proliferation. Thus, conditioned medium

derived from SPC-HA/TCR-HA AECII reduced the T cell response by almost 50%, whereas

proliferation of T cells cultured in AECII-conditioned medium from healthy control mice was

unaffected. In conclusion, these results strongly suggest that AECII derived from the inflamed

lung of SPC-HA/TCR-HA mice actively suppress T cell proliferation by - at least in part - the

release of soluble factors.

To identify AECII-derived factors that contribute to the suppression of T cell

proliferation we performed a comparative expression analysis for selected AECII expressed

genes. Components recently discussed in the context of lung immune regulation are the

pulmonary surfactant proteins (SP), which mediate primarily host defence functions and are

members of the collectin family of proteins (43). Specifically SP-A und SP-D were reported

to dampen T cell proliferation and interfere with T cell activation (44, 45). Other secretory

factors discussed to suppress T cell activation and proliferation are platelet factor 4 (PF-4,

CXCL4) and TGF-β (46, 47). Real-time RT-PCR analysis revealed an elevated expression of

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all four mediators in AECII under inflammatory conditions, as compared to AECII isolated

from healthy mice (Fig. 5D). Taken together, AECII from the diseased lung show increased

expression of genes that are discussed in the context of immune suppression and T cell

response modulation.

To further investigate the immuno-suppressive function of AECII in vivo we

transferred 1x106 CFSE-labelled naïve HA-specific CD4+ T cells into either SPC-HA, SPC-

HA/TCR-HA or BALB/c mice. After 5 days, we analyzed the in vivo proliferation of lung and

BLN CD4+ T cells (Fig. 5E). As observed before, HA-specific CD4+ T cells proliferated in

the lung and BLN of SPC-HA mice. Proliferation was antigen-specific, since transferred cells

did neither proliferate in the spleen of SPC-HA mice nor in wt mice lacking HA expression.

Strikingly, in line with our in vitro data transferred T cells exhibit reduced proliferative

response to epithelial self-antigen in the lungs of SPC-HA/TCR-HA mice. The suppressive

effect was even more pronounced in the lymph nodes draining the lung. These data together

with our finding that epithelial self-antigen exposure in the lung leads to the induction of

tissue-specific Tregs collectively suggest that innate and adaptive mechanisms, i.e. AECII-

derived immunosuppressive mediators and in vivo induced Tregs synergize to re-establish

immunological tolerance and suppress autoaggressive T cell responses in the lung.

Alveolar type II epithelial cells induce Foxp3 expression in CD4+ T cells

TGF-β is a potent molecule to modify T cell responses and several studies have shown that

TGF-β can induce Foxp3 expression in naïve CD4+ T cells (48-51). Since we observed a

coincidence of elevated TGF-β expression in AECII from diseased SPC-HA/TCR-HA mice

and the induction of Foxp3+CD4+ T cells specific for epithelial antigen, we reasoned that

AECII-derived TGF-β may contribute to the induction of Tregs in the lung. To get further

insight into the course of TGF-β induction upon inflammation we investigated the kinetic of

21

TGF-β induction in AECII. To this end, we isolated AECII from SPC-HA mice 1, 4 and 7

days after transfer of self-antigen specific CD4+ T cell (acute inflammation) as well as from

SPC-HA/TCR-HA mice (chronic inflammation). AECII from healthy SPC-HA mice served

as control for basal TGF-β expression under healthy conditions. Real-time RT-PCR data

revealed that TGF-β expression is induced in AECII shortly after CD4+ T cell recognition of

epithelial self-antigen. Expression levels steadily increased with time after transfer and

reached maximum levels in SPC-HA/TCR-HA mice (Fig. 6A).

In order to dissect in more detail whether TGF-β expression by AECII has an impact

on Tregs induction, we activated naïve CD4+CD25-Foxp3- T cells in the presence of AECII-

conditioned media derived from either SPC-HA, SPC-HA/TCR-HA or SPC-HA mice 7 days

after adoptive transfer of HA-specific T cells in the presence or absence of antibody blocking

TGF-β. As a control for in vitro generation of Foxp3+ Tregs naïve T cells were activated in

medium supplemented with recombinant TGF-β. As depicted in Fig. 6B and 6C, recombinant

TGF-β induced a conversion of CD4+CD25-Foxp3- T cells in Foxp3+ Tregs. Tregs induction

could be abolished by the addition of anti-TGF-β antibody. Whereas AECII conditioned

medium from SPC-HA mice did not induce Foxp3 expression in CD4+ T cells, T cell

stimulation in the presence of AECII conditioned media from both SPC-HA/TCR-HA as well

as adoptively transferred SPC-HA induces Foxp3 expression in CD4+ T cells (Fig. 6B, C).

Notably, Foxp3 expression in CD4+ T cells was significantly abrogated when antibodies

blocking TGF-β were added to AECII conditioned medium derived from the acutely inflamed

lung (SPC-HA mice 7 days after transfer). By contrast antibody to TGF-β did not affect

Foxp3 induction when T cells were stimulated in the presence of AECII conditioned media

from the chronically inflamed lung (SPC-HA/TCR-HA). Collectively, these data indicate that

CD4+ T cell recognition of epithelial self-antigen rapidly induces, besides other immune

modulating factors, TGF-β expression in AECII. Whereas in the early stage of inflammation

22

TGF-β is the dominant factor inducing the conversion of naïve CD4+CD25-Foxp3- T cells into

CD4+CD25highFoxp3+ Tregs, other so far unidentified AECII-derived factors can substitute for

and/or synergize with TGF-β in the latter phase of pulmonary inflammation and induce Foxp3

expression in CD4+ T cells independent of TGF-β. Thus, TGF-β secretion by AECII may

represent an immediate innate immune response to counterbalance tissue destructive cellular

immunity by the induction of Tregs that limit immuno-pathology.

