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Human Blood and Tonsil Plasmacytoid Dendritic Cells Display Similar Gene Expression Profiles but Exhibit Differential Type I IFN Responses to Influenza A Virus This information is current as Infection of September 28, 2021. Sindhu Vangeti, Jens Gertow, Meng Yu, Sang Liu, Faezzah Baharom, Saskia Scholz, Danielle Friberg, Magnus Starkhammar, Alexander Ahlberg and Anna Smed-Sörensen J Immunol published online 13 February 2019 Downloaded from http://www.jimmunol.org/content/early/2019/02/12/jimmun ol.1801191 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2019/02/12/jimmunol.180119 Material 1.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 13, 2019, doi:10.4049/jimmunol.1801191 The Journal of Immunology

Human Blood and Tonsil Plasmacytoid Dendritic Cells Display Similar Gene Expression Proﬁles but Exhibit Differential Type I IFN Responses to Inﬂuenza A Virus Infection

Sindhu Vangeti,* Jens Gertow,* Meng Yu,* Sang Liu,* Faezzah Baharom,* Saskia Scholz,* Danielle Friberg,† Magnus Starkhammar,‡ Alexander Ahlberg,x and Anna Smed-So¨rensen*

Inﬂuenza A virus (IAV) infection constitutes an annual health burden across the globe. Plasmacytoid dendritic cells (PDCs) are

central in antiviral defense because of their superior capacity to produce type I IFNs in response to viruses. Dendritic cells Downloaded from (DCs) differ depending on their anatomical location. However, only limited host-pathogen data are available from the initial site of infection in humans. In this study, we investigated how human tonsil PDCs, likely exposed to virus because of their location, responded to IAV infection compared with peripheral blood PDCs. In tonsils, unlike in blood, PDCs are the most frequent DC subset. Both tonsil and blood PDCs expressed several genes necessary for pathogen recognition and immune response, generally in a similar pattern. MxA, a protein that renders cells resistant to IAV infection, was detected in both tonsil and blood PDCs. How-

ever, despite steady-state MxA expression and contrary to previous reports, at high IAVconcentrations (typically cytopathic to other http://www.jimmunol.org/ immune cells), both tonsil and blood PDCs supported IAV infection. IAVexposure resulted in PDC maturation by upregulation of CD86 expression and IFN-a secretion. Interestingly, blood PDCs secreted 10-fold more IFN-a in response to IAV compared with tonsil PDCs. Tonsil PDCs also had a dampened cytokine response to puriﬁed TLR ligands compared with blood PDCs. Our ﬁndings suggest that tonsil PDCs may be less responsive to IAV than blood PDCs, highlighting the importance of studying immune cells at their proposed site of function. The Journal of Immunology, 2019, 202: 000–000.

cute respiratory infections are the fourth leading cause public health problem. Influenza A viruses (IAV) cause seasonal of deaths worldwide, accounting for over 3.9 million infections estimated to account for 500,000 deaths annually and by guest on September 28, 2021 A deaths annually (1, 2). In addition, respiratory infections predominantly affect infants, elderly, and immunocompromised are associated with significant morbidity, making them a marked individuals. In addition, the segmented genome of influenza viruses potentiates reassortment to generate highly patho- genic strains that can result in pandemics with high mortality. *Division of Immunology and Allergy, Department of Medicine Solna, Karolinska IAV is transmitted via inhalation of droplets containing virus, † Institutet, 171 64 Stockholm, Sweden; Department of Surgical Sciences, Uppsala aerosolized virus, and via contact with infected individuals (3). University, 751 85 Uppsala, Sweden; ‡Capio Ear, Nose and Throat Clinic Globen, 121 77 Johanneshov, Sweden; and xDivision of Ear, Nose and Throat Diseases, Following infection of the respiratory epithelium in the upper air- Department of Clinical Science, Intervention and Technology, Karolinska University ways, the lytic virus replicates rapidly and spreads to surrounding Hospital Huddinge, Huddinge, 141 86 Stockholm, Sweden cells. The innate immune system orchestrates the early events fol- ORCIDs: 0000-0003-3404-6878 (S.V.); 0000-0002-5663-7209 (J.G.); 0000-0002- lowing infection by releasing inflammatory mediators and chemo- 9798-6624 (M.Y.); 0000-0001-5554-912X (A.A.); 0000-0001-6966-7039 (A.S.-S.). kines to enhance cell recruitment to the site of infection, as well as Received for publication August 27, 2018. Accepted for publication January 25, 2019. by releasing antiviral molecules to protect surrounding healthy cells (4). Recognition of single-stranded viral RNA by intracellular This work was supported by grants (to A.S.-S.) from the Swedish Research Council (Vetenskapsradet),˚ the Swedish Heart-Lung Foundation, the Swedish Childhood pathogen recognition receptors triggers expression of type I IFNs, Cancer Fund, and Karolinska Institutet. which in turn induces expression of IFN-susceptible genes (ISGs), S.V., J.G., M.Y., S.L., F.B., and S.S. performed experiments. A.A., M.S., and D.F. which are important in antiviral defense (5–7). Central to bridging performed tonsillectomies and collected clinical data. S.V., J.G., S.L., and A.S.-S. the innate and adaptive arms of the immune system are dendritic analyzed data and prepared figures. S.V. and A.S.-S. designed the study and wrote the manuscript. All coauthors edited the manuscript. cells (DCs) (8, 9). Address correspondence and reprint requests to Dr. Anna Smed-So¨rensen, Division DCs are innate immune cells and professional APCs with the of Immunology and Allergy, Department of Medicine Solna, Karolinska Institutet, unique capacity to activate naive T cells and thereby initiate Karolinska University Hospital Solna, BioClinicum J7:30, Visionsgatan 4, 171 64 pathogen-specific adaptive immune responses, which are typically Stockholm, Sweden. E-mail address: [email protected] required to control and clear viral infections. The human immune The online version of this article contains supplemental material. system has two major groups of DCs, CD11c+ myeloid DCs Abbreviations used in this article: DC, dendritic cell; IAV, influenza A virus; ISG, + IFN-susceptible gene; MDC, myeloid DC; MOI, multiplicity of infection; NP, nu- (MDCs) and CD123 plasmacytoid DCs (PDCs), that have distinct cleoprotein; PDC, plasmacytoid DC; PRR, pattern recognition receptor; R10, RPMI characteristics with several overlapping functions (10–12). Over- 1640 medium supplemented with 10% FCS, 5 mM L-glutamine, and 100 U/ml all, MDCs, which can be further subdivided into CD141+ MDCs penicillin and streptomycin; TMC, tonsil mononuclear cell. (also known as cDC1) and CD1c+ MDCs (also known as cDC2), Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 are the more prevalent DCs and are found in blood and throughout

