Genomic and Functional Delineation of Dendritic Cells and Memory T Cells Derived from Grass Pollen-Allergic Patients and Healthy Individuals

Genomic and functional delineation of dendritic cells and memory T cells derived from grass pollen-allergic patients and healthy individuals

Malin Lindstedt1*,A˚ sa Schio¨ tt1,2*, Astrid Bengtsson1,3, Kristina Larsson1, Magnus Korsgren3, Lennart Greiff4 and Carl A. K. Borrebaeck1 Downloaded from https://academic.oup.com/intimm/article/17/4/401/680426 by guest on 28 September 2021 1Department of Immunotechnology, Lund University, PO Box 7031, S-220 07 Lund, Sweden 2SIK, Swedish Institute for Food and Biotechnology, S-223 70 Lund, Sweden 3Department of Clinical Pharmacology and 4Department of Otorhinolaryngology, Lund University Hospital, S-221 85 Lund, Sweden

Keywords: allergen, gene expression proﬁling, hay fever, Phleum,Th2 cells

Abstract Dendritic cells (DCIs) possess a potent ability to modulate and activate specific T-cell responses to allergens, which play a pivotal role in allergic inflammation by secreting cytokines and other mediators. However, the molecular mechanisms by which allergen-challenged DCs regulate specific T-cell responses are still not well characterized. This study aims at elucidating the molecular mechanisms underlying the DC–T-cell interaction during an allergic immune response to grass pollen, using a genomic and functional approach. Transcriptional analysis was performed on grass allergen Phleum pratense-stimulated DCs and on autologous memory CD41 T cells co-cultured with allergen-challenged DCs from healthy and allergic donors. DCs from the allergic donors were potent inducers of T-cell proliferation and Th2 polarization, as demonstrated by high IL-4, IL-5 and IL-13, and low IFN-c production. A gradual up-regulation of activation markers on both DCs and T cells was evident during the co-culture period, demonstrating an educational element of the DC–T-cell interaction. The global transcriptional analysis revealed a differential gene regulation in DCs and T cells derived from allergic donors after stimulation with allergen, as compared with the healthy donors. Peripheral memory CD41 T cells from healthy and allergic donors also responded differently after stimulation with allergen-loaded DCs with respect to cytokine production, proliferation, surface marker expression and gene transcription. We found up-regulated genes involved in Th2 cell biology, such as genes important for homing, adhesion, signaling and transcription, in addition to genes previously not described in the context of allergy. The panel of differentially expressed genes in the allergic group will form the basis for an increased understanding of the molecular mechanisms in allergy.

Introduction Allergic rhinitis is one of the most common atopic diseases, phils, basophils and mast cells. The systemic immune clinically characterized by inflammation of the nasal mucosa response in atopic diseases, such as allergic rhinitis, is also and symptoms such as rhinorrhea, sneezing and blockage demonstrated by data showing that local exposure of allergen, (1). The immune response in subjects with allergic rhinitis as i.e. in the nose, may produce distant inflammatory changes, well as in other atopic diseases is characterized by circulating such as increased bronchial expression of adhesion mole- activated Tcells producing Th2 cytokines such as IL-4, IL-5, IL- cules, bronchial eosinophilia and increase of serum IL-5 levels 9 and IL-13 (1, 2). The secretion of these cytokines plays (3–5). a major role in the inflammatory response in rhinitis, by Dendritic cells (DCs) are professional regulators of the triggering production, recruitment and activation of eosino- immune response to inhaled antigen which rapidly travel from

*These authors contributed equally to the experimental work of this article. Correspondence to: C. A. K. Borrebaeck; E-mail: [email protected] Received 17 December 2004, accepted 12 January 2005 Transmitting editor: K. Takatsu Advance Access publication 3 March 2005 402 Transcript analysis of Phl p-pulsed DCs and T cells the circulation following allergen exposure to the regional dermatitis, nasal polyposis and infections. The healthy donors lymph nodes for initiation or amplification of the allergic were skin prick test negative and matched with the allergic immune response (6). Recently, the direct effect of DCs in Th2 group with regards to gender and age. The study was sensitization was shown in transfer experiments during which approved by the local Ethics Committee. systemically administered allergen-loaded DCs induced Th2 immunity upon a subsequent aeroallergen challenge (7, 8). Allergen-specific IgE measurements The unique capacity of DCs to initiate and regulate allergic Timothy-specific plasma IgE concentrations in human serum immune responses clearly demonstrates the need to in- were determined with Pharmacia CAP System (Pharmacia vestigate the gene expression in DCs exposed to allergens Diagnostics AB, Uppsala, Sweden) according to the manu- and their functional properties. facturer’s instruction. The detection limit of the assay is 0.35 Global transcriptional analysis is a superior tool for predict- kUA lÿ1. ing verifiable phenotypic characteristics important for the understanding of disease mechanisms and, thus, for identi-