23

DISCUSSION

The mechanisms underlying the peripheral induction of Tregs are subject of intensive

investigation. However, the complex interplay of different cellular subsets, soluble factors,

inhibitory molecules and the overall micro-milieu needed for the in vivo conversion of naïve

into regulatory T cells is only partially understood. Here we suggest a new mechanism of T

cell tolerance induction in the periphery. We demonstrate that a specialized population of

AECII actively participates both in the initiation and regulation of CD4+ T cell mediated

autoimmune tissue attack in the lung. We show that recognition of MHC class II presented

self-antigen on lung resident AECII results in priming and proliferation of naïve CD4+ T cells,

followed by the acquisition of a regulatory Foxp3+ phenotype. Our data add new insights into

the antigen presenting and T cell priming capacity of AECII and in the complexity of

peripheral tolerance induction and points at a close collaboration of the innate and adaptive

immunity in balancing T cell reactivity to self-antigen in the lung.

It is well established that delivery of exogenous antigens to DCs under steady state

condition renders cognate naïve T cells tolerant. Furthermore, various studies suggested that

steady state DCs represent the most important APCs population able to induce T cell

tolerance to self-antigens (11, 52). Nevertheless, there is growing evidence that in addition to

DCs other APCs such as macrophages (53) and B cells (54-56) exhibit the capacity to induce

T cell tolerance in vivo. Focussing on AECII as non-immune cells in inducing peripheral

tolerance to self-antigen we demonstrate here that these epithelial cells expressing the neo-

self-antigen hemagglutinin in the lung of SPC-HA transgenic mice have antigen presenting

capacity and stimulate antigen-specific CD4+ T cell response in vitro and in vivo. CD4+ T cell

activation by AECII was found to be strictly MHC class II dependant, since prevention of

MHC class II/TCR interaction by the addition of blocking antibodies abrogated in vitro T cell

proliferation (Figure 3). Existence of an MHC class II antigen processing and presentation

24

machinery in AECII as well as MHC class II dependent CD4+ T cell activation by AECII was

further substantiated using a second antigen, exogenous OVA either applied to AECII in vitro

or in vivo followed by co-culture of these cells with corresponding OVA-specific CD4+

responder T cells (see Figure E2 in the online data supplement). In contrast to Lo and

colleagues who did not observe priming of naive CD4+ T cells by AECII (21), we could again

demonstrate priming of naive CD4+ T cells as indicated by antigen-specific T cell

proliferation not only with endogenous HA but also with exogenously applied OVA. This

may be attributed by the fact that we were using primary AECII in combination with either

HA- or OVA-specific naive CD4+ T cells, whereas Lo et al. determined T cell activation by

AECII by measurement of IL-2 secretion by OVA-specific T cell hybridomas or proliferation

of alloreactive CD4+ T cells.

Importantly, we observed that in SPC-HA mice depleted from DCs priming of self-reactive

CD4+ T cell still occurred in the lung, but was completely abolished in BLN, suggesting that

T cell priming can be mediated by AECII in the lung in a DCs and lymph node independent

way. This was further corroborated by blocking the egress of T cells from lymphatic organs

by the use of FTY720. Our data are well in line with data recently reported by Hagymasi and

colleagues who demonstrated that DCs depletion did not abrogate priming of naïve antigen-

specific CD4+ T cells specific for parenchymal self-antigen (57). Although the authors did not

further specify the cellular subset responsibly for priming of self-reactive T cells, they

elegantly proof that T cell reactivity to self-antigen can be induced in the absence of DCs.

Interestingly our data indicated an activation of AECII (30) and an antigen-specific

stimulatory capacity of naïve AECII expressing endogenous antigen. Furthermore we could

show an increased MHC II surface expression in the context of autoimmune CD4+ T cell

mediated lung inflammation (Fig. 5A). However, despite elevated MHC II expression

25

activated AECII derived from an autoimmune environment, i.e. in the absence of infection,

failed to induce T cell proliferation in vitro (Fig. 5B). We suggest that the failure of T cells to

proliferate can be attributed to the suppressive effect of factors over-expressed and released

by AECII isolated from the inflamed environment. These factors include SP-A, SP-D, PF-4

TGF-β (Fig. 5D) and certainly many others which may synergize to suppress T cell activation

and proliferation, but also induce the differentiation of Foxp3+ Tregs from naïve precursors.

Surfactant proteins play an important role in the innate immune defence of the

respiratory tract (43). Nevertheless, recent research has highlighted that these proteins not

only augment innate immune responses to invading microorganisms but also act on adaptive

immune functions like DCs maturation (31) and T cell proliferation (44, 45). Concerning PF-

4 it has been shown that its binding to T cells resulted in down-regulation of IL-2-release and

correlated with the inhibition of functions in activated T cells (46).