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801191 2 IAV SUSCEPTIBILITY OF HUMAN TONSIL AND BLOOD PDC the body lining the skin and mucosal surfaces (13). In contrast, assessed using Trypan Blue (Sigma-Aldrich) exclusion and manual PDCs are normally only found in blood and lymphoid tissue under counting using a light microscope or using an automated Countess cell steady-state conditions but are rapidly recruited to sites of infec- counter (Invitrogen). tion or inflammation. The vast majority of our knowledge on human DC enrichment DCs comes from work on human peripheral blood. However, it PDCs were enriched from tonsils and blood using the Diamond Plasmacytoid is becoming increasingly clear that the function of DCs and Dendritic Cell Isolation Kit II (Miltenyi Biotec). Briefly, tonsil mononuclear many immune cells differs depending on their anatomical location cells (TMCs) or PBMCs were labeled with a mixture of biotin-conjugated Abs (14–21), which highlights the importance of studying immune against lineage markers (CD3, CD14, CD16, CD19, CD20, and CD66), cells at the site where they exert their function. myeloid markers (CD13 and CD33), and Fc receptors (CD16, CD64, and ε PDCs have Ag-presenting functions (22–25) but are mainly Fc RI). The labeled non-PDCs were depleted with anti-biotin Micro Beads on magnetic columns, and enriched PDCs were collected from the eluate. known for their superior capacity to produce type I IFNs and can Isolated PDCs were counted and assessed for viability as well as purity by produce 100–1000-fold more type I IFNs in response to viruses flow cytometry. Highly pure (.95%), viable (.85%) PDCs were used for all than other cells (24, 26–28). Blood PDCs have been shown to be experiments. PDCs were cultured at 1 3 106 cells/ml in R10 supplemented resistant to IAV infection under experimental conditions in which with 1 ng/ml IL-3 (R&D Systems) and incubated at 37˚C and 5% CO2. CD1c+ MDCs were isolated from blood using the CD1c (BDCA-1)+ Den- MDCs are readily susceptible (29–32). However, IAV infection is dritic Cell Isolation Kit (Miltenyi Biotec). typically restricted to the airways and lungs (33, 34), and it is unclear whether PDCs residing in tissue that is exposed to virus Infection of PDCs with IAV during human IAV infection behave similarly to blood PDCs. To IAV strain X-31 (from Influenza A/Aichi/2/68; H3N2) was propa- address this, we investigated PDCs from human palatine tonsils. gated in chicken eggs, purified, and concentrated on sucrose gradi- Downloaded from Tonsils are a rich source of tissue-resident DCs (35, 36) and a ents (Virapur). The 50% tissue culture-infective dose was determined comparatively accessible sampling site, making them a good by monitoring cytopathic effects on monolayers of MDCK cells in the presence of trypsin. The original virus stock (3 3 109 infectious source for studies of respiratory DCs, which are likely among units/ml) was diluted to obtain a multiplicity of infection (MOI) ranging the first APCs exposed to IAV (25, 37). from 0.6 to 6.0 (i.e., 600,000–6,000,000 infectious particles of IAV per In this study, we investigated how PDCs from human tonsils 1,000,000 PDCs), and infection was carried out in the absence of trypsin. As a replication control, 20 mM NH Cl (Sigma-Aldrich), which blocks responded to IAV infection compared with PDCs isolated from 4 http://www.jimmunol.org/ blood. We found that both tonsil and blood PDCs were susceptible endosomal acidification and thereby viral fusion, was added before addition of IAV. to IAV infection. However, viral protein production was only detectable after PDCs were exposed to virus concentrations that are Stimulation with TLR ligands typically cytopathic to other immune cells. The requirement for Purified PDCs were stimulated with 1 ng/ml CpG class C oligonucleotide high virus doses is likely related to the constitutive expression of (ODN 2395; InvivoGen) or 3M-019 (7/8L; gift from Dr. R. Seder, National MxA in both tonsil and blood PDCs, a protein that renders cells Institutes of Health) for 18 h in R10 supplemented with 1 ng/ml IL-3 largely resistant to IAV infection. Although both blood and tonsil (R&D Systems) and incubated at 37˚C and 5% CO2. TLR concentra- PDCs matured and secreted large quantities of IFN-a in response tions were optimized for maturation without loss of viability. to IAV infection, we found that blood PDCs produced 10-fold Flow cytometry analysis by guest on September 28, 2021 more IFN-a than tonsil PDCs. Blood PDCs were also superior at Phenotypic analysis was performed on TMCs and PBMCs using Live/Dead producing IFN-a,TNF-a, and IL-6 in response to purified TLR Blue; lineage markers CD3 (SP34-2 and SK7; BD Biosciences), CD20 ligands as compared with tonsil PDCs. This suggests that tonsil (L27; BD), CD56 (HCD56; BD Biosciences), HLA-DR (TU36; Life PDCs may be less responsive than PDCs isolated from blood, Technologies), CD14 (M5E2; BD Biosciences), CD16 (3GE; BioLegend), which in turn may affect the local immune response to IAV. CD11c (B-Ly6; BD Biosciences), CD1c (AD5-8E7; Miltenyi Biotec), CD141 (AD5-14H12; Miltenyi Biotec), Clec9a (8F9; Miltenyi Biotec), CD123 (7G3; BD Biosciences), and CD303 (AC144; Miltenyi Biotec); and Materials and Methods maturation marker CD86 (2331; BD Biosciences). Cells were fixed with Ethics statement 1% paraformaldehyde for flow cytometry. In infection experiments, intracellular staining was performed on surface-stained PDCs after fixation Informed consent was obtained from all volunteers following verbal and and permeabilization using a staining buffer set (eBioscience) and staining written information. The study was approved by the local Ethical Review with an anti–nucleoprotein (NP) mAb (431; Abcam). Samples were ac- Board at Karolinska Institutet (Stockholm, Sweden) and performed quired on an LSRFortessa flow cytometer (BD Biosciences) and analyzed according to the Declaration of Helsinki. using FlowJo software (Tree Star). Subjects and sample materials ELISA We obtained palatine tonsils from 27 patients undergoing routine tonsil- Tonsil and blood PDCs were suspended in R10 supplemented with IL-3 at a lectomies to treat recurrent tonsillitis, tonsillar hypertrophy, and/or ob- density of 1 3 106 cells/ml and exposed to IAV. Culture supernatants were structive sleep apnea. Informed consent was obtained from all patients. collected at different time points and frozen. IFN-a ELISA was performed Patients were between 2 and 68 y of age. Tonsils were transported from using standard kit instructions with the commercially available Human surgery at the Karolinska University Hospital Huddinge and Capio Ear, IFN-a All Subtype ELISA kit (PBL Assay Science). TNF-a and IL-6 Nose and Throat Clinic in Stockholm, Sweden, at room temperature in ELISA were performed using commercially available DuoSet develop- complete medium: RPMI 1640 (Sigma-Aldrich) medium supplemented ment kits (R&D Systems). with 10% FCS, 5 mM L-glutamine, and 100 U/ml penicillin and streptomycin (all Invitrogen) (R10). Buffy coats were obtained from healthy Western blot blood donors at the Karolinska University Hospital blood bank. Tonsil and blood PDC lysates were collected after 24 h of stimulation Isolation of mononuclear cells from tonsils and blood in RIPA lysis and extraction buffer (Sigma-Aldrich), separated by SDS-PAGE, and transferred to a PVDF membrane (GE Healthcare) using Tonsils were washed with R10, cleaned, and rid of charred/cauterized standard protocols. Immunoblotting was performed using primary Abs sections. The tissue was then mechanically disrupted and filtered sequen- against MxA (Genentech) and the housekeeping gene GAPDH (Santa tially through 100- and 70- mm nylon mesh filters multiple times to obtain a Cruz Biotechnology). Immunoreactive bands were detected using HRP- single-cell suspension. Mononuclear cells were obtained from tonsil conjugated IgG (Thermo Fisher Scientific). Western blots were developed suspensions and buffy coats by density gradient centrifugation us- using ECL reagents (GE Healthcare), visualized on a Bio-Rad ChemiDoc ing Ficoll-Paque Plus (GE Healthcare) after centrifugation at 900 3 g at XRS+ imaging system, and quantified using the inbuilt Image Lab room temperature for 25 min as previously described (7, 38). Viability was Touch software. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. PDCs are the most frequent DC subset in human tonsils. (A and B) DCs were identified from tonsils (A) or blood (B) using flow cytometry. Among live, HLA-DR+, lineage (CD3/CD20/CD56)2 cells that do not express CD14 or CD16, DCs were identified as CD11c+ MDCs or CD123+ PDCs (white). CD11c+ MDCs were further subdivided into CD1c+ MDCs (gray) or CD141+ MDCs (black). Contour plots show one representative donor of n =27 (tonsil) and n = 12 (blood). (C and D) Pie charts show the relative distribution of the three DC subsets among the DCs identified in tonsils (n = 27) and blood (n = 12). (E) The graph shows the frequency of CD141+ MDCs, CD1c+ MDCs, and PDCs in tonsils (circles) and blood (squares) among live, lineage2, HLA-DR+ cells from individual donors. Lines indicate median frequency of each subset. The Kruskal–Wallis test was used to assess differences in the frequency of DC subsets found in tonsil versus blood, with the Dunn multiple comparisons test. Differences were considered statistically significant at p , 0.05 or NS at p $ 0.05. *p , 0.05, ****p , 0.0001.

TaqMan low-density array analysis Statistical analysis Total RNA was extracted from pure populations of MACS-sorted tonsil Data were analyzed using GraphPad Prism version 6.0 (GraphPad Soft- and blood PDCs using the RNeasy Mini Kit (QIAGEN) according to the ware). Statistical significance was assessed using nonparametric tests. manufacturer’s instructions. RNA quantity and quality were assessed Differences between tonsil and blood PDCs were assessed using the Mann– using an RNA 6000 Pico chip on the 2100 Bioanalyzer (Agilent Whitney U test (at 95% confidence intervals) or one-way ANOVAwith the Technologies). cDNA was synthesized using a high-capacity cDNA Kruskal–Wallis test and the Dunn test to correct for multiple comparisons. reverse transcription kit (Applied Biosystems). Forty-eight TaqMan For comparisons between exposure conditions, two-way ANOVA with gene expression assays (Supplemental Table I) were preconfigured on a the Sidak multiple comparisons test was applied. Other tests were per- 384-well format microfluidic card (Applied Biosystems). The quanti- formed as indicated. Data were considered significant at p , 0.05. tative RT-PCR amplifications were performed using a TaqMan PreAmp Master Mix Kit and TaqMan Gene Expression Master Mix (both Ap- plied Biosystems) according to the manufacturer’s instructions on the Results QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). PDCs are the most frequent DC subset in human tonsils Relative gene quantities were obtained using the comparative cycle thresholdmethodafternormalizationtotheHPRT1geneasanen- Recent studies have made it evident that immune cells, including dogenous control. DCs, vary depending on anatomical location (11, 14–16, 39). IAV 4 IAV SUSCEPTIBILITY OF HUMAN TONSIL AND BLOOD PDC infection is initiated in the upper respiratory tract, which prompted the most pronounced difference in the distribution of DC subsets us to study DCs in the tonsils, a relatively accessible tissue in the between the two compartments was the relative abundance of human airways that is exposed to virus during IAV infection (40, PDCs in tonsils compared with blood (Fig. 1E). 41). Tonsils are a good source of mucosal immune cells, including Next, we isolated highly pure and relatively large numbers of DCs (36, 38, 42). We confirmed the presence of the major human PDCs from human tonsils using negative depletion of non-PDCs DC subsets (CD123+ PDCs, CD141+ MDCs, and CD1c+ MDCs) from TMCs with magnetic beads (Fig. 2A, 2B). In parallel, in tonsils and blood from healthy individuals using flow cytometry PDCs were isolated from blood using the same protocol as out- (Fig. 1A, 1B, Supplemental Fig. 1A, 1B). In line with previous lined in Fig. 2A and as previously described (22, 30, 47). Isolated reports (25, 42–46), we found that PDCs were the most frequent tonsil and blood PDCs with high and comparable purity (.95%) DC subset in tonsils (Fig. 1C), whereas CD1c+ MDCs were the were obtained (Fig. 2B, 2D). On average, tonsils yielded 1.23 3 most frequent DCs in peripheral blood (Fig. 1D). We found no 106 PDCs from 1 3 109 TMCs, and 0.87 3 106 blood PDCs were statistically significant difference in the frequency of CD141+ isolated from 1 3 109 PBMCs (median values; Fig. 2C). Both MDCs in tonsils compared with blood, whereas CD1c+ MDCs isolated tonsil and blood PDCs displayed high viability (mean: were more frequent in blood than in tonsils (Fig. 1E). However, 85%; Fig. 2E). Notably, blood PDCs expressed higher levels of Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Isolation of highly purified tonsil PDCs. (A) Surgically excised human tonsils were mechanically disrupted using forceps, filtered, and washed in PBS to obtain a single-cell suspension. TMCs were isolated by Ficoll density gradient centrifugation, and subsequently tonsil PDCs were isolated using negative selection, with magnetic beads removing non-PDCs. (B) Purity and viability of the isolated tonsil PDCs were determined by flow cytometry as assessed by lack of lineage markers (CD3, CD14, CD20, and CD56), low expression of live/dead marker, and high expression of HLA-DR+, CD123+, and CD303+ cells. Dot plots show one representative tonsil donor of 24, and numbers in each plot indicate the frequency of cells in the depicted gate. (C) The number of tonsil and blood PDCs isolated from TMCs (blue circles) and PBMCs (red squares), respectively. Graph shows data from individual donors of tonsil PDCs (n = 24) or blood PDCs (n = 22). Lines indicate median cell yield. (D) The purity of PDCs isolated from tonsils and blood as determined by Trypan Blue exclusion and manual counting. Graph shows percentage of live, Trypan Blue2 PDCs isolated from individual donors’ tonsils (n = 24) or blood (n = 22). Lines indicate median purity. (E) Viability of isolated tonsil PDCs (n = 24) and blood PDCs (n = 22) was .80% at the time of isolation as assessed by Trypan Blue exclusion and manual counting. Lines indicate median viability (88.7% for tonsil PDCs and 84.4% for blood PDCs). (F) The maturation status of freshly isolated PDCs was further characterized by assessing the mean fluorescence intensity (MFI) of CD86 expression. Blood PDCs (red squares, n = 8) were significantly more mature than tonsil PDCs (blue circles, n = 8) at the time of isolation. Lines indicate median MFI values. The Mann–Whitney U test was used to assess statistical differences in MFI values. **p , 0.01. The Journal of Immunology 5