Basophil degranulation assay Downloaded from https://academic.oup.com/intimm/article/17/4/401/680426 by guest on 28 September 2021 fication of potential novel drug targets. Recent studies have aimed at screening atopic and healthy subjects for differen- The BD FastImmune assay was performed according to the tially expressed transcripts and proteins in PBMC or biopsies manufacturer’s instructions (Becton Dickinson, San Jose, CA, (9, 10). However, PBMC as well as biopsies from nasal or USA). The statistical significance was detemined by Student’s bronchial airways consist of a complex mixture of cells, which t-test. makes comparative analyses very difficult. Furthermore, con- tribution from a specific cell type to the transcriptional Generation of monocyte-derived dendritic cells signature becomes impossible to delineate, and it is therefore Monocytes were purified from peripheral blood obtained desirable to first isolate the different cell types from blood or during pollen season and differentiated into monocyte-derived biopsies before characterization of their specific expression dendritic cells (MoDCs) using R5 culture medium [RPMI 1640, (11) (Lindstedt et al., unpublished data). A few studies have 2mML-glutamine (Sigma–Aldrich, St Louis, MO, USA), 5% used high-density microarrays to study the differential ex- fetal bovine serum (Hyclone Laboratories, Logan, UT, USA) ÿ1 pression of in vitro-generated Th1 and Th2 cells from humans and 50 lgml gentamicin] supplemented with recombinant (12) and mice (13). However, in this study we are taking a step human granulocyte macrophage colony-stimulating factor further, i.e. to monitor the changes in gene expression in (Novartis, Basel, Switzerland) and recombinant human IL-4 effector Tcells, following stimulation with allergen-pulsed DCs. (R&D Systems, Minneapolis, MN, USA) as previously de- This was performed without the addition of polarizing scribed (11). MoDCs were either stimulated with 25 lgmlÿ1 cytokines, utilizing cells from both healthy and atopic in- endotoxin-free Phl p extract, a generous gift from Peter Adler dividuals. In addition, we screened the transcriptional profiles Wu¨rtzen (ALK-Abello, Hørsholm, Denmark), for 48 h or left of allergen-stimulated DCs from healthy and allergic subjects unstimulated. Cell samples of DCs were collected for micro- to monitor their distinct response to the same antigens. array analysis at 48 h after grass pollen stimulation. In the present study, our aim was to identify molecular markers on DCs and T cells that are differentially expressed T-cell purification protocols between allergic and healthy donors. For the transcriptional + profiling, only donors who were either healthy or had ongoing CD4 T cells were purified by negative selection from allergic rhinitis, as defined by characteristic nasal symptoms peripheral whole blood obtained from autologous allergic and non-allergic donors 9 days after the initial blood sampling, in combination with positive skin prick test, high levels of + specific IgE, allergen-induced T 2 cytokine production, T-cell using the CD4 T-cell isolation MACS kit (Miltenyi Biotec, h Bergisch Gladbach, Germany). Memory CD27ÿCD4+ T cells proliferation, and basophil degranulation, were studied. We + could demonstrate a clear differential transcriptional profile were isolated by first incubating the CD4 cells with anti- in DCs from allergic individuals in response to allergen as CD45RA mAb, anti-glycophorin A mAb (DakoCytomation, compared with the healthy controls. Furthermore, allergen- Glostrup, Denmark) and microbead-conjugated anti-CD27 pulsed DCs induced differential gene expression in memory antibodies (Miltenyi Biotec) for 15 min at 4C. Thereafter, T cells derived from allergic and healthy donors. The present microbead-conjugated goat anti-mouse antibodies (Miltenyi data delineate genomic and functional differences in allergic Biotec) were added for 15 min at 4C to deplete naive cells and to enrich the CD27ÿ population. The isolated memory individuals in relation to mechanisms involved in DC–T-cell + + interactions and may act as a starting point for selecting CD4 T cells contained <1% contaminating CD45RA T cells. potential targets for therapy. Proliferation assay Methods Proliferation assays were performed with enriched CD27ÿCD4+ memory T cells by co-culturing with autologous Patient inclusion criteria DCs that were either unstimulated or had been pulsed with Phl Allergic donors participating in this project tested positive for p 48 h prior to the T-cell isolation. The assays were performed grass pollen allergen [Phleum pratense (Phl p)] in skin prick with a DC/T cell ratio of 1/20, using R5 culture medium, test. They also had a clinical history of strictly seasonal allergic supplemented with 2 3 10ÿ5 M 2-mercaptoethanol (Merck, rhinitis and displayed symptoms associated with seasonal Whitehouse, NJ, USA). After 7 days, 2.5 lCi mlÿ1 [3H]thymi- allergen exposure. Patient exclusion criteria were asthma, dine (Amersham Biosciences, Uppsala, Sweden) was added Transcript analysis of Phl p-pulsed DCs and T cells 403 to each well for 16 h. Thymidine incorporation was measured in Results a beta scintillation counter (Matrix 96 Direct beta counter, The allergic patient inclusion criteria were history and symp- Packard, Meriden, CT, USA). Samples were run in four toms of rhinitis of grass pollen exposure, positive skin prick test replicates and the statistical significance was detemined by and elevated levels of grass pollen-specific IgE in serum (Table Student’s t test. 1). Furthermore, to confirm that the allergic donors were

+ actually responding to the allergen, a basophil degranulation Cytokine production by memory CD4 T cells test was performed (Fig. 1A). This tool has proven to be highly T cells were co-cultured with autologous unstimulated or efﬁcient for detection of IgE-mediated allergy to inhalant allergen-stimulated DCs for 7 days. For intracellular cytokine allergens (14). Altogether, the differences between healthy detection, cells were incubated with phorbol myristate acetate and allergic donors, in terms of responsiveness to grass pollen (50 ng mlÿ1), ionomycin (500 ng mlÿ1) and brefeldin A (10 lg allergen, were highly signiﬁcant (P < 0.005). mlÿ1) (all from Sigma–Aldrich) for 5 h, washed twice in PBS Culturing blood monocytes from healthy and allergic donors