TGF-β is a potent, suppressive cytokine critically involved in the induction of

tolerance and the regulation of immune responses (58). This is best illustrated by the onset of

a severe autoimmune-like syndrome in TGF-β-/- mice, characterized by the spontaneous and

progressive, multiorgan infiltration of mononuclear cells and pathogenic autoantibodies (59).

Recently, it has been shown that TGF-β promotes Tregs expansion as well as generation of

Foxp3+ induced Tregs from naïve CD4+CD25- T cells, although the underlying molecular

mechanisms remain ill-defined (48-51). We show here elevated expression of TGF-β in

AECII of diseased mice. TGF-β induction was already evident very short after T cell antigen

recognition in the lung and reached maximum level in AECII derived from SPC-HA/TCR-

HA mice suffering from chronic pulmonary inflammation (Fig. 6A). Additionally, we could

show that components secreted by AECII from both acutely inflamed as well as chronically

inflamed lungs but not from the healthy environment induced the expression of Foxp3 in

activated CD4+CD25- T cells (Fig 6B). Furthermore, we demonstrated that the conversion of

26

CD4+CD25-Foxp3- T cells in Foxp3+ Tregs could partially be abolished by adding anti-TGF-β

antibody AECII conditioned medium derived from acutely inflamed SPC-HA mice indicating

a TGF-β dependent mechanism of Tregs induction. Strikingly, anti-TGF-β antibody could not

interfere with Foxp3 induction in T cells cultured in AECII conditioned media derived from

chronically inflamed SPC-HA/TCR-HA mice suggesting an alternative pathway of Tregs

induction in addition to the TGF-β dependent mechanism. Further studies are needed to

identify potential candidates that are involved in the conversion of naïve T cells into Foxp3+

Tregs, although it might be speculated that multiple AECII derived factors synergize in Tregs

induction thereby making it difficult to identify single factors that induce Tregs development

in the context of pulmonary inflammation.

In addition to AECII derived soluble factors that suppress T cell proliferation and

induce the expression of Foxp3 in CD4+ T cells, T cell tolerance to epithelial self-antigen may

also be attributed to the fact that AECII lack the expression of the costimulatory molecules

CD80 and CD86 (see Figure E3 in the online data supplement). This was also evident in

activated AECII that showed elevated surface expression of MHC II. It has been shown that

priming of T cells by APCs lacking costimulatory molecules resulted in incomplete

stimulation of naïve T cells and as a consequence in clonal deletion and peripheral tolerance

(60-62). Therefore, direct priming of T cells by AECII in the lung may also contribute to

peripheral tolerance induction, a mechanism also recently proposed by Lo and colleagues

(21). On the other hand it has been shown that functional T cell activation can occur in the

absence of costimulation provided high abundance of TCR ligands and continued presence of

MHC presented antigen (63, 64). Since we observe a conversion of naïve CD4+ T cells into

Foxp3+ Tregs but not a deletion of self-reactive T cells, we suggest that deletion of self-

reactive T cells as a consequence of incomplete T cell activation may only occur to a minor

extend.

27

In conclusion, CD4+ T cell recognition of alveolar epithelial self-antigen induces

multifaceted processes of immune-activation and immune-modulation in the lung. On the

basis of our findings, we propose the following model of AECII cell function in re-

establishing peripheral T cell tolerance to pulmonary self-antigen (Fig. 7): Under normal

conditions (i.e. in mice that are not depleted from DCs) we suppose classical uptake and

cross-presentation of epithelial derived self-antigen by pulmonary DCs and priming of auto-

reactive CD4+ T cell in BLN. This is in line with the observation of massive proliferation of

adoptively transferred HA-specific CD4+ T cells in BLN of SPC-HA mice (Fig 2A).

Subsequently, activated CD4+ T cell recognize their corresponding antigen on AECII in the

lung and initiate inflammation. In addition to this DCs-dependent priming of T cells in the

BLN, data obtained by depleting DCs or FTY720 treatment indicate unconventional priming

of T cells reactive to epithelial self-antigen directly through AECII. In both cases self-antigen

recognition by specific CD4+ T cells in the lung results in the activation of AECII which

subsequently over-express a variety of immuno-modulatory factors including SP-A and SP-D,

PF-4 as well as TGF-β. Via the secretion of these and certainly other factors AECII interfere

with adaptive immunity by both directly suppressing the proliferation of T cells and indirectly

by the conversion of naïve CD4+Foxp3- to CD4+Foxp3+ Tregs. These induced Tregs

synergize with soluble, AECII derived mediators in suppressing autoimmune tissue attack and

restoring immunological self-tolerance in the lung.

ACKNOWLEDGMENTS

We thank Silvia Prettin and Angelika Hoehne for excellent technical assistance and Lothar

Groebe for expert cell sorting.

28

FIGURE LEGENDS

Figure 1 HA-specific CD4+ T cells from SPC-HA/TCR-HA mice express elevated level of

Tregs-specific molecules. (A) Lymphocytes were isolated from lung and BLN derived from

diseased SPC-HA/TCR-HA and healthy TCR-HA transgenic mice. CD4+ HA-specific (6.5+)

T cell were analyzed for GITR, CD103, PD-1 and CTLA-4 expression by FACS. (B)

Lymphocytes were isolated from lung, spleen, BLN and MLN derived from diseased SPC-

HA/TCR-HA and healthy TCR-HA transgenic mice. CD4+ HA-specific (6.5+) T cell were

analyzed for Foxp3 expression by FACS. Expression level of indicated marker molecules are

depicted in percentage SPC-HA/TCR-HA vs. TCR-HA [xy% (xy%)]. Results are

representative of those obtained in two independent experiments.