CD86, a maturation marker, at the time of isolation compared with PDCs only expressed CD86 (Fig. 3C). Blood PDCs expressed tonsil PDCs (Fig. 2F). To characterize the inherent differences significantly higher levels of the maturation marker CD86 than between tonsil and blood PDCs, we next examined differences in tonsil PDCs, affirming findings from flow cytometry (Fig. 2F). their gene expression profile. We also determined the expression of TLRs on PDCs and, as expected, both tonsil and blood PDCs expressed high levels of Tonsil and blood PDCs display comparable gene TLR7 and TLR9, the endosomal PRRs that recognize viral RNA expression profiles and DNA, respectively (Fig. 3D). At steady state, tonsil and blood We isolated RNA from highly pure (.95%) tonsil and blood PDCs also expressed detectable levels of IFNA1, IFNB1, IL-6, PDCs (n = 4 each) and performed gene expression analysis using and TNF, with blood PDCs expressing significantly higher a custom-designed TaqMan low-density array (Supplemental levels of IFNA1, IL-6, and TNF than tonsil PDCs (Fig. 3E). We Table I). We analyzed expression of 47 selected genes compris- also assessed expression of signaling molecules and adaptors ing phenotypic markers, pattern recognition receptors (PRRs), downstream of the TLRs. Tonsil and blood PDCs expressed signaling molecules, cytokines, and maturation markers to identify high levels of MYD88 and IRF7 (Fig. 3F). IRF3, TRAF3,and potential differences between tonsil and blood PDCs (Fig. 3A). TRAF6 were also expressed to a small extent compared with In general, the pattern of gene expression was very similar be- IRF7. Finally, tonsil and blood PDCs expressed high levels tween tonsil and blood PDCs (Fig. 3A), as has also been previ- of IFNAR1 and detectable levels of RIG-I. Collectively, the ously reported (48). The purity of the isolated PDCs was verified expression profile indicated overall similarity in pattern be- by their selective expression of the phenotypic marker CD303 tween the tonsil and blood PDCs, with blood PDCs displaying

(BDCA2) and the absence of lymphocyte (CD20 and CD3D) and higher levels of expression of the maturation marker CD86 Downloaded from MDC markers (CD1a, CD1c, and XCR1) (Fig. 3B). Among the and several genes important for type I IFN responses than maturation markers assayed, freshly isolated tonsil and blood tonsil PDCs. http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Gene expression proﬁle of tonsil and blood PDCs. (A) Tonsil and blood PDCs (n = 4 each) were isolated and used to evaluate steady-state expression of 47 relevant genes (Supplemental Table I). Color changes within a row indicate expression levels relative to the median of the sample population. The color bar ranges from 23.5 (red) to 3.5 (green) and represents high and low expression levels, respectively. (B–G) Relative gene quantities were obtained using the comparative cycle threshold method after normalization to the HPRT1 gene as an endogenous control. (B) Phenotypic marker gene expression indicates high purity of PDCs by the high expression of BDCA2 (CD303) and the absence of phenotypic markers CD1a (MDCs), CD1c (CD1c MDCs), XCR1 (CD141 MDCs), SLC11A1 (monocytes), CD3 (T cells), and MS4A1 (CD20; B cells). Tonsil and blood PDCs express high levels of maturation marker CD86 (C); PRRs TLR7 and TLR9 (D); cytokines IFNA1, IFNA2, IL-6, and TNF (E); signaling molecules MYD88, IRF3, and IRF7 (F); and IFN-a receptor IFNAR1 (G). Statistical differences between expression levels in tonsil and blood PDCs were assessed using two-way ANOVAwith the Sidak multiple comparisons test. Differences were considered statistically signiﬁcant at p , 0.05 or NS at p $ 0.05. *p , 0.05, **p , 0.01, ****p , 0.0001. 6 IAV SUSCEPTIBILITY OF HUMAN TONSIL AND BLOOD PDC Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Tonsil and blood PDCs are susceptible to IAV infection in vitro. (A) Isolated tonsil and blood PDCs were exposed to increasing MOI of IAV and incubated at 37˚C for 12 h. As controls, PDCs were cultured with no virus or infected at the highest virus dose in the presence of NH4Cl to prevent viral fusion and replication by neutralizing endosomal acidiﬁcation. Plots show CD123+ tonsil (upper row) or blood (lower row) PDCs and their expression of IAV NP after 12 h of exposure as determined by intracellular staining and ﬂow cytometry from one representative donor of n = 27 (tonsils) and n =18 (blood). Numbers in the plots indicate the frequency of NP+ PDCs in the depicted gate. (B and C) The frequency of NP+ PDCs after exposure with IAVat an MOI of 6.0 over time was determined at 2, 6, 12, 18, and 24 h in both tonsil PDCs (blue circles, n = 27) (B) and blood PDCs (red squares, n = 18) (C). Graphs in (B) and (C) show data from PDCs isolated from individual donors, and lines indicate median frequency of NP+ PDCs at each time point. Differences at various time points were assessed using the Kruskal–Wallis test with the Dunn multiple comparisons test and (Figure legend continues) The Journal of Immunology 7