and fixed with 2% PFA for 15 min. Cells were permeabilized for 7 days with granulocyte macrophage colony-stimulating Downloaded from https://academic.oup.com/intimm/article/17/4/401/680426 by guest on 28 September 2021 with 0.5% saponin/PBS (Sigma–Aldrich) and stained with IL-4 factor and IL-4 resulted consistently in CD1a+, CD14ÿ, CD80ÿ PE (Becton Dickinson), IFN-c FITC and IL-13 PE antibodies and CD86ÿ immature DCs (data not shown). To investigate (Pharmingen, San Diego, CA, USA) for 30 min at 4C. whether Phl p could induce maturational reprograming of Thereafter, cells were washed twice in 0.5% saponin/PBS, these DCs, cells were stimulated with Phl p extract for 48 h and re-suspended in 1% BSA/PBS and analyzed with a FACScan analyzed phenotypically. However, no up-regulation of activa- (Becton Dickinson). For soluble cytokine detection, the human tion and co-stimulatory markers was detected. In addition, DC Th1/Th2 cytokine bead array kit was used, according to the supernatants were analyzed for the production of inflamma- manufacturer’s instruction (Becton Dickinson). tory cytokines, such as IL-1b, tumor necrosis factor-a (TNF-a), IL-6 and IL-12. Again, no detectable cytokine production was Preparation of cRNA and gene chip hybridization induced by the allergens (data not shown). Despite the fact Phl p-stimulated DCs and Tcells from allergic and non-allergic that no phenotypical changes were observed in DCs individuals were co-cultured (1/20) for 7 days as above. stimulated with allergen, samples were collected after 48 h During the last 5 h, Tcells were activated with plate bound anti- of culture in the presence of Phl p and snap frozen for global CD3 mAb (10 lgmlÿ1) (OKT3, Ortho Biotech, Raritan, NJ, transcriptional analysis. + USA) and soluble anti-CD28 mAb (0.5 lgmlÿ1) (Pharmingen). Autologous memory CD4 Tcells were co-cultured for 7 days Cell cultures from allergic donors A4–A6 and non-allergic with unstimulated and Phl p-stimulated DCs. The expression of donors H1, H3 and H7 were lyzed in TRIzol Reagent co-stimulatory markers CD80 and CD86 on Phl p-stimulated (Invitrogen, Paisley, UK) and stored at ÿ20C until further DCs was analyzed during the T-cell co-culture period, on days RNA isolation. Fragmentation, hybridization and scanning 0, 3, 5 and 7 (Fig. 1B). Of note, both unstimulated and Phl p- of the human U133A arrays were performed according to stimulated DCs derived from healthy and allergic individuals the manufacturer’s protocol (Affymetrix Inc., Santa Clara, up-regulated the CD80 and CD86 following the cognate T-cell CA, USA) and as previously described (11). For the DC-co- interaction. Since DCs from both healthy and allergic subjects cultured T cells, preparation of labeled cRNA was per- displayed a similar kinetics of activation, this clearly demon- formed according to the small-sample labeling protocol vII strated an antigen-independent activation of DCs, induced (Affymetrix Inc.). only by the presence of T cells in the culture. Even more intriguing, the gated CD3+ memory T cells Microarray data analysis evidently also up-regulated the co-stimulatory markers CD80 and CD86 as well as MHC class II molecules after DC contact The analysis was performed at the Swegene MicroArray (Fig. 1C). This CD80, CD86 and HLA-DR expression on Resource Center (MARC) at Lund University, which is an Affymetrix Service Provider. MARC is a Swedish reference Table 1. Donor parameters laboratory for DNA microarray analysis and is minimum information about a microarray experiment compliant. The Donor History Skin prick Scorea (0–6) Specific IgE fluorescence intensities were analyzed, using the Microarray test (kUA lÿ1) Suite Software 5.0 (Affymetrix Inc.) and scaled to a target value H1 ÿ 0 <0.35 of 100. Further data analysis was performed with Gene- H2 2 <0.35 TM ÿ Spring 5.0 software (Silicon Genetics, Redwood City, CA, H3 ÿ 0 <0.35 USA). The median of the signal intensity in three samples in H4 ÿ 0 <0.35 each group was calculated and thereafter, based on fold H5 ÿ 0 <0.35 change, genes were selected that were either up- or down- H6 ÿ 2 <0.35 H7 ÿ 0 <0.35 regulated in the allergic donors A4–A6 compared with the non- A1 Rhinitis + 2 1.16 allergic donors H1, H3 and H7. In the up-regulated list of A2 Rhinitis + 0.5 36.4 genes, the transcripts were denoted present (P) in A4–A6. A3 Rhinitis + 3 55.2 Converserly, the down-regulated transcripts were denoted A4 Rhinitis + 2.5 >100 A5 Rhinitis + 1 24.7 present (P) in H1, H3 and H7. Primary expression data were A6 Rhinitis + 4.5 12.8 submitted to the Array Express database (http://www.ebi. ac.uk/arrayexpress/). aSymptoms of rhinitis at time of blood sampling. 404 Transcript analysis of Phl p-pulsed DCs and T cells memory T cells was progressively up-regulated during the 7- donors and allergic donors. A4–A6 displayed a clear Th2profile day culture period. In this case, however, a clear difference in after stimulation, with the production of IL-4, IL-5 and IL-13. No T-cell response between the allergic and healthy donors was differences in IFN-c production could be detected between the observed. healthy and allergic group. DCs were also compared for their The capacity of allergen-stimulated DCs to regulate the ability to induce memory T-cell proliferation (Fig. 3). T cells cytokine production of autologous memory Tcells was explored stimulated with Phl p-pulsed DCs from the allergic donors with flow cytometry after 7 days of culture (Fig. 2). The displayed a significantly higher level (P < 0.05) of proliferation production of cytokines varied considerably between the as compared with T cells cultured with unstimulated DCs. Downloaded from https://academic.oup.com/intimm/article/17/4/401/680426 by guest on 28 September 2021