Figure 2 Induction of Tregs in SPC-HA mice after adoptive transfer of HA-specific CD4+ T

cells. (A) Lymphocytes were isolated from lung, BLN and spleen derived from adoptively

transferred transgenic SPC-HA mice and BALB/c control mice. CD4+ HA-specific (6.5+) T

cell were analyzed by FACS for loss of CFSE dye to assess their proliferation in transgenic

and non-transgenic mice. (B) CD4+ HA-specific (6.5+) T cells from lung and BLN derived

from adoptively transferred transgenic SPC-HA mice were analyzed for loss of CFSE dye and

the expression of GITR, CTLA-4, CD103 and PD-1. Naïve CD4+ HA-specific T cell derived

from TCR-HA mice where included as a control. Expression levels of indicated marker

molecules are depicted in percentage SPC-HA vs. TCR-HA [xy% (xy%)]. Data obtained in

one out of two experiments with similar results are shown.

Figure 3 Alveolar type II epithelial cells present endogenous self-antigen and induce antigen

specific CD4+ T cell proliferation. (A) 1x105 freshly isolated AECII from BALB/c and SPC-

29

HA transgenic mice were co-cultured with 2.5x104 immature, bone marrow derived DCs

isolated from BALB/c. After 48 h 2.5x105 naïve CD4+ T cells from TCR-HA mice were

added and incubated for further 48 h. (B) 1x105 freshly isolated AECII from BALB/c and

SPC-HA transgenic mice were co-cultured with 2.5x105 naïve CD4+ T cells from TCR-HA

mice for 48 h. (C and D) AECII and HA-specific CD4+ T cells were co-cultured as in (B),

only that AECII were pre-incubated with an antibody blocking MHC class II (C) and

additionally loaded extracellular with HA peptide (D). Proliferation of responder T cells was

determined by 3[H]-thymidine incorporation. Note that the scaling is different in A-D to

emphasize differences in T cell proliferation observed with AECII cells from SPC-HA and

control mice. Data presented are representative for two or three independent experiments with

similar results. Results are expressed as mean counts per minute ± SD of triplicate wells. (A)

AECII/DCs/T cell co-culture: SPC-HA vs. BALB/c P<0.0001; (B) AECII/T cell co-culture:

SPC-HA vs. BALB/c P<0.0014; (C) AECII/T cell co-culture/MHCII block: SPC-HA vs.

SPC/HA with MHCII block: P=0.018; (D) AECII/T cell co-culture with peptide/MHCII

block: SPC-HA vs. SPC-HA with MHCII block: P<0.0001.

Figure 4 AECII are able to induce self-antigen specific CD4+ T cell proliferation in the lung.

SPC-HA/DTR transgenic mice were injected i.t. with 100 ng DT or PBS as a control followed

by adoptive transfer of CFSE labelled CD4+ HA-specific (6.5+) T cells following the

experimental setup drafted in (A). (B) One day after instillation, lungs and lung draining

lymphnodes (BLN) were analyzed for the presence of CD11c+ MHC IIhigh cells by FACS. (C)

4 days after adoptive transfer lymphocytes from lungs and BLN were re-isolated and CD4+

HA-specific (6.5+) T cells were analyzed CFSE loss as indicator of proliferation. (D)

Schematic drawing of the experimental setup. SPC-HA transgenic mice were injected i.p.

daily with FTY720 or equal volume of sterile PBS until mice were sacrificed for analysis.

30

Two days after the first administration of FTY720 mice were received adoptive transfer of

CFSE labelled HA-specific (6.5+) CD4+ T cells. (e) Lymphocytes from lung and BLN of

FTY720 and PBS treated SPC-HA mice were isolated. CD4+ HA-specific (6.5+) T cells were

analyzed for loss of CFSE dye by FACS. Data depicted are representative of those obtained in

two independent experiments each with similar outcome.

Figure 5 Immunosuppressive function of AECII. (A) AECII from diseased SPC-HA/TCR-

HA and healthy SPC-HA control mice were isolated and analyzed for surface expression of

MHC II molecules by FACS. (B) 1 x 105 freshly isolated AECII from SPC-HA/TCR-HA,

SPC-HA and BALB/c mice were co-cultured with 2.5 x 105 CD4+ T cells derived from TCR-

HA mice for 48h. T cell proliferation was quantified by 3[H]-thymidine incorporation. (C) 1 x

105 naïve CD4+ T cells were stimulated with 1 μg/ml α-CD3 (plate bound) in the presence of

conditioned media derived from AECII isolated from the lung of BALB/c, SPC-HA or SPC-

HA/TCR-HA transgenic mice. IMDM medium served as internal control. After 48 h at 37 °C,

proliferation of responder T cells was determined by 3[H]-thymidine incorporation. One

representative experiment out of three is shown. Values indicate mean counts per minute of

triplicate wells ± SD. AECII/T cell co-culture: SPC-HA vs. SPC-HA/TCR-HA P<0.0002;

conditioned media AECII/T cell culture: SPC-HA vs. SPC-HA/TCR-HA P<0.001; IMDM vs.

