Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation

Mast cells regulate CD4 1 T-cell differentiation in the absence of antigen presentation Hector Rodriguez Cetina Biefer, MD, a,b * Timm Heinbokel, MD, a,c * Hirofumi Uehara, MD, d * Virginia Camacho, BSc, e * Koichiro Minami, MD, a Yeqi Nian, MD, a Suresh Koduru, PhD, f Rachid El Fatimy, PhD, g Ionita Ghiran, MD, h Alexander J. Trachtenberg, MSc, i Miguel A. de la Fuente, PhD, j Haruhito Azuma, MD, PhD, d Omid Akbari, PhD, k Stefan G. Tullius, MD, PhD, a Anju Vasudevan, PhD, l and Abdallah Elkhal, PhD a Boston and Belmont, Mass, Zurich, Switzerland, Berlin, Germany, Osaka, Japan, Hyderabad, India, Valladolid, Spain, and Los Angeles, Calif Background: Given their unique capacity for antigen uptake, processing, and presentation, antigen-presenting cells (APCs) are critical for initiating and regulating innate and adaptive immune responses. We have previously shown the role of nicotinamide adenine dinucleotide (NAD 1 ) in T-cell differentiation independently of the cytokine milieu, whereas the precise mechanisms remained unknown. Objective: The objective of this study is to further dissect the mechanism of actions of NAD 1 and determine the effect of APCs on NAD 1 -mediated T-cell activation. Methods: Isolated dendritic cells and bone marrow–derived mast cells (MCs) were used to characterize the mechanisms of action of NAD 1 on CD4 1 T-cell fate in vitro. Furthermore, NAD 1 -mediated CD4 1 T-cell differentiation was investigated in vivo by using wild-type C57BL/6, MC 2/2 , MHC class II 2/2 , Wiskott-Aldrich syndrome protein (WASP) 2/2 , 5C.C7 recombination-activating gene 2 (Rag2) 2/2 , and CD11b-DTR transgenic mice. Finally, we tested the physiologic effect of NAD 1 on the systemic immune response in the context of Listeria monocytogenes infection. Results: Our in vivo and in vitro findings indicate that after NAD 1 administration, MCs exclusively promote CD4 1 T-cell differentiation, both in the absence of antigen and independently of major APCs. Moreover, we found that MCs mediated CD4 1 T-cell differentiation independently of MHC II and T-cell receptor signaling machinery. More importantly, although treatment with NAD 1 resulted in decreased MHC II expression on CD11c 1 cells, MC-mediated CD4 1 T-cell differentiation rendered mice resistant to administration of lethal doses of L monocytogenes. Conclusions: Collectively, our study unravels a novel cellular and molecular pathway that regulates innate and adaptive immunity through MCs exclusively and underscores the therapeutic potential of NAD 1 in the context of primary immunodeficiencies and antimicrobial resistance. (J Allergy Clin Immunol 2018;142:1894-908.) Key words: Nicotinamide adenine dinucleotide, mast cells, T cells, antigen presentation, MHC, T-cell receptor, CD4 1 T-cell differentiation, dendritic cells, macrophages, Listeria monocytogenes, cytokine Antigen-presenting cells (APCs) play a central role in regulation of the innate and adaptive immune responses. 1 APCs have the ability to capture, process, and present antigens through their MHC cell-surface molecules to the T-cell receptor (TCR) to mount an MHC-restricted immune response. 2-6 APCs include a myriad of immune cells, such as B cells, neutrophils, macrophages, eosinophils, basophils, and dendritic cells (DCs). Among these populations, DCs are considered the major APCs bridging innate and adaptive immune responses. 7,8 The mode of action of DCs is mediated through at least 3 signals: (1) TCR activation, (2) activation of costimulatory molecules, and (3) secretion of chemokines and proinflammatory cytokines. 8 Indeed, depletion of CD11c 1 DCs has been shown to alter cytotoxic T-lymphocyte responses to infection, as well as CD4 1 T-cell activation and antibody production. 9 In addition, DCs can also regulate innate and adaptive immune responses by recognizing pathogen-associated molecular patterns (PAMPs), such as microbial nucleic acids, lipoproteins, and carbohydrates, or damage-associated molecular patterns (DAMPs) released from injured cells through intracellular or surfaceexpressed pattern recognition receptors (PRRs). 10-14 Although considered ‘‘atypical’’ APCs, Mast cells (MCs) have been described mainly for their role in allergic and autoimmune responses. 15,16 It is well established that MCs are important effector cells in IgE-mediated allergic inflammation, and MCs are also recognized to influence innate and adaptive immune responses. 