Fig. 1. Flow cytometric analysis of basophils, DCs and T cells after allergen challenge. (A) Allergic individuals displayed higher levels of surface CD63 expression, a measure of basophil degranulation, after allergen challenge, as compared with the healthy individuals. Gates were set to include viable HLA-DRÿ/CD123+ cells. Allergic donors are shown in gray bars while healthy donors are shown in black bars. (B) Allergen stimulation was not sufﬁcient to induce DC activation and, evidently, DCs required contact with autologous T cells for maturation. DCs from both healthy and allergic subjects were either left unstimulated (open diamond) or cultured with Phl p for 48 h (0 day) (closed square) prior to co-culture with CD27ÿ-enriched CD4+ memory T cells. The kinetics of up-regulation of CD80 and CD86 on DCs were assayed on days 0, 3, 5 and 7. (C) Net expression on memory T cells induced by allergen-loaded DCs from allergic (closed square) and healthy donors (open diamond), i.e. with expression induced by unstimulated DCs subtracted. Gates were set to include only viable CD3+ cells. The average values are represented in (B) and (C) of the healthy and allergic donors 6 standard deviations.

Fig. 2. IL-4, IL-5, IL-13 and IFN-c production by CD27ÿ-enriched memory CD4+ T cells after stimulation with allergen-loaded DCs. DCs were stimulated with grass pollen extract or medium alone for 48 h. Isolated memory T cells were thereafter co-cultured with DCs for 7 days and the intracellular cytokine production was analyzed by flow cytometry. Results represent net cytokine production induced by allergen-loaded DCs subtracted by the production induced by the unstimulated DCs. Atopic donors are shown in gray bars while healthy donors are shown in white bars. Transcript analysis of Phl p-pulsed DCs and T cells 405 To further dissect the molecular mechanisms involved in the DC/T cell response to allergen, high-density oligonucleotide arrays (with >12,500 transcripts) were used to produce a comprehensive picture of gene regulation following allergen stimulation in both DCs and memory T cells. Only the donors exhibiting either complete absence of allergic response or a strong ongoing response were chosen for transcriptional analysis. This selection was based on the overall results of the skin prick test, specific IgE levels, allergen-induced Th2 cytokine production, T-cell proliferation and basophil degranulation. Consequently, cells from the allergic donors A4– Fig. 3. Proliferation of CD27ÿ-enriched memory CD4+ T cells after A6, as well as the healthy donors H1, H3 and H7, were stimulation with allergen-loaded DCs. DCs were either left unstimu- selected for transcriptional analysis. The differential transcrip- lated or cultured with Phl p for 48 h prior to co-culture with memory T tional profiles of DCs challenged for 48 h with Phl p were cells for 7 days. Results shown represent the average of four replicate Downloaded from https://academic.oup.com/intimm/article/17/4/401/680426 by guest on 28 September 2021 compared between the above donors (Fig. 4A). In addition, we cultures 6 standard deviation, and the proliferation induced by unstimulated DCs are subtracted for each donor. The statistical monitored the gene regulation in response to Phl p-pulsed significance was detemined by Student’s t test (*P < 0.05, **P < DCs in the expanding memory T cells from the same 0.005, ***P < 0.0005). Atopic donors are shown in gray bars while individuals (Fig. 4B). A selection of the differentially expressed healthy donors are shown in white bars. genes is displayed in Fig. 4, functionally grouped according to their gene ontology. The global transcriptional analysis of Phl p- challenged DCs clearly revealed that grass pollen induced allergic individuals. These results reveal differential transcrip- differential transcript profiles in DCs from allergic as compared tional profiles relevant for both a basic understanding of the with healthy donors. Accordingly, many genes coding for allergic reaction as well as for the quest of finding novel adhesion and signaling molecules were differentially ex- therapeutic targets. pressed. For example, in DCs derived from allergic donors The lack of up-regulation of the co-stimulatory molecules TNFRSF11A (RANK), SPN (CD43), CDH2 and LAMC1 were CD80 and CD86 on DCs after Phl p stimulation was intriguing, up-regulated, whereas IL2RG, IL7R, IL8RB, CD209L and especially since both CD80 and CD86 were induced after CLEC1 were found to be down-regulated. Other examples of T-cell contact in both healthy and allergic individuals. Sur- genes differentially regulated were the up-regulated mem- prisingly little is still known about the role of DCs in human Th2 brane tetraspanin TM4SF2 and the down-regulated TM4SF7 effector choice and there is a great need for further in- and TM9SF4. The divergence of transcription in allergen- vestigations. Interestingly, it was recently shown in a mice pulsed DC/T cells in healthy and allergic individuals was also model that DCs can discriminate between Th1- and Th2- extensive and may explain many of the important molecular polarizing antigens even when presented simultaneously (17). features of the interaction between DCs and T cells in the Both antigens were internalized by discrete pathways and allergic response. Among signaling molecules and receptors entered non-overlapping cellular compartments. Furthermore, IL1A, IL1RAP, HRH4, MS4A2 (FCER1B) and FCGR2B were up- many maturation-associated changes in DCs were reportedly regulated in the allergic