SPC-HA/TCR-HA P<0.0004. (D) AECII were isolated from the lung of SPC-HA mice (n = 3)

and SPC-HA/TCR-HA mice (n = 3). Cells were subjected to quantitative real-time RT-PCR

analyses. mRNA expression levels of SP-A, SP-D, PF-4 and TGF-β were analyzed in real-

time RT-PCR assays. RPS9 served as internal control (not shown). Relative mRNA quantities

were normalized with respect to expression levels in AECII isolated from SPC-HA mice (fold

change = 1). (E) Lymphocytes from adoptively transferred transgenic SPC-HA, SPC-

HA/TCR-HA and BALB/c control mice were isolated from lung, BLN and spleen. CD4+ HA-

31

specific (6.5+) T cell were analyzed by FACS for the loss of CFSE dye to assess their

proliferation in transgenic, double-transgenic and non-transgenic mice. Depicted results are

representative for two independent experiments with similar outcome.

Figure 6 Elevated expression of TGF-β in AECII from the inflamed lung and induction of

Foxp3+ expression in CD4+ T cells. (A) AECII were isolated from the lung of SPC-HA/TCR-

HA double transgenic mice or SPC-HA mice one (n = 3), four (n = 3) and seven (n = 3) days

after adoptive transfer of HA-specific CD4+ T cells. Cells were subjected to quantitative real-

time RT-PCR analyses. mRNA expression level of TGF-β was quantified and results were

normalized to the housekeeping control RPS9. Relative mRNA amounts were normalized

with respect to expression levels in AECII isolated from SPC-HA mice not receiving CD4+ T

cell transfer (fold change = 1). (B) 2 x 105 MACS sorted CD4+CD25- T cells from the spleen

of BALB/c mice were stimulated with 1μg/ml soluble α-CD3 in the presence of 5 x 105

irradiated syngeneic APCS. Cells were either cultured in IMDM with or without 2ng/ml TGF-

β or 20 mg/ml α-TGF-β or in AECII conditioned media in the presence or absence of α-TGF-

β for 5 days. After 5 days cells were analyzed for CD4, CD25 and Foxp3 expression by

FACS. Percentage of CD4+CD25highFoxp3+ T cells after in vitro culture is depicted.

Representative results from three independent experiments are shown. (C) Diagram

summarizes the percentage of CD4+CD25highFoxp3+ T cells after culture as described in (B).

Figure 7 Model of AECII function in re-establishing peripheral T cell tolerance against

alveolar self-antigen. Details are discussed in the text.

32

Reference List

1. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance

maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25).

Breakdown of a single mechanism of self-tolerance causes various autoimmune

diseases. J Immunol. 1995;155:1151-1164.

2. Shevach EM. CD4+ CD25+ suppressor T cells: more questions than answers. Nat Rev

Immunol. 2002;2:389-400.

3. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function

of CD4+CD25+ regulatory T cells. Nat Immunol. 2003;4:330-336.

4. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the

transcription factor Foxp3. Science. 2003;299:1057-1061.

5. Cottrez F, Hurst SD, Coffman RL, Groux H. T regulatory cells 1 inhibit a Th2-specific

response in vivo. J Immunol. 2000;165:4848-4853.

6. Groux H, O'Garra A, Bigler M, et al. A CD4+ T-cell subset inhibits antigen-specific T-

cell responses and prevents colitis. Nature. 1997;389:737-742.

7. Chen Y, Kuchroo VK, Inobe J, Hafler DA, Weiner HL. Regulatory T cell clones induced

by oral tolerance: suppression of autoimmune encephalomyelitis. Science.

1994;265:1237-1240.

33

8. Weiner HL. Induction and mechanism of action of transforming growth factor-beta-

secreting Th3 regulatory cells. Immunol Rev. 2001;182:207-214.

9. Azukizawa H, Kosaka H, Sano S, et al. Induction of T-cell-mediated skin disease specific

for antigen transgenically expressed in keratinocytes. Eur J Immunol. 2003;33:1879-

1888.

10. Mayerova D, Parke EA, Bursch LS, Odumade OA, Hogquist KA. Langerhans cells

activate naive self-antigen-specific CD8 T cells in the steady state. Immunity.

2004;21:391-400.

11. Scheinecker C, McHugh R, Shevach EM, Germain RN. Constitutive presentation of a

natural tissue autoantigen exclusively by dendritic cells in the draining lymph node. J

Exp Med. 2002;196:1079-1090.

12. Vezys V, Olson S, Lefrancois L. Expression of intestine-specific antigen reveals novel

pathways of CD8 T cell tolerance induction. Immunity. 2000;12:505-514.

13. Lefrancois L, Puddington L. Intestinal and pulmonary mucosal T cells: local heroes fight

to maintain the status quo. Annu Rev Immunol. 2006;24:681-704.

14. Fehrenbach H. Alveolar epithelial type II cell: defender of the alveolus revisited. Respir

Res. 2001;2:33-46.

15. Mason RJ. Biology of alveolar type II cells. Respirology. 2006;11 Suppl:S12-S15.

34

16. Singh G, Katyal SL, Brown WE, Collins DL, Mason RJ. Pulmonary lysozyme--a

secretory protein of type II pneumocytes in the rat. Am Rev Respir Dis.