Tonsil and blood PDCs are susceptible to IAV infection in vitro Blood PDCs secrete 10-fold more IFN-a in response to IAV PDCs have generally been considered resistant to virus infection, than tonsil PDCs owing to constitutive expression of IRF7 allowing for rapid in- Potent type I IFN production in response to viral pathogens like duction of type I IFNs (49). To assess if the reported resistance of IAV is a hallmark of PDCs. We quantified IFN-a secretion in PDCs to IAV infection in vitro was dependent on the viral dose, supernatants collected at different time points from tonsil and we exposed tonsil and blood PDCs to increasing MOI of IAV for blood PDCs exposed to IAV by ELISA. Both tonsil and blood 12 h. We quantified infection using intracellular staining for the PDCs secreted readily detectable amounts of IFN-a in response to influenza viral NP and flow cytometry. As previously described, IAV already at 2 h, with peak concentrations detected in the su- blood PDCs were largely resistant to IAV infection at virus con- pernatant at 12 h of exposure (Fig. 6A, 6B). Exposing PDCs to centrations (MOI 0.6–1.5 as determined in MDCK cells) known to IAV in the presence of NH4Cl resulted in detectable but signifi- readily infect other cells, including human blood CD1c+ MDCs cantly lower concentrations of secreted IFN-a (Fig. 6A, 6B). (Supplemental Fig. 2) (30). Tonsil PDCs were also largely resis- Although PDCs from both tissues could potently produce IFN-a in tant to IAV infection at an MOI of 0.6. However, with escalating response to IAV, it was striking that blood PDCs produced 10-fold doses of IAV we observed a dose-dependent increase in the fre- more IFN-a at 12 h as compared with tonsil PDCs (median values at quency of NP+ tonsil and blood PDCs (Fig. 4A, Supplemental peak production: 21,498 pg/ml, n = 9, and 2,424 pg/ml, n = 12, Fig. 3). The appearance of NP+ PDCs was a result of new respectively; Fig. 6C). Interestingly, the disparity was not limited to viral protein production rather than virus binding to and/or an IAV MOI of 6.0. At lower MOI, blood PDCs produced ∼15-fold uptake by the PDCs, because we did not observe NP+ PDCs more IFN-a than tonsil PDCs (Supplemental Fig. 3E). As secretion Downloaded from when the virus was added in the presence of NH4Cl, which of IFN-a induces an antiviral state and thus confers protection to prevents endosomal acidification and inhibits viral fusion and bystander cells from virus infection (50–52), it is possible that the replication. higher IFN-a production by blood PDCs (detected already after 2 h) We next exposed tonsil and blood PDCs to IAVat an MOI of 6.0 could partly contribute to the trend of lower infection observed and determined the frequency of IAV infection at different time at early time points in blood PDCs compared with tonsil PDCs points. The frequency of NP+ tonsil and blood PDCs steadily in- (Fig. 4B, 4C). creased for a period of 12 h (Fig. 4B, 4C). After 18 h, tonsil PDCs Another possible explanation for differences in susceptibility to http://www.jimmunol.org/ from some donors showed a continued increase in the frequency IAV infection could be differential expression of the ISG MxA. of NP+ cells, whereas at 24 h the frequency, on average, dropped MxA, when overexpressed, renders cells resistant to IAV infection to a level similar to that seen at 2 h (Fig. 4B). In contrast, PDCs (53). All cells have the capacity to upregulate MxA in response to isolated from blood appeared more homogenous, in that the fre- type I IFNs. However, blood PDCs, unlike other cells, have been quency of IAV-infected PDCs dropped in all donors after 12 h of shown to express MxA already at steady state (30, 54). To de- IAV exposure (Fig. 4C). As expected, the viability of both tonsil termine if tonsil PDCs also constitutively expressed MxA and to and blood PDCs was gradually reduced over time when cells were compare if and how MxA levels in tonsil and blood PDCs changed kept in culture, and PDC cell death was significantly accelerated in response to IAV exposure, we performed Western blots on ly- by guest on September 28, 2021 (p , 0.0001) in the presence of replicating IAV (Fig. 4D, 4E). sates collected from tonsil and blood PDCs before and after ex- Also, blood PDC viability displayed a steeper decline as compared posure to 6.0 MOI IAV for 18 h. We found that blood PDCs and with tonsil PDCs. Taken together, our data show that both tonsil tonsil PDCs expressed detectable and comparable levels of MxA and blood PDCs were susceptible to IAV infection, and the re- at steady state immediately after isolation (Fig. 6D). The ex- duced frequency of NP+ PDCs at later time points was likely a pression of MxA was further augmented by exposure to IAV in result of death of infected PDCs induced by the cytopathic nature both tonsil and blood PDCs (Fig. 6D, 6E). of IAV. Blood PDCs respond more potently to TLR stimulus than Tonsil and blood PDCs mature in response to IAV exposure tonsil PDCs DCs respond to pathogens and/or stimulation by undergoing We next addressed whether the difference in IFN-a secretion maturation and upregulate costimulatory molecules like CD86 that between blood and tonsil PDCs was also apparent when the cells are required for efficient DC–T cell interactions. We analyzed the were exposed to other stimuli than IAV. We stimulated tonsil and cell surface expression of CD86 over time on tonsil and blood blood PDCs with TLR ligands CpG (TLR9 agonist) and 7/8L PDCs in response to IAV exposure (Fig. 5A). We found that both (TLR7/8 agonist). Both tonsil and blood PDCs upregulated tonsil and blood PDCs upregulated CD86 in response to IAV, and CD86 expression when exposed to CpG and 7/8L (Fig. 7A) and the expression significantly increased over time as compared with also secreted IFN-a (Fig. 7B). Although the IFN-a response was unstimulated PDCs (Fig. 5A–C). CD86 upregulation by IAV was significantly lower with the TLR ligands than when PDCs were dependent on viral fusion, as no or very limited CD86 upregula- exposed to IAV (Fig. 6C), we observed a similar trend, namely tion was detected on PDCs exposed to IAV in the presence of that blood PDCs produced significantly more IFN-a in response to NH4Cl (Fig. 5A). Blood PDCs also expressed at least 2-fold CpG compared with tonsil PDCs (Fig. 7B). higher CD86 than tonsil PDCs in response to IAV exposure at all This led us to ask if the differential response between blood time points (Fig. 5D), suggesting a dampened response to IAV in and tonsil PDCs was restricted to IFN-a production or was in- tonsil PDCs. dicative of a more general profile of PDC cytokine responses after

were considered statistically significant at p , 0.05. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. (D and E) PDC viability was determined at each time point of IAV exposure using Trypan Blue exclusion and manual counting. Graphs show mean viability 6 SD of tonsil PDCs (n = 27) (D) and blood PDCs (n = 18) (E) in cells cultured with no virus (empty) or in the presence of IAV MOI 6.0 (filled). (D and E) Differences in viability between IAV-exposed and -unexposed PDCs were assessed with two-way ANOVA using the Sidak multiple comparisons test and were considered statistically significant at p , 0.05. *p , 0.05, **p , 0.01, ****p , 0.0001. 8 IAV SUSCEPTIBILITY OF HUMAN TONSIL AND BLOOD PDC

stimulation. We therefore measured TNF-a and IL-6 in supernatants from PDCs exposed to IAVor TLR ligands at different time points. We found that blood PDCs secreted signiﬁcantly higher levels of TNF-a and IL-6 as compared with tonsil PDCs when exposed to IAV or a TLR ligand (Fig. 7C, 7D). In summary, our data show that both tonsil and blood PDCs secreted IFN-a, TNF-a, and IL-6 in response to different TLR stimuli but with different potencies, supporting the growing literature that the tissue microenvironment alters the functional capacity of immune cells.

Discussion In this study, we described that both human tonsil and blood PDCs, despite constitutive expression of the antiviral protein MxA, were susceptible to IAV infection when exposed to increasing doses of virus. As seen in other cell types, exposure to IAV in vitro and subsequent viral replication and NP production were accompanied by gradual cell death over time. However, PDC cultures remained sufﬁciently viable over 24 h to allow characterization of PDC responses to IAV infection. We found that both tonsil and blood Downloaded from PDCs matured and secreted IFN-a in response to IAV. Notably, at peak levels, blood PDCs secreted 10-fold more IFN-a as compared with tonsil PDCs in response to IAV. This difference was not unique to IAV but was also observed following PDC stimulation with puriﬁed TLR ligands, resulting in secretion of higher con-