group, whereas IL1R2, IL7R, LTA, LTB, only seen with the Th1-inducing antigen, which is in agreement LTBP1, MRC1 and IGSF6 were down-regulated. It was evident with our finding that allergen stimulation did not induce any that well-established marker genes for a cellular Th2 response phenotypical changes in DCs. We observed that the allergen- or atopic disease, such as IL-9 (15), TNFRSF8 (CD30) (16) and stimulated DCs need the presence of T cells for their VEGF (9), were differentially expressed in healthy and allergic maturational process, which then could be mediated either donors as well as genes not previously described in indirectly by mediators or directly by a cognate interaction. the context of allergy, such as TNFRSF11B, BCL-3, INSR This phenomenon is likely to enable allergen-challenged DCs and CD1E. to maintain their activity levels within defined limits in tissues of To confirm the differences in cytokine production in the allergic inflammation, such as nasal and bronchial airways, healthy and allergic in vitro cell systems, we performed and postpones the maturational trigger until an effector T-cell cytometric bead array analysis of memory T-cell supernatants contact is established. 2 days after collecting samples for transcriptional profiling Our kinetic study also clearly showed that T cells became (Fig. 5). TNF-a was highly expressed in the healthy group, activated after stimulation with allergen-loaded DCs from whereas more IL-4 and IL-5 were produced in the allergic allergic donors. A gradual increase of CD86, CD80 and HLA- group, demonstrating an expected skewing of effector T-cell DR expression on T cells was evident. CD86 expression on T responses. cells has been shown in vitro to be induced by long-term CD3 and IL-2R stimulation (18) and down-regulated when the cells become quiescent. Such CD86+ T cells represent effector Discussion memory cells, which are functional in co-stimulating other We have studied factors of importance for the interaction resting Tcells into proliferation and cytokine production. These between DCs and memory T cells during an immune response results are intriguing since it indicates that T cells can relevant to allergic airway inflammation, such as in allergic themselves amplify and regulate their own responses through rhinitis and asthma. Furthermore, we analyzed the regulatory proteins mainly referred to as antigen-presenting cell markers. mechanisms of DCs in response to Phl p, in both healthy and The differences observed between allergic and healthy donors 406 Transcript analysis of Phl p-pulsed DCs and T cells Downloaded from https://academic.oup.com/intimm/article/17/4/401/680426 by guest on 28 September 2021 Transcript analysis of Phl p-pulsed DCs and T cells 407 in the present study demonstrate that an antigen-specific disease (19). Chemokines directly affect the retention and signal is required for the T cells to become CD80, CD86 and relocation of DCs and Tcells in the allergic inflammation. Thus, HLA-DR positive. a breakdown of the underlying control mechanisms of Experience from our previous investigations has demon- leukocyte migration might contribute to the atopic disease strated the importance of having clearly defined patient (20). Interestingly, in the present study, a wide range of parameters as well as purified cell populations for the chemokines were found to be differentially expressed in Phl p- transcriptional profiling. Consequently, using isolated cell challenged DC/T cells from allergic as compared with healthy populations, we could demonstrate that Phl p clearly induced individuals, suggesting a highly regulated migratory response differential transcriptional profiles in both DCs and memory in the two panels of donors. In the allergic donors, the T cells from allergic and healthy donors (Fig. 6). Many chemokines CCL17 (TARC) and CCL22 (MDC), active on + transcriptional changes associated with functions in the CCR4 Th2 cells, were both expressed, as well as CCL2 (MCP- allergic immune reaction were identified, in addition to novel 1), which plays an important role in the activation of mast molecular mechanisms. cells and induction of histidine decarboxylase mRNAs (21). The presence of specialized T cells at the site of in- Interestingly, the allergic donors also displayed 2.5-fold Downloaded from https://academic.oup.com/intimm/article/17/4/401/680426 by guest on 28 September 2021 flammation is associated with the pathology of allergic airway increased transcript levels of histidine decarboxylase, as compared with the healthy group (data not shown). CCL17 and CCL22 levels have also been demonstrated to increase in bronchoalveolar lavage after allergen challenge in patients with allergic pulmonary inflammation (22), and our data suggest that memory Th2 cells and DCs were the main sources of chemokines in that study. In addition, CXCR4 mRNA, a chemotactic component involved in allergic airway disease (23), was detected in the allergic individuals, suggesting a switch in highly specific migratory properties. Fig. 5. Production of soluble TNF-a, IL-4 and IL-5 by memory T cells Furthermore, a diverse set of chemokines were down- after stimulation with Phl p-loaded DCs. Supernatants were harvested regulated in the allergic group, among others CXCL13 (BLC) 48 h after collecting samples for microarray analysis. White bars and CCL18 (DC-CK1), which have previously been shown to correspond to cytokine levels in the healthy donors, whereas gray bars correspond to atopic donors. Results shown represent average be down-regulated after CD40–CD40 ligand interaction (24, cytokine levels (in ng) in cell culture supernatants 6 standard 25), and XCL1 (lymphotactin), which is expressed in activated deviation. Th1 cells (26).