1988;138:1261-1267.

17. Strunk RC, Eidlen DM, Mason RJ. Pulmonary alveolar type II epithelial cells synthesize

and secrete proteins of the classical and alternative complement pathways. J Clin

Invest. 1988;81:1419-1426.

18. Cunningham AC, Zhang JG, Moy JV, Ali S, Kirby JA. A comparison of the antigen-

presenting capabilities of class II MHC-expressing human lung epithelial and

endothelial cells. Immunology. 1997;91:458-463.

19. Zissel G, Ernst M, Rabe K, et al. Human alveolar epithelial cells type II are capable of

regulating T-cell activity. J Investig Med. 2000;48:66-75.

20. Debbabi H, Ghosh S, Kamath AB, et al. Primary type II alveolar epithelial cells present

microbial antigens to antigen-specific CD4+ T cells. Am J Physiol Lung Cell Mol

Physiol. 2005;289:L274-L279.

21. Lo B, Hansen S, Evans K, Heath JK, Wright JR. Alveolar Epithelial Type II Cells Induce

T Cell Tolerance to Specific Antigen. J Immunol. 2008;180:881-888.

22. Lee SH, Goswami S, Grudo A, et al. Antielastin autoimmunity in tobacco smoking-

induced emphysema. Nat Med. 2007;13:567-569.

35

23. Taraseviciene-Stewart L, Scerbavicius R, Choe KH, et al. An animal model of

autoimmune emphysema. Am J Respir Crit Care Med. 2005;171:734-742.

24. Bruder D, Westendorf AM, Geffers R, et al. CD4 T Lymphocyte-mediated lung disease:

steady state between pathological and tolerogenic immune reactions. Am J Respir Crit

Care Med. 2004;170:1145-1152.

25. Gereke M, Groebe L, Prettin S, et al. Alveolar type II epithelial cells: pneumocytes with

regulatory properties. 16th European Congress of Immunology -ECI. Sept. 06-09,

2006; Paris, France.

26. Gereke M, Buer J, Bruder D. Collaboration of the innate and adaptive immune system

leads to immunological tolerance in the lung. 37. Annual Meeting of the German

Society for Immunology. Sept. 05-08, 2007; Heidelberg, Germany.

27. Gereke M, Jung S, Buer J et al. Alveolar epithelial self antigen display promotes the

peripheral induction of Foxp3+ regulatory T cells. Joint Annual Meeting of

Immunology of the Austrian and German Society. Sept. 03-06, 2008; Vienna, Austria.

28. Kirberg J, Baron A, Jakob S, Rolink A, Karjalainen K, von BH. Thymic selection of

CD8+ single positive cells with a class II major histocompatibility complex-restricted

receptor. J Exp Med. 1994;180:25-34.

29. Jung S, Unutmaz D, Wong P, et al. In vivo depletion of CD11c(+) dendritic cells

abrogates priming of CD8(+) T cells by exogenous cell-associated antigens. Immunity.

2002;17:211-220.

36

30. Gereke M, Grobe L, Prettin S, et al. Phenotypic alterations in type II alveolar epithelial

cells in CD4+ T cell mediated lung inflammation. Respir Res. 2007;8:47.

31. Brinker KG, Martin E, Borron P, et al. Surfactant protein D enhances bacterial antigen

presentation by bone marrow-derived dendritic cells. Am J Physiol Lung Cell Mol

Physiol. 2001;281:L1453-L1463.

32. Gavin MA, Clarke SR, Negrou E, Gallegos A, Rudensky A. Homeostasis and anergy of

CD4(+)CD25(+) suppressor T cells in vivo. Nat Immunol. 2002;3:33-41.

33. Huehn J, Siegmund K, Lehmann JC, et al. Developmental stage, phenotype, and

migration distinguish naive- and effector/memory-like CD4+ regulatory T cells. J Exp

Med. 2004;199:303-313.

34. Lechner O, Lauber J, Franzke A, Sarukhan A, von BH, Buer J. Fingerprints of anergic T

cells. Curr Biol. 2001;11:587-595.

35. Lehmann J, Huehn J, de la RM, et al. Expression of the integrin alpha Ebeta 7 identifies

unique subsets of CD25+ as well as. Proc Natl Acad Sci U S A. 2002;99:13031-13036.

36. Takahashi T, Tagami T, Yamazaki S, et al. Immunologic self-tolerance maintained by

CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-

associated antigen 4. J Exp Med. 2000;192:303-310.

37. Uraushihara K, Kanai T, Ko K, et al. Regulation of murine inflammatory bowel disease

by CD25+ and. J Immunol. 2003;171:708-716.

37

38. Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+CD25+

T regulatory cells. Nat Immunol. 2003;4:337-342.

39. Landsman L, Varol C, Jung S. Distinct differentiation potential of blood monocyte

subsets in the lung. J Immunol. 2007;178:2000-2007.

40. van Rijt LS, Jung S, Kleinjan A, et al. In vivo depletion of lung CD11c+ dendritic cells

during allergen challenge abrogates the characteristic features of asthma. J Exp Med.

2005;201:981-991.

41. Matloubian M, Lo CG, Cinamon G, et al. Lymphocyte egress from thymus and peripheral

lymphoid organs is dependent on S1P receptor 1. Nature. 2004;427:355-360.