centrations of IFN-a as well as TNF and IL-6 from blood PDCs http://www.jimmunol.org/ compared with tonsil PDCs. Collectively, our data suggest that human PDCs isolated from different anatomical locations are differentially primed in their ability to respond to virus infections like IAV, which in turn may impact the nature and magnitude of local immune responses mounted. The role of DCs in innate immune defense and in the initiation of adaptive immune responses has been studied extensively since their discovery in the 1970s (10, 11, 55–64). PDCs display several properties that distinguish them from other (myeloid) DC subsets, by guest on September 28, 2021 in particular the large amounts of type I IFNs they produce during virus infection (65, 66). Type I IFN signaling induces transcription of a large number of ISGs, promoting a general antiviral, protective state (67). Recent methodological advances have established that PDCs, previously known to be transcriptionally distinct from MDC subsets (68–72), actually arise from CD33+ pre-DCs (73). Inter- estingly, recent data suggest that the prototypic DC functions of naive T cell activation and IL-12 secretion are lacking in bona fide PDCs and are a consequence of contamination by pre-DCs, which express CD33, CD45RA, CX3CR1, and CD2, in addition to CD123 FIGURE 5. Tonsil and blood PDCs mature upon in vitro IAV infection. (73). The PDCs described in this study are bona fide PDCs that do (A) The surface expression of the costimulatory molecule CD86 was de- not express CD33 (data not shown) and produce large amounts of termined using flow cytometry on tonsil (upper row) and blood (lower row) IFN-a upon stimulation. PDCs over time after exposure to IAV MOI 6.0. Histograms show CD86 In this study, we used human tonsils as a source of tissue-resident expression on tonsil (blue) or blood (red) PDCs exposed to no virus (filled DCs from the respiratory tract. The tonsils comprise pharyngeal or histogram), MOI IAV 6.0 (solid line), or NH4Cl plus IAV MOI 6.0 (dashed adenoid, palatine, tubal, and lingual tonsils that line the pharynx line) from one representative donor of n = 27 (tonsil) and n = 18 (blood). (36, 38, 42) and are constantly exposed to respiratory pathogens B C ( and ) Graphs show the mean fluorescence intensity (MFI) of CD86 and foreign Ags (74). Tonsillectomy is a routine procedure typi- expression on PDCs after 2, 6, 12, and 18 h of exposure with IAV MOI 6.0 cally performed to remove enlarged tonsils to relieve conditions of in both tonsil PDCs (blue circles, n = 18) (B) and blood PDCs (red squares, n = 16) (C). Graphs show data from PDCs isolated from individual donors indicating CD86 MFI of PDCs at each time point. Median CD86 MFI values for no-virus controls are displayed as empty blue circles (tonsil) or red squares (blood) connected by dashed lines. Differences between the tonsil (blue, n = 18) and blood PDCs (red, n = 16) exposed to IAV MOI 6.0 level of PDC maturation at different time points were assessed using the (filled circles and squares) and PDCs unexposed to virus (straight lines, Kruskal–Wallis test and were considered statistically significant at n = 8–11, depending on availability of CD86 MFI data at time of PDC p , 0.05 (asterisks above plot). Asterisks below the plots refer to statistical isolation). The graph illustrates the fold differences between the response differences between IAV-exposed and -unexposed PDCs, assessed with of tonsil and blood PDCs over time. Differences in MFI values between two-way ANOVA using the Sidak multiple comparisons test, which were tonsil and blood PDCs were assessed with two-way ANOVA using the considered statistically significant at p , 0.05. **p , 0.01, ***p , 0.001, Sidak multiple comparisons test and were considered statistically signifi- ****p , 0.0001. (D) Graphs show overlaid median CD86 MFI values for cant at p , 0.05. ****p , 0.0001. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 6. PDCs isolated from peripheral blood secrete 10-fold more IFN-a in response to IAV than tonsil PDCs. (A and B)23 105 tonsil (blue) (A)or blood PDCs (red) (B) were suspended in R10 supplemented with IL-3 at a density of 1 3 106 cells/ml and exposed to 6.0 MOI of IAV in the absence (filled) by guest on September 28, 2021 or presence (striped) of 20 mM NH4Cl or not exposed to virus (white) for 2–24 h. Supernatants were collected at different time points, and the concentration of IFN-a (picograms per milliliter) secreted by the PDCs was determined by ELISA. Graphs show median 6 SD values for n = 12 donors (tonsils) and n =9 (blood). Differences in IFN-a values between PDC exposure conditions were assessed with the Friedman test with the Dunn multiple comparisons test and were considered statistically significant at p , 0.05. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. (C) Graph shows mean 6 SD IFN-a (picograms per milliliter) secreted from tonsil PDCs (blue circles) and blood PDCs (red squares) after exposure to IAV MOI 6.0 at different time points. Two-way ANOVA using the Sidak multiple comparisons test was performed to analyze statistical differences between tonsil and blood PDCs, and differences were considered statistically significant at p , 0.05. **p , 0.01, ****p , 0.0001. (D) Tonsil and blood PDCs express MxA at steady state. Lysates from TMCs, isolated tonsil PDCs, and blood PDCs were assayed for MxA expression using Western blot at steady state as well as after 12 h of IAV exposure. Figure shows a representative Western blot of MxA with GAPDH as a loading control. (E) Bar graph shows the mean relative MxA expression 6 SD (normalized to GAPDH) in tonsil and blood PDCs isolated from three donors. Differences between the expression levels of MxA in tonsil and blood PDCs were assessed using two-way ANOVA using the Sidak multiple comparisons test and were considered statistically significant at p , 0.05. *p , 0.05. recurrent tonsillitis, tonsillar hypertrophy, and obstructive sleep PDCs displayed a higher expression than tonsil PDCs of MyD88 apnea (75). Often the underlying cause of enlarged tonsils is not and IRF3, both key mediators in type I IFN signaling. IFN-a investigated in great detail. For the current study, we used palatine production by PDCs mediates antiviral protection in many virus tonsils from patients that showed no sign of ongoing infection and infections, including measles virus infection, in a MyD88- were afebrile and otherwise healthy. We did not find a correlation dependent fashion downstream of TLR7 and 9 (49, 50, 77, 78). with the age of the patients or the underlying cause for tonsil- Increased IRF3 expression on human PDCs has previously been lectomy to our findings (data not shown). The tonsillar tissue shown to augment IFN-a levels in patients with systemic lupus obtained was disrupted using mechanical methods to yield a single- erythematosus, albeit only in a population where IRF5 muta- cell suspension (38). We have not used enzymatic digestion, which tions have been linked to their development of systemic lupus may cleave off surface epitopes or activate the cells, and PDCs in erythematosus (53). In addition, the expression of RIG-I, previ- particular, during the processing stage. Although enzymatic digestion ously unreported in tonsil PDCs, is of particular interest because it has been used to successfully isolate T cells from tonsils, it has been suggests that tonsil PDCs are also capable of pathogen sensing showntoresultinloweryieldandhigheractivationofcells(76). via cytosolic receptors and could further augment existing IFN In this article, we showed that tonsil and blood PDCs overall responses. To understand the mechanism underlying the differ- displayed a similar pattern of expression of genes related to viral ence in tonsil and blood PDC responses, further studies are re- recognition and signaling. However, we observed a significant quired, preferably with side-by-side comparisons of PDCs from functional difference downstream of PRRs, most notably the different anatomical locations in the same individual, using amount of IFN-a produced by blood and tonsil PDCs in response RNA sequencing and epigenetics to better delineate whether on- to IAV. This may partly be explained by the observation that blood togeny or environment drives the observed cell subset difference. 10 IAV SUSCEPTIBILITY OF HUMAN TONSIL AND BLOOD PDC Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Tonsil and blood PDCs respond to in vitro stimulation with TLR ligands. (A) Expression of CD86 was determined using flow cytometry on tonsil (upper row) and blood (lower row) PDCs over time after exposure to TLR ligands. Histograms show CD86 expression in response to CpG (solid line) or TLR 7/8 ligand (dashed line) as compared with unstimulated control (filled histogram) from one representative donor of n = 4 (tonsil, blue) and n =4 (blood, red). (B–D) Cytokine production of 2 3 105 tonsil and blood PDCs (at a density of 1 3 106 cells/ml) in response to IAVor TLR ligands at 2, 12, and 18 h of exposure were quantified using ELISAs. Graphs show mean 6 SD IFN-a (picograms per milliliter) (B), TNF-a (picograms per milliliter) (C), and IL-6 (picograms per milliliter) (D) secreted from tonsil (blue) and blood PDCs (red) under different conditions over time. Differences in cytokine levels produced by tonsil and blood PDCs were assessed with two-way ANOVA using the Tukey multiple comparisons test and were considered statistically significant at p , 0.05. *p , 0.05, ****p , 0.0001.