Fig. 6. Potential mechanisms involved in the speciﬁc memory T cell response induced by allergen-triggered DCs. Gene names in red text represent up-regulated genes in the atopic donors, whereas the genes in green are down-regulated.

Fig. 4. Differential transcript profiles of Phl p-stimulated DCs (A) and their effect on transcription in autologous CD4+ memory T cells (B), from allergic (A4–A6) and healthy donors (H1, H3, H7). Genes were considered differentially expressed if a change of at least 1.8 fold-change in expression level was observed and the raw values were above 10 in signal intensity. Also, all genes had to be denoted as present in the triplicate samples. The level of flourescent intensity is indicated by color; high signal (>500) is yellow, medium signal (100–500) is red and low signal (<100) is blue. GenBank accession numbers as well as short gene name are indicated and positive fold change represents higher signal intensity in the allergic group, as compared with the healthy group. Based on information from the Gene OntologyTM Consortium, genes are grouped according to their known functions: (i) adhesion, migration, cell surface, (ii) miscellaneous, (iii) signaling and transcriptional regulation, (iv) apoptosis and cell cycle, (v) transport and (vi) cytokines, cytokine receptors and growth factors. 408 Transcript analysis of Phl p-pulsed DCs and T cells Two cytokines expressed in inflammatory airway diseases, recognition and initiation of the allergic immune response is IL-17 (27) and leukemia inhibitory factor (28), were up- mediated by DCs (6) and in the present study we report the regulated in the DC-stimulated T cells of the present allergic presence of specific gene regulation in Phl p-pulsed DCs donors. Furthermore, IL-9 and BCL-3 were both up-regulated derived from atopic donors. We have also followed the impact in the DC/T cells of these subjects, as compared with the of these DCs on gene transcription in memory T cells, re- healthy donors. IL-9 has been shown to regulate BCL-3 flecting the first recognition of allergens in tissues and the expression, and thus nuclear factor-jB (NF-kB) activity, in subsequent process of T-cell activation in tissues of allergic activated Tcells and mast cells and represses TNF-dependent rhinitis, respectively. The data demonstrate a complexed im- transcriptional activation, suggesting different regulatory munoregulatory network of potential gene products, com- pathways for the NF-kB transcription factors (29). Interestingly, prising both known as well as previously not described we observed reduced TNF-a in the DC/T cell co-culture components, which will pave the way for further functional supernatants derived from the allergic donors, as compared studies on the molecular mechanisms of allergy. with the healthy donors. Adenosine as well as adenosine metabolites and their Downloaded from https://academic.oup.com/intimm/article/17/4/401/680426 by guest on 28 September 2021 receptors have powerful immunoregulatory effects and evi- Acknowledgements dence is accumulating on their role as potent features of We would like to thank Ann-Charlotte Olsson and Charlotte Cervin- allergic inflammation. The allergen-challenged DC/T cells Hoberg for expert laboratory assistance. This work was supported by displayed up-regulated mRNA levels of two of these receptors, grants from the European Commission and National Science Council. namely adenosine A2b receptor (ADORA2B) and purinergic receptor P2X (P2RX5). Recently, an adenosine–IL-13 amplification pathway was described in IL-13 transgenic mice, Abbreviations defining a clear role for adenosine in Th2-mediated inflamma- DC dendritic cells tion in respiratory tissues (30). The transcription of ADORA2B MARC MicroArray Resource Center MoDC monocyte-derived dendritic cells in the allergic setting of this study suggests that the human DC/ NF-kB nuclear factor-kB T cells stimulated with allergen contribute to the response Phl p Phleum pratense induced by adenosine. In addition, signaling through ATP TNF tumor necrosis factor receptors such as P2RX5, in parallel to LPS-induced DC maturation, results in the inhibition of IL-6, IL-12, TNF-a and IL- References 1b production and thus lower IFN-c and higher IL-4 and IL-5 production by DC-co-cultured Tcells (31), in addition to strong 1 Borish, L. 2003. Allergic rhinitis: systemic inflammation and implications for management. J. Allergy Clin. Immunol. 