8,16 From a the Division of Transplant Surgery and Transplantation Surgery Research Laboratory and g the Department of Neurology, Center for Neurologic Diseases, Initiative for RNA Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston; b the Clinic for Cardiovascular Surgery, University Hospital Zurich; c the Department of Nephrology, Charite Universitaetsmedizin Berlin; d the Department of Urology, Osaka Medical College; e the Flow Cytometry Core Facility and h the Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Stem Cell Institute, Boston; f the School of Medical Sciences, University of Hyderabad; i StART Families, Boston; j Instituto de Biolog ıa y Gen etica Molecular, University of Valladolid; k the Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles; and l the Angiogenesis and Brain Development Laboratory, Division of Basic Neuroscience, McLean Hospital, Harvard Medical School, Belmont. *These authors contributed equally and are co-first authors. Supported by National Institutes of Health grants R01NS073635 and R01MH110438 (to A.V.), R01 HL096795 and U01 HL126497 (to I.G.), and R01AG039449 (to S.G.T.). H.R.C.B. was supported by the Swiss Society of Cardiac Surgery. M.A.d.l.F was supported by FIS-ISCIII (grant PI10/02 511) and Fundaci on Ram on Areces (CIVP16A1843). Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest. Received for publication June 30, 2017; revised December 19, 2017; accepted for publication January 28, 2018. Available online February 20, 2018. Corresponding author: Abdallah Elkhal, PhD, Department of Surgery, Division of Transplant Surgery, Brigham and Women’s Hospital, 221 Longwood Ave, Boston, MA 02115. E-mail: [email protected]. The CrossMark symbol notifies online readers when updates have been made to the article such as errata or minor corrections 0091-6749 Ó2018 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). https://doi.org/10.1016/j.jaci.2018.01.038 1894 Abbreviations used APC: Antigen-presenting cell BMMC: Bone marrow–derived mast cell DAMP: Damage-associated molecular pattern DC: Dendritic cell EAE: Experimental autoimmune encephalomyelitis LPA: Lysophosphatidic acid MC: Mast cell NAD 1 : Nicotinamide adenine dinucleotide PAMP: Pathogen-associated molecular pattern PRR: Pattern recognition receptor Rag2: Recombination-activating gene 2 TCR: T-cell receptor WASP: Wiskott-Aldrich syndrome protein WT: Wild-type MC-deficient mice have been shown to exhibit an altered CD4 1 T-cell response to infection and in experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis, suggesting that MCs play a role in mediating T-cell responses. 17-20 Like DCs, MCs can directly present antigens to T cells in vitro, inducing an antigen-specific clonal expansion of T-cell populations, and are known to express costimulatory molecules and secrete a myriad of chemokines and proinflammatory cytokines. 8,15,16,21 However, MCs express MHC class II intracellularly rather than at the cell surface. 22 Moreover, MCs have been shown to promote T-cell activation in an antigen-independent manner, and no direct evidence has been provided thus far on the capacity of direct antigen presentation to the TCR. 8,23 Thus the mechanisms by which MCs regulate T-cell responses remain unclear and are yet to be determined. Recently, we have shown the role of nicotinamide adenine dinucleotide (NAD 1 ), a cofactor found in all living cells and nutrients, in T-cell fate regulation. 24,25 We have demonstrated that NAD 1 wasabletoregulateCD4 1 T-cell differentiation through a novel pathway that is independent of the cytokine environment and well-established transcription factors. 25 More recently, we have reported the unique immunosuppressive properties mediated by NAD 1 through systemic IL-10 cytokine production. 24 Although we characterized the role of NAD 1 in regulating T-cell fate, the precise mechanisms of action remain largely unknown. Here we show that after NAD 1 administration, MCs are able to induce exclusively CD4 1 T-cell differentiation in vitro and in vivo in the absence of antigen and major APCs. Furthermore, we demonstrate that MC-driven CD4 1 T-cell differentiation was independent of MHC class II or TCR activation. Furthermore, when assessing the functional effect of MC-mediated CD4 1 T-cell differentiation, we observed that treatment with NAD 1 resulted in profound alterations in innate and adaptive immunity and survival outcome after Listeria monocytogenes infection. Collectively, our study unravels a new cellular and molecular pathway regulating innate and adaptive immune responses that is mediated exclusively by MCs. METHODS Animals and diphtheria toxin treatment Eightto 10-week-old wild-type (WT) C57BL/6 (B6, H2 b ) mice were purchased from Charles River Laboratories (Wilmington, Mass). MC 2/2 (WBB6F1/J-Kit W /Kit W-v /J [Kit W /Kit W-v ] and Kit W-sh /HNihrJaeBsmJ [kit Wsh /Kit Wsh ]), MHC class II 2/2 (B6.129S-H2dlAb1-Ea), Wiskott-Aldrich syndrome