An important study pursuing this avenue showed that transcriptional used as an indirect correlate of type I IFNs in human tissues because regulation of DC subsets across tissues was more conserved than in most cells it is exclusively expressed after exposure to type I that observed between different DC subsets from the same ana- IFNs (53, 55, 82, 83). The exception is human blood PDCs, which, tomical location, implying that ontogeny may override tissue mi- unlike other cell types, constitutively express MxA (50, 53, 67). croenvironment in influencing human DC behavior (79). However, This could explain why PDCs in general are not very susceptible to to what extent tissue microenvironment gains importance during IAV infection in vitro at concentrations that readily infect blood diseased state (e.g., IAV infection) is currently largely unknown. CD1c+ MDCs. At higher MOI, MxA may be saturated, allowing the In response to IAV, both tonsil and blood PDCs produced large virus to replicate within the PDCs as we observed. Further studies amounts of IFN-a, with peak IFN-a production observed after 12 h are required to see if MxA overexpression or MxA knockdown, or of IAV exposure, and importantly, blood PDCs produced 10-fold alternatively dose response experiments using recombinant IFN-a, more IFN-a compared with tonsil PDCs. Possibly as a consequence can modulate PDC maturation and susceptibility to IAV infection. of the higher IFN-a secretion, blood PDCs also induced a higher In this study, we used a relatively high concentration of IAV for expression of the ISG MxA after IAV exposure compared with in vitro infection experiments, which raises the question of cor- tonsil PDCs. MxA has been widely studied for its antiviral pro- relation with human infections. Because IAV is a lytic virus, each tection (53, 80, 81). MxA can bind the IAV PB2 protein and IAV lysis cycle can release up to 104 progeny virions per infected cell, NP in the cytoplasm of IAV-exposed cells and can subsequently suggesting that cells along the infected mucosa can be locally inhibit transcription of viral genes. MxA has also successfully been exposed to relatively high doses of virus (84). Also, we observed a The Journal of Immunology 11 gradual drop in viability of PDCs after exposure to IAVat an MOI of References 6.0, suggesting that PDCs were sensitive to the cytopathic effects of 1. World Health Organization. 2017. The top 10 causes of death. World Health the virus despite producing large quantities of IFN-a. This may be a Organization, Geneva, Switzerland. Available at: https://www.who.int/news-room/ fact-sheets/detail/the-top-10-causes-of-death. reflection of IFN-a more efficiently protecting bystander cells in a 2. Global Burden of Disease Study 2015 HIV Collaborators. 2016. Estimates paracrine manner rather than the cells secreting the cytokine (32, 85). of global, regional, and national incidence, prevalence, and mortality of HIV, Recent and current studies are increasingly highlighting the 1980-2015: the Global Burden of Disease Study 2015. [Published erratum appears in 2016 Lancet HIV 3: e408.] Lancet HIV 3: e361–e387. significance of studying immune cells and responses at the site of 3. Cowling, B. J., D. K. Ip, V. J. Fang, P. Suntarattiwong, S. J. Olsen, J. Levy, infection (86–89). Our study describes the susceptibility and re- T. M. Uyeki, G. M. Leung, J. S. Malik Peiris, T. Chotpitayasunondh, et al. 2013. Aerosol transmission is an important mode of influenza A virus spread. Nat. sponse of human PDCs from two different anatomical compart- Commun. 4: 1935. ments to in vitro IAV infection. Differences in the functional 4. Pulendran, B., and M. S. Maddur. 2015. Innate immune sensing and response to capacity of circulating blood PDCs compared with mucosal PDCs influenza. Curr. Top. Microbiol. Immunol. 386: 23–71. 5. Iwasaki, A., E. F. Foxman, and R. D. Molony. 2017. Early local immune de- raise the question of what role tonsil PDCs play in mucosal im- fences in the respiratory tract. Nat. Rev. Immunol. 17: 7–20. munity. One possibility is that the effective disparity in IFN-a 6. Iwasaki, A., and P. S. Pillai. 2014. Innate immunity to influenza virus infection. concentration is overcome by PDCs being relatively more abun- Nat. Rev. Immunol. 14: 315–328. 7. Baharom, F., S. Thomas, A. Bieder, M. Hellme´r, J. Volz, K. J. Sandgren, dant in tonsils compared with blood. Also, upon exposure to G. M. McInerney, G. B. Karlsson Hedestam, I. Mellman, and A. Smed-So¨rensen. IAV in vitro, blood PDCs displayed a steeper decline in viability 2015. Protection of human myeloid dendritic cell subsets against influenza A virus infection is differentially regulated upon TLR stimulation. J. Immunol. than tonsil PDCs. Perhaps tonsil PDCs, which are more abundant, 194: 4422–4430. comparatively less potent at IFN-a production, but capable of 8. Hristova, M., A. Habibovic, C. Veith, Y. M. Janssen-Heininger, A. E. Dixon, resisting cytopathic effects of IAV for a longer duration (as M. Geiszt, and A. van der Vliet. 2016. Airway epithelial dual oxidase 1 mediates Downloaded from allergen-induced IL-33 secretion and activation of type 2 immune responses. compared with blood PDCs), are necessary to supplement the quicker J. Allergy Clin. Immunol. 137: 1545–1556.e11. loss of blood PDCs. Tonsils also contain blood vessels, and it is also 9. van Vliet, S. J., J. den Dunnen, S. I. Gringhuis, T. B. Geijtenbeek, and Y. van likely that the mononuclear cell suspensions obtained from tonsils Kooyk. 2007. Innate signaling and regulation of dendritic cell immunity. Curr. Opin. Immunol. 19: 435–440. also contain a few PDCs that arrive from blood, a challenge inherent 10. Pulendran, B., H. Tang, and T. L. Denning. 2008. Division of labor, plasticity, to studying immune cells obtained from tissues and one currently and crosstalk between dendritic cell subsets. Curr. Opin. Immunol. 20: 61–67. 11. Granot, T., T. Senda, D. J. Carpenter, N. Matsuoka, J. Weiner, C. L. Gordon, without a viable solution. Another consideration during human IAV M. Miron, B. V. Kumar, A. Griesemer, S. H. Ho, et al. 2017. Dendritic http://www.jimmunol.org/ infection is the recruitment of PDCs to the nasopharynx (90), which cells display subset and tissue-specific maturation dynamics over human life. may enhance the antiviral response mounted against IAV infection Immunity 46: 504–515. 12. Baharom, F., G. Rankin, A. Blomberg, and A. Smed-So¨rensen. 2017. Human at the initial site of infection. lung mononuclear phagocytes in health and disease. Front. Immunol. 8: 499. Alternatively, there may be a lower requirement for IFN-a in 13. Iwasaki, A. 2007. Mucosal dendritic cells. Annu. Rev. Immunol. 25: 381–418. tissues like tonsils that are constantly exposed to foreign agents, as 14. Baharom, F., S. Thomas, G. Rankin, R. Lepzien, J. Pourazar, A. F. Behndig, C. Ahlm, A. Blomberg, and A. Smed-So¨ rensen. 2016. Dendritic cells and has been previously speculated (91). Dampened responses in the monocytes with distinct inflammatory responses reside in lung mucosa of tonsils (PDCs in particular) would therefore prevent excessive healthy humans. J. Immunol. 196: 4498–4509. 15. Yu, Y. R., D. F. Hotten, Y. Malakhau, E. Volker, A. J. Ghio, P. W. Noble, inflammation. A few studies on human primary PDCs have pre- M. Kraft, J. W. Hollingsworth, M. D. Gunn, and R. M. Tighe. 2016. Flow viously demonstrated dampened IFN-a production by negative cytometric analysis of myeloid cells in human blood, bronchoalveolar lavage, by guest on September 28, 2021 regulation of PDC functions. Cross-linking of the Ig-like transcript and lung tissues. Am. J. Respir. Cell Mol. Biol. 54: 13–24. ε 16. Desch, A. N., S. L. Gibbings, R. Goyal, R. Kolde, J. Bednarek, T. Bruno, ILT7 and the transmembrane adapter Fc RIg on primary human J. E. Slansky, J. Jacobelli, R. Mason, Y. Ito, et al. 2016. Flow cytometric analysis PDCs inhibited IFN-a production (92). CD303 (BDCA-2) can of mononuclear phagocytes in nondiseased human lung and lung-draining lymph also directly bind FcεRIg and activated a novel BCR-like sig- nodes. Am. J. Respir. Crit. Care Med. 193: 614–626. 17. von Garnier, C., L. Filgueira, M. Wikstrom, M. Smith, J. A. Thomas, naling pathway suppressing IFN-a production (93, 94). Either of D. H. Strickland, P. G. Holt, and P. A. Stumbles. 2005. Anatomical location the inhibitory pathways may be responsible for dampened IFN-a determines the distribution and function of dendritic cells and other APCs in the respiratory tract. J. Immunol. 175: 1609–1618. production by tonsil PDCs via mechanisms currently unknown. 18. Chen, K., J. M. Wang, R. Yuan, X. Yi, L. Li, W. Gong, T. Yang, L. Li, and S. Su. Blood PDCs, in contrast, are very potent at producing IFN-a in 2016. Tissue-resident dendritic cells and diseases involving dendritic cell response to virus infection, suggesting that, when activated, they malfunction. Int. Immunopharmacol. 34: 1–15. 19. Da Silva, C., C. Wagner, J. Bonnardel, J. P. Gorvel, and H. Lelouard. 2017. rapidly secrete large amounts of IFN-a into the blood circulation, The Peyer’s patch mononuclear phagocyte system at steady state and during which is highly relevant during infections associated with viremia, infection. Front. Immunol. 8: 1254. in which the virus is detectable in blood, like dengue, measles, 20. Lutz, M. B., H. Strobl, G. Schuler, and N. Romani. 2017. GM-CSF monocyte- derived cells and Langerhans cells as part of the dendritic cell family. Front. hepatitis B/C, or HIV (67). However, IAV infections can, in rare Immunol. 8: 1388. cases, be associated with viremia, and typically the virus remains 21. Munro, D. A. D., and J. Hughes. 2017. The origins and functions of tissue- restricted to tissues of the upper respiratory tract. In this context, resident macrophages in kidney development. Front. Physiol. 8: 837. 22. Di Pucchio, T., B. Chatterjee, A. Smed-So¨rensen, S. Clayton, A. Palazzo, IFN-a produced by mucosal tissue-resident PDCs may be of M. Montes, Y. Xue, I. Mellman, J. Banchereau, and J. E. Connolly. 2008. Direct greater relevance in the context of in vivo viral infection than that proteasome-independent cross-presentation of viral antigen by plasmacytoid dendritic cells on major histocompatibility complex class I. Nat. Immunol. 9: produced by PDCs in circulation. These speculations merit further 551–557. investigation, and our study highlights the relevance of studies of 23. Matsui, T., J. E. Connolly, M. Michnevitz, D. Chaussabel, C. I. Yu, C. Glaser, immune cells at the site where they are expected to exert their S. Tindle, M. Pypaert, H. Freitas, B. Piqueras, et al. 2009. CD2 distinguishes two subsets of human plasmacytoid dendritic cells with distinct phenotype and function to better characterize the immune mediators in play functions. J. Immunol. 182: 6815–6823. during infection. 24. Fonteneau, J. F., M. Gilliet, M. Larsson, I. Dasilva, C. Mu¨nz, Y. J. Liu, and N. Bhardwaj. 2003. Activation of influenza virus-specific CD4+ and CD8+ T cells: a new role for plasmacytoid dendritic cells in adaptive immunity. Blood 101: 3520–3526. Acknowledgments 25. Segura, E., M. Durand, and S. Amigorena. 2013. Similar antigen cross- We thank the patients who have contributed clinical material to this study. presentation capacity and phagocytic functions in all freshly isolated human We thank Julia Volz and Thomas Buters for technical assistance. lymphoid organ-resident dendritic cells. J. Exp. Med. 210: 1035–1047. 26. Liu, Y. J. 2005. IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu. Rev. Immunol. 23: 275–306. 27. Kadowaki, N., S. Antonenko, J. Y. Lau, and Y. J. Liu. 2000. Natural interferon Disclosures alpha/beta-producing cells link innate and adaptive immunity. J. Exp. Med. 192: The authors have no financial conflicts of interest. 219–226. 12 IAV SUSCEPTIBILITY OF HUMAN TONSIL AND BLOOD PDC