112:1021. up-regulation of CXCR4 and CCL22 production (32). 2 Togias, A. 2004. Systemic effects of local allergic disease. J. Inhibition of the endo/lysosomal enzyme cathepsin L in Allergy Clin. Immunol. 113:S8. Leishmania major-infected BALB/c mice potentiates Th2 3 Braunstahl, G. J., Overbeek, S. E., Fokkens, W. J. et al. 2001. responses with up-regulated IL-4 production (33). Our find- Segmental bronchoprovocation in allergic rhinitis patients affects ings of CTSL (cathepsin L) down-regulation in allergic donors mast cell and basophil numbers in nasal and bronchial mucosa. Am. J. Respir. Crit. Care Med. 164:858. suggest that cathepsin L has a function in the degradation of 4 Braunstahl, G. J., Overbeek, S. E., Kleinjan, A., Prins, J. B., endocytosed antigens leading toward a Th1-type instead of Hoogsteden, H. C. and Fokkens, W. J. 2001. Nasal allergen aTh2-type response. Similarly, up-regulation of LAMP1 and provocation induces adhesion molecule expression and tissue LAPTM4B, mRNAs encoding for two lysosomal proteins, in eosinophilia in upper and lower airways. J. Allergy Clin. Immunol. 107:469. DCs stimulated with Phl p in allergic donors suggests that they 5 Greiff, L., Andersson, M., Svensson, C., Linden, M., Wollmer, P. and respond differently in terms of endocytosis and processing of Persson, C. G. 1999. Demonstration of bronchial eosinophil activity the same antigen. in seasonal allergic rhinitis by induced plasma exudation Of note, it was evident that markers expressed by other cells combined with induced sputum. Thorax 54:33. involved in the allergic reactions were also present in our DC/T 6 Lambrecht, B. N. and Hammad, H. 2003. Taking our breath away: dendritic cells in the pathogenesis of asthma. Nat. Rev. Immunol. cells, such as prostaglandin receptor EP3 (PTGER3) involved 3:994. in IgE/allergen-induced degranulation in mast cells (34) and 7 Graffi, S. J., Dekan, G., Stingl, G. and Epstein, M. M. 2002. transforming growth factor-a, induced by IL-4, IL-13 and Systemic administration of antigen-pulsed dendritic cells induces allergen Der p in bronchial epithelial cells (35). This suggests experimental allergic asthma in mice upon aerosol antigen rechallenge. Clin. Immunol. 103:176. that DCs and T cells may be more involved in other processes 8 Hammad, H., Lambrecht, B. N., Pochard, P. et al. 2002. Monocyte- of allergic inflammation than previously acknowledged. derived dendritic cells induce a house dust mite-specific Th2 In summary, we have studied allergen-pulsed DCs, derived allergic inflammation in the lung of humanized SCID mice: involvement of CCR7. J. Immunol. 169:1524. from allergic and healthy donors, and their interaction with Th cells, without the addition of exogenous lymphokines. This 9 Benson, M., Carlsson, B., Carlsson, L. M., Wennergren, G. and Cardell, L. O. 2002. Increased expression of vascular endothelial probably more closely mirrors an allergic immune response, growth factor-A in seasonal allergic rhinitis. Cytokine 20:268. as compared with T-cell polarization in vitro studied under 10 Heishi, M., Kagaya, S., Katsunuma, T. et al. 2002. High-density the influence of e.g. IL-4 and IL-12. The rational is that al- oligonucleotide array analysis of mRNA transcripts in peripheral lergic rhinitis is associated with various local and systemic blood cells of severe atopic dermatitis patients. Int. Arch. Allergy Immunol. 129:57. inflammatory processes and that several cell types, such as 11 Lindstedt, M., Johansson-Lindbom, B. and Borrebaeck, C. A. 2002. airway epithelial cells, mast cells, basophils, eosinophils and Global reprogramming of dendritic cells in response to a concerted infiltrating mononuclear cells, are involved (36). However, the action of inflammatory mediators. Int. Immunol. 14:1203. Transcript analysis of Phl p-pulsed DCs and T cells 409 12 Rogge, L., Bianchi, E., Biffi, M. et al. 2000. Transcript imaging of the 24 Vulcano, M., Struyf, S., Scapini, P. et al. 2003. Unique regulation of development of human T helper cells using oligonucleotide arrays. CCL18 production by maturing dendritic cells. J. Immunol. 170: Nat. Genet. 25:96. 3843. 13 Chtanova, T., Kemp, R. A., Sutherland, A. P., Ronchese, F. and 25 Vissers, J. L., Hartgers, F. C., Lindhout, E., Figdor, C. G. and Mackay, C. R. 2001. Gene microarrays reveal extensive differential Adema, G. J. 2001. BLC (CXCL13) is expressed by different gene expression in both CD4(+) and CD8(+) type 1 and type 2 dendritic cell subsets in vitro and in vivo. Eur. J. Immunol. 