42. Klingenberg R, Nofer JR, Rudling M, et al. Sphingosine-1-Phosphate Analogue FTY720

Causes Lymphocyte Redistribution and Hypercholesterolemia in ApoE-Deficient

Mice. Arterioscler Thromb Vasc Biol. 2007.

43. Wright JR. Immunoregulatory functions of surfactant proteins. Nat Rev Immunol.

2005;5:58-68.

44. Borron P, McCormack FX, Elhalwagi BM, et al. Surfactant protein A inhibits T cell

proliferation via its collagen-like tail and a 210-kDa receptor. Am J Physiol.

1998;275:L679-L686.

38

45. Borron PJ, Mostaghel EA, Doyle C, Walsh ES, Heyzer-Williams MG, Wright JR.

Pulmonary surfactant proteins A and D directly suppress CD3+/CD4+ cell function:

evidence for two shared mechanisms. J Immunol. 2002;169:5844-5850.

46. Fleischer J, Grage-Griebenow E, Kasper B, et al. Platelet factor 4 inhibits proliferation

and cytokine release of activated human T cells. J Immunol. 2002;169:770-777.

47. Tiemessen MM, Kunzmann S, Schmidt-Weber CB, et al. Transforming growth factor-

beta inhibits human antigen-specific CD4+ T cell proliferation without modulating the

cytokine response. Int Immunol. 2003;15:1495-1504.

48. Chen W, Jin W, Hardegen N, et al. Conversion of peripheral CD4+. J Exp Med.

2003;198:1875-1886.

49. Cobbold SP, Castejon R, Adams E, et al. Induction of foxP3+ regulatory T cells in the

periphery of T cell receptor transgenic mice tolerized to transplants. J Immunol.

2004;172:6003-6010.

50. Coombes JL, Siddiqui KR, rancibia-Carcamo CV, et al. A functionally specialized

population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-

beta and retinoic acid-dependent mechanism. J Exp Med. 2007;204:1757-1764.

51. Rao PE, Petrone AL, Ponath PD. Differentiation and expansion of T cells with regulatory

function from human peripheral lymphocytes by stimulation in the presence of TGF-

{beta}. J Immunol. 2005;174:1446-1455.

39

52. Belz GT, Behrens GM, Smith CM, et al. The CD8alpha(+) dendritic cell is responsible

for inducing peripheral self-tolerance to tissue-associated antigens. J Exp Med.

2002;196:1099-1104.

53. Miyazaki T, Suzuki G, Yamamura K. The role of macrophages in antigen presentation

and T cell tolerance. Int Immunol. 1993;5:1023-1033.

54. Eynon EE, Parker DC. Small B cells as antigen-presenting cells in the induction of

tolerance to soluble protein antigens. J Exp Med. 1992;175:131-138.

55. Fuchs EJ, Matzinger P. B cells turn off virgin but not memory T cells. Science.

1992;258:1156-1159.

56. Reichardt P, Dornbach B, Rong S, et al. Naive B cells generate regulatory T cells in the

presence of a mature immunologic synapse. Blood. 2007;110:1519-1529.

57. Hagymasi AT, Slaiby AM, Mihalyo MA, et al. Steady state dendritic cells present

parenchymal self-antigen and contribute to, but are not essential for, tolerization of

naive and Th1 effector CD4 cells. J Immunol. 2007;179:1524-1531.

58. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factor-beta

regulation of immune responses. Annu Rev Immunol. 2006;24:99-146.

59. Kulkarni AB, Huh CG, Becker D, et al. Transforming growth factor beta 1 null mutation

in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci

U S A. 1993;90:770-774.

40

60. Bonifaz L, Bonnyay D, Mahnke K, Rivera M, Nussenzweig MC, Steinman RM. Efficient

targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state

leads to antigen presentation on major histocompatibility complex class I products and

peripheral CD8+ T cell tolerance. J Exp Med. 2002;196:1627-1638.

61. Finkelman FD, Lees A, Birnbaum R, Gause WC, Morris SC. Dendritic cells can present

antigen in vivo in a tolerogenic or immunogenic fashion. J Immunol. 1996;157:1406-

1414.

62. Heath WR, Carbone FR. Cross-presentation, dendritic cells, tolerance and immunity.

Annu Rev Immunol. 2001;19:47-64.

63. Kundig TM, Shahinian A, Kawai K, et al. Duration of TCR stimulation determines

costimulatory requirement of T cells. Immunity. 1996;5:41-52.

64. Viola A, Lanzavecchia A. T cell activation determined by T cell receptor number and

tunable thresholds. Science. 1996;273:104-106.

lung BLN spleen MLN

Foxp3

B26,2%(5,0%)

13,8%(9,4%)

22,5%(15,2%)

11,2%(9,5%)

SPC-HA/TCR-HA TCR-HA

CTLA-4 CD103 GITR PD-1

lung

BLN

A

13,3%(3,8%)

35,6%(8,75%)

29,9%(4,8%)

13,9%(4,4%)

6,3%(2,5%)

14,1%(3,7%)

8,6%(3,2%)

51,3%(12,1%)