28. Siegal, F. P., N. Kadowaki, M. Shodell, P. A. Fitzgerald-Bocarsly, K. Shah, 56. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, S. Ho, S. Antonenko, and Y. J. Liu. 1999. The nature of the principal type 1 and R. M. Steinman. 1992. Generation of large numbers of dendritic cells from interferon-producing cells in human blood. Science 284: 1835–1837. mouse bone marrow cultures supplemented with granulocyte/macrophage 29. Cella, M., F. Facchetti, A. Lanzavecchia, and M. Colonna. 2000. Plasmacytoid colony-stimulating factor. J. Exp. Med. 176: 1693–1702. dendritic cells activated by influenza virus and CD40L drive a potent TH1 57. Steinman, R. M. 1991. The dendritic cell system and its role in immunogenicity. polarization. Nat. Immunol. 1: 305–310. Annu. Rev. Immunol. 9: 271–296. 30. Smed-So¨rensen, A., C. Chalouni, B. Chatterjee, L. Cohn, P. Blattmann, 58. Freudenthal, P. S., and R. M. Steinman. 1990. The distinct surface of human N. Nakamura, L. Delamarre, and I. Mellman. 2012. Influenza A virus infection blood dendritic cells, as observed after an improved isolation method. Proc. Natl. of human primary dendritic cells impairs their ability to cross-present antigen to Acad. Sci. USA 87: 7698–7702. CD8 T cells. PLoS Pathog. 8: e1002572. 59. Steinman, R. M., G. Kaplan, M. D. Witmer, and Z. A. Cohn. 1979. Identification 31. Cella, M., D. Jarrossay, F. Facchetti, O. Alebardi, H. Nakajima, A. Lanzavecchia, of a novel cell type in peripheral lymphoid organs of mice. V. Purification of and M. Colonna. 1999. Plasmacytoid monocytes migrate to inflamed lymph spleen dendritic cells, new surface markers, and maintenance in vitro. J. Exp. nodes and produce large amounts of type I interferon. Nat. Med. 5: 919–923. Med. 149: 1–16. 32. Thitithanyanont, A., A. Engering, P. Ekchariyawat, S. Wiboon-ut, 60. Steinman, R. M., J. C. Adams, and Z. A. Cohn. 1975. Identification of a novel A. Limsalakpetch, K. Yongvanitchit, U. Kum-Arb, W. Kanchongkittiphon, cell type in peripheral lymphoid organs of mice. IV. Identification and distri- P. Utaisincharoen, S. Sirisinha, et al. 2007. High susceptibility of human den- bution in mouse spleen. J. Exp. Med. 141: 804–820. dritic cells to avian influenza H5N1 virus infection and protection by IFN-alpha 61. Steinman, R. M., and Z. A. Cohn. 1973. Identification of a novel cell type in and TLR ligands. J. Immunol. 179: 5220–5227. peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distri- 33. Stramer, S. L., C. Collins, T. Nugent, X. Wang, M. Fuschino, J. W. Heitman, bution. J. Exp. Med. 137: 1142–1162. J. Law, D. E. Krysztof, N. Kiely, D. Todd, et al; NHLBI Retrovirus Epidemi- 62. Steinman, R. M., and Z. A. Cohn. 1974. Identification of a novel cell type in ology Donor Study-II (REDS-II). 2012. Sensitive detection assays for influenza peripheral lymphoid organs of mice. II. Functional properties in vitro. J. Exp. RNA do not reveal viremia in US blood donors. J. Infect. Dis. 205: 886–894. Med. 139: 380–397. 34. Taubenberger, J. K., and D. M. Morens. 2008. The pathology of influenza virus 63. Shortman, K., and Y. J. Liu. 2002. Mouse and human dendritic cell subtypes. infections. Annu. Rev. Pathol. 3: 499–522. Nat. Rev. Immunol. 2: 151–161. 35. McClory, S., T. Hughes, A. G. Freud, E. L. Briercheck, C. Martin, A. J. Trimboli, 64. Worbs, T., S. I. Hammerschmidt, and R. Fo¨rster. 2017. Dendritic cell migration J. Yu, X. Zhang, G. Leone, G. Nuovo, and M. A. Caligiuri. 2012. Evidence for a in health and disease. Nat. Rev. Immunol. 17: 30–48. Downloaded from stepwise program of extrathymic T cell development within the human tonsil. 65. Szabo, A., Z. Magyarics, K. Pazmandi, L. Gopcsa, E. Rajnavolgyi, and A. Bacsi. J. Clin. Invest. 122: 1403–1415. 2014. TLR ligands upregulate RIG-I expression in human plasmacytoid dendritic 36. Micheletti, A., G. Finotti, F. Calzetti, S. Lonardi, E. Zoratti, M. Bugatti, cells in a type I IFN-independent manner. Immunol. Cell Biol. 92: 671–678. S. Stefini, W. Vermi, and M. A. Cassatella. 2016. slanDCs/M-DC8+ cells con- 66. O¨ sterlund, P., M. Strengell, L. P. Sarin, M. M. Poranen, R. Fagerlund, K. Meleń, stitute a distinct subset of dendritic cells in human tonsils [corrected]. [Published and I. Julkunen. 2012. Incoming influenza A virus evades early host recognition, erratum appears in 2016 Oncotarget 7: 66323.] Oncotarget 7: 161–175. while influenza B virus induces interferon expression directly upon entry. 37. Elpek, K. G., A. Bellemare-Pelletier, D. Malhotra, E. D. Reynoso, V. Lukacs- J. Virol. 86: 11183–11193.

Kornek, R. H. DeKruyff, and S. J. Turley. 2011. Lymphoid organ-resident den- 67. Schneider, W. M., M. D. Chevillotte, and C. M. Rice. 2014. Interferon- http://www.jimmunol.org/ dritic cells exhibit unique transcriptional fingerprints based on subset and site. PLoS stimulated genes: a complex web of host defenses. Annu. Rev. Immunol. 32: One 6: e23921. 513–545. 38. Johnston, A., S. L. Sigurdardottir, and J. J. Ryon. 2009. Isolation of mononuclear 68. Merad, M., P. Sathe, J. Helft, J. Miller, and A. Mortha. 2013. The dendritic cell cells from tonsillar tissue. Curr. Protoc. Immunol. 86: 7.8.1–7.8.4. lineage: ontogeny and function of dendritic cells and their subsets in the steady 39. Sichien, D., B. N. Lambrecht, M. Guilliams, and C. L. Scott. 2017. Development state and the inflamed setting. Annu. Rev. Immunol. 31: 563–604. of conventional dendritic cells: from common bone marrow progenitors to 69. Onai, N., K. Kurabayashi, M. Hosoi-Amaike, N. Toyama-Sorimachi, multiple subsets in peripheral tissues. Mucosal Immunol. 10: 831–844. K. Matsushima, K. Inaba, and T. Ohteki. 2013. A clonogenic progenitor with 40. Drago, L., S. Esposito, E. De Vecchi, P. Marchisio, F. Blasi, E. Baggi, prominent plasmacytoid dendritic cell developmental potential. Immunity 38: P. Capaccio, and L. Pignataro. 2008. Detection of respiratory viruses and atyp- 943–957. ical bacteria in children’s tonsils and adenoids. J. Clin. Microbiol. 46: 369–370. 70. Schlitzer, A., V. Sivakamasundari, J. Chen, H. R. Sumatoh, J. Schreuder, J. Lum, 41. Jartti, T., O. Palomares, M. Waris, O. Tastan, R. Nieminen, T. Puhakka, B. Malleret, S. Zhang, A. Larbi, F. Zolezzi, et al. 2015. Identification of cDC1-