31:1544. T cells. J. Immunol. 167:3057. 26 Nagai, S., Hashimoto, S., Yamashita, T. et al. 2001. Comprehensive 14 Sanz, M. L., Sanchez, G., Gamboa, P. M. et al. 2001. Allergen- gene expression profile of human activated T(h)1- and T(h)2- induced basophil activation: CD63 cell expression detected by polarized cells. Int. Immunol. 13:367. flow cytometry in patients allergic to Dermatophagoides ptero- 27 Laan, M., Palmberg, L., Larsson, K. and Linden, A. 2002. Free, nyssinus and Lolium perenne. Clin. Exp. Allergy 31:1007. soluble interleukin-17 protein during severe inflammation in human 15 Erpenbeck, V. J., Hohlfeld, J. M., Volkmann, B. et al. 2003. airways. Eur. Respir. J. 19:534. Segmental allergen challenge in patients with atopic asthma leads 28 Knight, D. 2001. Leukaemia inhibitory factor (LIF): a cytokine of to increased IL-9 expression in bronchoalveolar lavage fluid emerging importance in chronic airway inflammation. Pulm. lymphocytes. J. Allergy Clin. Immunol. 111:1319. Pharmacol. Ther. 14:169. 16 Del Prete, G., De Carli, M., D’Elios, M. M. et al. 1995. CD30- 29 Richard, M., Louahed, J., Demoulin, J. B. and Renauld, J. C. 1999. Downloaded from https://academic.oup.com/intimm/article/17/4/401/680426 by guest on 28 September 2021 mediated signaling promotes the development of human T helper Interleukin-9 regulates NF-kappaB activity through BCL3 gene type 2-like T cells. J. Exp. Med. 182:1655. induction. Blood 93:4318. 17 Cervi, L., MacDonald, A. S., Kane, C., Dzierszinski, F. and Pearce, 30 Blackburn, M. R., Lee, C. G., Young, H. W. et al. 2003. Adenosine E. J. 2004. Cutting edge: dendritic cells copulsed with microbial mediates IL-13-induced inflammation and remodeling in the lung and helminth antigens undergo modified maturation, segregate and interacts in an IL-13-adenosine amplification pathway. J. Clin. the antigens to distinct intracellular compartments, and concur- Investig. 112:332. rently induce microbe-specific Th1 and helminth-specific th2 31 la Sala, A., Ferrari, D., Corinti, S., Cavani, A., Di Virgilio, F. and responses. J. Immunol. 172:2016. Girolomoni, G. 2001. Extracellular ATP induces a distorted 18 Jeannin, P., Herbault, N., Delneste, Y. et al. 1999. Human effector maturation of dendritic cells and inhibits their capacity to initiate memory T cells express CD86: a functional role in naive T cell Th1 responses. J. Immunol. 166:1611. priming. J. Immunol. 162:2044. 32 la Sala, A., Sebastiani, S., Ferrari, D. et al. 2002. Dendritic cells 19 Larche, M., Robinson, D. S. and Kay, A. B. 2003. The role of exposed to extracellular adenosine triphosphate acquire the T lymphocytes in the pathogenesis of asthma. J. Allergy Clin. migratory properties of mature cells and show a reduced capacity Immunol. 111:450; quiz 464. to attract type 1 T lymphocytes. Blood 99:1715. 20 Moser, B., Wolf, M., Walz, A. and Loetscher, P. 2004. Chemokines: 33 Zhang, T., Maekawa, Y., Sakai, T. et al. 2001. Treatment with multiple levels of leukocyte migration control. Trends Immunol. cathepsin L inhibitor potentiates Th2-type immune response in 25:75. Leishmania major-infected BALB/c mice. Int. Immunol. 13:975. 21 Conti, P. and DiGioacchino, M. 2001. MCP-1 and RANTES are 34 Gomi, K., Zhu, F. G. and Marshall, J. S. 2000. Prostaglandin E2 mediators of acute and chronic inflammation. Allergy Asthma Proc. selectively enhances the IgE-mediated production of IL-6 and 22:133. granulocyte-macrophage colony-stimulating factor by mast cells 22 Bochner, B. S., Hudson, S. A., Xiao, H. Q. and Liu, M. C. 2003. through an EP1/EP3-dependent mechanism. J. Immunol. 165: Release of both CCR4-active and CXCR3-active chemokines 6545. during human allergic pulmonary late-phase reactions. J. Allergy 35 Lordan, J. L., Bucchieri, F., Richter, A. et al. 2002. Cooperative Clin. Immunol. 112:930. effects of Th2 cytokines and allergen on normal and asthmatic 23 Gonzalo, J. A., Lloyd, C. M., Peled, A., Delaney, T., Coyle, A. J. and bronchial epithelial cells. J. Immunol. 169:407. Gutierrez-Ramos, J. C. 2000. Critical involvement of the chemotac- 36 Howarth, P. H., Salagean, M. and Dokic, D. 2000. Allergic tic axis CXCR4/stromal cell-derived factor-1 alpha in the inflamma- rhinitis: not purely a histamine-related disease. Allergy 55 tory component of allergic airway disease. J. Immunol. 165:499. (Suppl. 64) :7.