Figure 1

CFSE

lung

BLN

sple

en

72,3%

62,1%

93,9%

BALB/c

95,3%

90,3%

95,7%

6,1%

37,9%

27,7%

4,7%

9,7%

4,3%SPC-HAA

CFSEPD-1 CFSEPD-1

CFSE

FoxP

3

Foxp3

BLN

CFSE

Foxp

3

Foxp3

lung

CFSE

CTL

A-4

CTLA-4

CFSE

CD

103

CD103

CFSE

GIT

R

GITR

PD-1

PD-1

CFSE

GIT

R

GITR

CFSE

CD

103

CD103

CFSEC

TLA

-4CTLA-4

B47,42%(8,88%)

(34,53%)88,71%

(7,17%)65,90%

(12,15%)45,36%

(1,35%)

(1,75%)

(6,99%)

5,35%

13,41%

3,72%

42,70%

(18,39%)

(4,54%)

(10,90%)

70,45%

33,38%

Figure 2naive CD4+6.5+ T cells transferred CD4+6.5+ T cells

60

50

40

SPC‐HABALB/c

P< 0.0001A

n [cpm

] 103

8

10

12

14SPC‐HA

BALB/cP= 0.0014

B

[cpm

] 103

**

10

30

20

Prolife

ration

2

4

6

8

Prolife

ration

 

AECII DC AECII+DC+CD4+ T cells

CD4+ T cells AECII AECII+CD4+ T cells

CD4+ T cells

SPC‐HA

3

10C

03

100

SPC‐HA

DP= 0.014

P= 0 018 P< 0.0001*BALB/c

eration [cpm

] 10

4

6

8

feration

 [cpm

] 1

40

60

80 BALB/cP= 0.018 P  0.0001

*

AECII AECII+CD4+ T cells

CD4+ T cellsAECII+CD4+ T cells

Prolife

2

AECII AECII+CD4+ T cells

AECII+CD4+ T cells

CD4+ T cells

Proli

20

*

*

+CD4 T cells +CD4 T cells+αMHCII

+CD4 T cells+Peptid

CD4 T cells+Peptid+αMHCII

Figure 3

day 0

100ng DTx/PBS i.t.

6.5+ CD4+CFSE+

transfer i.v.

day 1

FACS analysis/proliferation

day 4

SPC-HA/DTR SPC-HA/DTR SPC-HA/DTR

A

C

CFSE

lung

BLN

+ D

Tx+

DTx

+ PB

S+

PBS

8,1% 91,9%

45,6%

66,6%

66,8%

54,4%

32,4%

33,2%

B

MH

C II

CD11c

6,48% 1,10%

0,24% 0,07%

SPC-HA/DTR + PBS SPC-HA/DTR + DTx

lung

BLN

D

day -2

dailyFTY720/PBS i.p.

6.5+ CD4+CFSE+

transfer i.v.

day 0

FACS analysis/proliferation

day 5

SPC-HA SPC-HA SPC-HA

E

CFSE

67,5% 32,5%lung

94,9% 5,1%

67,9% 32,1%

96,8% 3,2%

BLN

+ PB

S+

PBS

+ FT

Y720

+ FT

Y720

Figure 4

AECII AECII+CD4+ T cells

CD4+ T cells

2

4

6

8

10

12

Prol

ifera

tion

[cpm

] 103 SPC-HA

BALB/c

SPC-HA/TCR-HA

P= 0.0002

*

BA78,9% MFI 27691,4% MFI 381

MHC II

SPC-HA/TCR-HASPC-HA

Figure 5

Prol

ifera

tion

[cpm

] 103

2

4

6

8

10

12

14

BALB/c SPC-HA SPC-HA/TCR-HA IMDM

P = 0.001 P = 0.0004

*

C

88,0%

94,9%

97,3%

CFSE

SPC-HA/TCR-HA

lung

BLN

sple

en

75,6%

63,4%

95,8%

BALB/c

94,3%

90,9%

94,9%12,0%

5,1%

2,7% 4,2%

36,6%

24,4%

5,7%

9,1%

5,1%SPC-HA

0,5 1,51,0 2,52,0fold change

2,271,0

SP-D

0,5 1,51,0 2,52,0fold change

2,381,0

SP-A

1,02,26

TGF-β

0,5 1,51,0 2,52,0fold change

PF-4

4,851,0

1 2 3 4 5 6fold change

D

E

SPC-HA/TCR-HA

SPC-HA

TGF-β

0,5 1,5 2,51,0 2,0

1

1,2

1,56

1,97

2,26SPC-HA/TCR-HA

SPC-HA 7 days post transfer

SPC-HA 4 days post transfer

SPC-HA 1 day post transfer

SPC-HA

A

C

Figure 6

CD4

Foxp

3

18,2% 8,7%

17,0% 19,0%

3,4% 3,9%

2,2% 56,4% 4,0%

IMD

M

SPC-H

ASPC

-HA

7 dayspost transfer

SPC-H

A/

TCR

-HA

+TGF-ß +TGF-β+α-TGF-β

+α-TGF-β

+α-TGF-β

+α-TGF-β

B

IMDM SPC-HA SPC-HA/TCR-HA

SPC-HA 7 dayspost transfer

5

10

15

20

25

perc

enta

geof

Fox

p3+

cells

[%] w/o α-TGF-β

+ α-TGF-β

antigen uptake

Dendritic cell

naÏve CD4+ T cell

effector CD4+ T cell

TGF-beta

IL-2/INF-gamma

PF-4

SP-D

SP-A

Tregs

alveolar type II epithelial cell

Figure 7