B. Ru¨ckert, A. Aab, T. Vuorinen, T. Allander, et al. 2014. Distinct regulation of and cDC2-committed DC progenitors reveals early lineage priming at the by guest on September 28, 2021 tonsillar immune response in virus infection. Allergy 69: 658–667. common DC progenitor stage in the bone marrow. Nat. Immunol. 16: 718–728. 42. Durand, M., and E. Segura. 2016. Dendritic cell subset purification from human 71. Lee, J., G. Breton, T. Y. Oliveira, Y. J. Zhou, A. Aljoufi, S. Puhr, M. J. Cameron, tonsils and lymph nodes. Methods Mol. Biol. 1423: 89–99. R. P. Se´kaly, M. C. Nussenzweig, and K. Liu. 2015. Restricted dendritic cell and 43. Rescigno, M. 2013. Plasmacytoid DCs are gentle guardians of tonsillar epithe- monocyte progenitors in human cord blood and bone marrow. J. Exp. Med. 212: lium. Eur. J. Immunol. 43: 1142–1146. 385–399. 44. Stent, G., J. C. Reece, D. C. Baylis, K. Ivinson, G. Paukovics, M. Thomson, and 72. Breton, G., J. Lee, Y. J. Zhou, J. J. Schreiber, T. Keler, S. Puhr, N. Anandasabapathy, P. U. Cameron. 2002. Heterogeneity of freshly isolated human tonsil dendritic S. Schlesinger, M. Caskey, K. Liu, and M. C. Nussenzweig. 2015. Circulating cells demonstrated by intracellular markers, phagocytosis, and membrane dye precursors of human CD1c+ and CD141+ dendritic cells. J. Exp. Med. 212: transfer. Cytometry 48: 167–176. 401–413. 45. Segura, E., and S. Amigorena. 2014. Cross-presentation by human dendritic cell 73. See, P., C. A. Dutertre, J. Chen, P. Gu¨nther, N. McGovern, S. E. Irac, subsets. Immunol. Lett. 158: 73–78. M. Gunawan, M. Beyer, K. Ha¨ndler, K. Duan, et al. 2017. Mapping the human 46. Segura, E., and J. A. Villadangos. 2011. A modular and combinatorial view of DC lineage through the integration of high-dimensional techniques. Science 356: the antigen cross-presentation pathway in dendritic cells. Traffic 12: 1677–1685. eaag3009. 47. Smed-So¨rensen, A., K. Lore´, J. Vasudevan, M. K. Louder, J. Andersson, 74. Perry, M. E. 1994. The specialised structure of crypt epithelium in the human J. R. Mascola, A. L. Spetz, and R. A. Koup. 2005. Differential susceptibility to palatine tonsil and its functional significance. J. Anat. 185: 111–127. human immunodeficiency virus type 1 infection of myeloid and plasmacytoid 75. Hallenstal,˚ N., O. Sunnergren, E. Ericsson, C. Hemlin, A. C. HesseńSo¨derman, dendritic cells. J. Virol. 79: 8861–8869. P. Nerfeldt, E. Odhagen, M. Ryding, and J. Stalfors. 2017. Tonsil surgery in 48. Lindstedt, M., K. Lundberg, and C. A. K. Borrebaeck. 2005. Gene family Sweden 2013-2015. Indications, surgical methods and patient-reported outcomes clustering identifies functionally associated subsets of human in vivo blood and from the National Tonsil Surgery Register. Acta Otolaryngol. 137: 1096–1103. tonsillar dendritic cells. J. Immunol. 175: 4839–4846. 76. Weigel, C., K. Geißler, R. Markwart, K. Schubert, I. Rubio, O. Guntinas-Lichius, 49. Honda, K., H. Yanai, T. Mizutani, H. Negishi, N. Shimada, N. Suzuki, Y. Ohba, and R. P. Requardt. 2015. Isolation of viable and functional T-cells from human A. Takaoka, W. C. Yeh, and T. Taniguchi. 2004. Role of a transductional- palatine tonsils. J. Immunol. Methods 427: 66–72. transcriptional processor complex involving MyD88 and IRF-7 in Toll-like 77. Beignon, A. S., K. McKenna, M. Skoberne, O. Manches, I. DaSilva, receptor signaling. Proc. Natl. Acad. Sci. USA 101: 15416–15421. D. G. Kavanagh, M. Larsson, R. J. Gorelick, J. D. Lifson, and N. Bhardwaj. 50. Fitzgerald-Bocarsly, P., J. Dai, and S. Singh. 2008. Plasmacytoid dendritic cells 2005. Endocytosis of HIV-1 activates plasmacytoid dendritic cells via toll-like and type I IFN: 50 years of convergent history. Cytokine Growth Factor Rev. 19: receptor-viral RNA interactions. J. Clin. Invest. 115: 3265–3275. 3–19. 78. Wang, J. P., P. Liu, E. Latz, D. T. Golenbock, R. W. Finberg, and D. H. Libraty. 51. Thomas, J. M., Z. Pos, J. Reinboth, R. Y. Wang, E. Wang, G. M. Frank, P. Lusso, 2006. Flavivirus activation of plasmacytoid dendritic cells delineates key elements G. Trinchieri, H. J. Alter, F. M. Marincola, and E. Thomas. 2014. Differential of TLR7 signaling beyond endosomal recognition. J. Immunol. 177: 7114–7121. responses of plasmacytoid dendritic cells to influenza virus and distinct viral 79. Heidkamp, G. F., J. Sander, C. H. K. Lehmann, L. Heger, N. Eissing, pathogens. J. Virol. 88: 10758–10766. A. Baranska, J. J. Lu¨hr, A. Hoffmann, K. C. Reimer, A. Lux, et al. 2016. Human 52. Darnell, J. E., Jr. 1998. Studies of IFN-induced transcriptional activation uncover lymphoid organ dendritic cell identity is predominantly dictated by ontogeny, not the Jak-Stat pathway. J. Interferon Cytokine Res. 18: 549–554. tissue microenvironment. Sci. Immunol. 1: eaai7677. 53. Haller, O., and G. Kochs. 2011. Human MxA protein: an interferon-induced 80. Zimmermann, P., B. Ma¨nz, O. Haller, M. Schwemmle, and G. Kochs. 2011. The dynamin-like GTPase with broad antiviral activity. J. Interferon Cytokine Res. viral nucleoprotein determines Mx sensitivity of influenza A viruses. J. Virol. 85: 31: 79–87. 8133–8140. 54. Ghoreishi, M., C. Vera Kellet, and J. P. Dutz. 2012. Type 1 IFN-induced protein 81. Haller, O., S. Gao, A. von der Malsburg, O. Daumke, and G. Kochs. 2010. MxA and plasmacytoid dendritic cells in lesions of morphea. Exp. Dermatol. 21: Dynamin-like MxA GTPase: structural insights into oligomerization and im- 417–419. plications for antiviral activity. J. Biol. Chem. 285: 28419–28424. 55. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of 82. Horisberger, M. A. 1992. Interferon-induced human protein MxA is a GTPase immunity. Nature 392: 245–252. which binds transiently to cellular proteins. J. Virol. 66: 4705–4709. The Journal of Immunology 13

83. Horisberger, M. A. 1995. Interferons, Mx genes, and resistance to influenza 90. Gill, M. A., K. Long, T. Kwon, L. Muniz, A. Mejias, J. Connolly, L. Roy, virus. Am. J. Respir. Crit. Care Med. 152: S67–S71. J. Banchereau, and O. Ramilo. 2008. Differential recruitment of dendritic cells 84. Stray, S. J., and G. M. Air. 2001. Apoptosis by influenza viruses correlates with and monocytes to respiratory mucosal sites in children with influenza virus or efficiency of viral mRNA synthesis. Virus Res. 77: 3–17. respiratory syncytial virus infection. J. Infect. Dis. 198: 1667–1676. 85.Ciancanelli,M.J.,L.Abel,S.Y.Zhang,and J. L. Casanova. 2016. Host genetics of 91.Polak,M.E.,N.J.Borthwick,F.G.Gabriel,M.J.Jager,andI.A.Cree. severe influenza: from mouse Mx1 to human IRF7. Curr. Opin. Immunol. 38: 109–120. 2008. Activation of tonsil dendritic cells with immuno-adjuvants. BMC 86. GeurtsvanKessel, C. H., I. M. Bergen, F. Muskens, L. Boon, H. C. Hoogsteden, Immunol. 9: 10. A. D. Osterhaus, G. F. Rimmelzwaan, and B. N. Lambrecht. 2009. Both con- 92. Cao, W., D. B. Rosen, T. Ito, L. Bover, M. Bao, G. Watanabe, Z. Yao, L. Zhang, ventional and interferon killer dendritic cells have antigen-presenting capacity L. L. Lanier, and Y. J. Liu. 2006. Plasmacytoid dendritic cell-specific receptor during influenza virus infection. PLoS One 4: e7187. ILT7-Fc epsilonRI gamma inhibits Toll-like receptor-induced interferon pro- 87. Snelgrove, R. J., A. Godlee, and T. Hussell. 2011. Airway immune homeostasis and duction. J. Exp. Med. 203: 1399–1405. implications for influenza-induced inflammation. Trends Immunol. 32: 328–334. 93. Janovec, V., B. Aouar, A. Font-Haro, T. Hofman, K. Trejbalova, J. Weber, 88. Athale, S., R. Banchereau, L. Thompson-Snipes, Y. Wang, K. Palucka, V. Pascual, L. Chaperot, J. Plumas, D. Olive, P. Dubreuil, et al. 2018. The MEK1/2-ERK and J. Banchereau. 2017. Influenza vaccines differentially regulate the interferon pathway inhibits type I IFN production in plasmacytoid dendritic cells. Front. response in human dendritic cell subsets. Sci. Transl. Med. 9: eaaf9194. Immunol. 9: 364. 89. To, K. K., J. F. Chan, and K. Y. Yuen. 2014. Viral lung infections: epidemiology, 94. Cao, W., L. Zhang, D. B. Rosen, L. Bover, G. Watanabe, M. Bao, L. L. Lanier, and virology, clinical features, and management of avian influenza A(H7N9). Curr. Y. J. Liu. 2007. BDCA2/Fc epsilon RI gamma complex signals through a novel Opin. Pulm. Med. 20: 225–232. BCR-like pathway in human plasmacytoid dendritic cells. PLoS Biol. 5: e248. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 Supplementary figure 1.

Supplementary figure 1. Monocytes and dendritic cells can be identified in human tonsils and blood. Gating strategy used to identify dendritic cells and monocytes in human (A) tonsil and (B) peripheral blood mononuclear cells.

Supplementary figure 2.

Supplementary figure 2. Blood CD1c+ MDCs are readily susceptible to IAV infection in vitro in a dose-dependent manner. (A) CD1c+ MDCs were isolated from human blood and exposed to increasing MOI of IAV, similar to Figure 4A. Plots show CD1c+ blood MDCs and their expression of IAV nucleoprotein (NP) after 12 hours of exposure as determined by intracellular staining and flow cytometry from one representative donor of n=2. Numbers in the plots indicate the frequency of NP+ CD1c+ MDCs in the depicted gate. (B) Bar graph shows frequency of NP+ CD1c+ blood MDCs after exposure to IAV at an MOI of 0.6, 1.5, 3.0 and

6.0 for 12 hours (n=2). (C) Bar graph represents the viability of blood CD1c+ MDC upon exposure to increasing MOI of IAV for 12 hours (n=2). (D) Bar graph shows the MFI of CD86 expression on blood CD1c+ MDC after 12 hours of exposure to increasing MOI of IAV (n=2).

Supplementary figure 3.

Supplementary figure 3. Tonsil and blood PDCs are susceptible to IAV infection in vitro in a dose-dependent manner. (A-B) Bar graph shows frequency of NP+ PDCs after exposure to IAV at an MOI of 0.6, 1.5, 3.0 and 6.0 after 12 hours of exposure, in both tonsil (blue, n=2) and blood PDCs (red, n=2), correspond to Figure 4A. Tonsil and blood PDCs were seeded at a density of 1x106 cells/mL in R10 supplemented with IL-3. (C-D) Bar graph represents the viability of tonsil (blue, n=2) and blood PDCs (red, n=2), upon exposure to increasing MOI of

IAV for 12 hours. (E) Graph shows median±SD IFNα (pg/mL) secreted from tonsil PDCs (blue circles, n=2) and blood PDCs (red squares, n=2) after exposure to IAV MOI 6.0 at different time points. The graph illustrates the fold differences between the IFNα response of tonsil and blood PDCs to increasing concentrations of IAV exposure, which varies between 15-fold at

MOIs of 0.6-3.0, to 10-fold at MOI 6.0, where peak IFNα production was observed. Supplementary table I.

Supplementary table I. TaqMan gene expression assays used for TLDA.