protein (WASP) 2/2 (B6.129S6-Was tm1Sbs /J), and CD11b-DTR (B6.FVB-Tg[ITGAM-DTR/EGFP]34Lan/J) mice were purchased from Jackson Laboratory (Bar Harbor, Me). Recombination-activating gene 2 (Rag2) 2/2 gc 2/2 (B10; B6-Rag2 tm1Fwa II2rg tm1Wjl ), Rag2 2/2 , and 5C.C7 Rag2 2/2 mice (both on B10.A background) were purchased from Taconic Biosciences (Albany, NY). For CD11b 1 cell depletion with diphtheria toxin treatment, CD11b-DTR transgenic mice weighing 25 to 30 g were injected with diphtheria toxin (25 ng/g body weight; Sigma-Aldrich, St Louis, Mo) 24 hours before and 72 hours after beginning NAD 1 or PBS administration. Isolation of mouse naive CD4 1 CD44 2 CD62L 1 T cells and DCs Single-cell leukocyte suspensions were obtained from spleens of 8to 10-week-old C57BL/6 mice, and naive CD4 1 CD44 2 CD62L 1 T cells were isolated by means of flow cytometry, as described previously. 25 For isolation of CD11c 1 DCs, single-cell leukocyte suspensions were obtained from spleens of 8to 10-week-old C57BL/6 WT mice. CD11c 1 DCs were then isolated with the EasySep Mouse CD11c Positive Selection Kit (STEMCELL Technologies, Vancouver, British Columbia, Canada), according to the manufacturer’s protocol, followed by cell sorting (CD11c 1 CD11b 1 cells). L monocytogenes infection L monocytogenes bacteria (ATCC #35152) were cultured overnight at 378C in Brain Heart Infusion (Teknova, Hollister, CA) with gentle agitation. Eightto 10-week-old WT and MC 2/2 mice were infected intraperitoneally with 0.1 mL of a solution containing 1 310 7 colony-forming units (nonlethal dose) or 1310 8 colony-forming units (lethal dose) of viable L monocytogenes cells in 0.01 mol/L PBS (pH 7.4). Weight loss and survival after infection were monitored. Before infection, mice were pretreated daily for a period of 5 days with NAD 1 (40 mg administered intraperitoneally) or pretreated 5 days before infection and continuously treated daily after infection. Cultivation of bone marrow–derived mast cells Bone marrow–derived mast cells (BMMCs) from 8to 10-week-old C57BL/6J WT mice were obtained by culturing bone marrow cells from femurs and tibias. In short, mice were killed by means of cervical dislocation, intact femurs and tibias were removed, and bone marrow cells were harvested by means of repeated flushing with sterile media. BM cells were cultured in WEHI-3–conditioned medium (containing IL-3) for 90 days, at which time the cells were greater than 95% c-Kit high FcεRIa high , as determined by using flow cytometric analysis with PE-Cy7 anti-mouse FcεRIa(clone MAR-1; eBioscience, San Diego, Calif) and ef450 anti-mouse c-Kit/CD117 (clone 2B8; eBioscience, San Diego, Calif). Human MC line LAD-2 culture The human MC line LAD-2 was a generous gift from Dr A. Kirshenbaum (National Institutes of Health/National Institute of Allergy and Infectious Diseases). LAD-2 MCs were cultured in serum-free media (StemPro-34 SFM; Life Technologies, Grand Island, NY) supplemented with 2 mmol/L L-glutamine, 100 U/mL penicillin, 50 mg/mL streptomycin, and 100 ng/mL recombinant stem cell factor. LAD-2 cells were tested periodically for expression of Kit and FcεRI by using flow cytometry. Cell culture Isolated naive CD4 1 T cells or CD11c 1 DCs (1 310 6 cells per well) were cultured in 48-well flat-bottom plates in 0.5 mL of complete RPMI 1640 medium supplemented with 10% FCS, 200 mmol/L L-glutamine, 100 U/mL penicillin/streptomycin, and 4.5 g/L glucose in the presence of 10 mg/mL plate-bound anti-mouse a-CD3 (17A2) and 2 mg/mL soluble a-CD28 (37.51). NAD 1 (catalog no. N3014; Sigma-Aldrich) was diluted in PBS and J ALLERGY CLIN IMMUNOL VOLUME 142, NUMBER 6 RODRIGUEZ CETINA BIEFER ET AL 1895 J ALLERGY CLIN IMMUNOL DECEMBER 2018 1896 RODRIGUEZ CETINA BIEFER ET AL added as indicated. LPS was added at a concentration of 1 mg/mL. All recombinant cytokines and antibodies were purchased from eBioscience. After the indicated day of culture, supernatants and cells were collected and analyzed by means of ELISA and flow cytometry, respectively. Coculture of mouse naive CD4 1 T cells and BMMCs in transwell systems Noncontacting cocultured cells were prepared as follows: isolated naive CD4 1 CD44 2 CD62L 1 T cells were plated on the bottom of the 24-well transwell cell culture system (Costar, Cambridge, Mass). BMMCs were cocultured at a ratio of 1:100 in the upper transwell compartment. Cells were stimulated with NAD 1 (500 mmol/L) or PBS as a control. Naive CD4 1 T cells were cultured in complete media only or in the presence of 10 mg/mL plate-bound anti-mouse a-CD3 (clone 17A2) and 2 mg/mL soluble a-CD28 (clone 37.51). For cell-cell contact experiments, BMMCs and naive CD4 1 CD44 2 CD62L 1 T cells were cocultured (at a ratio of 1:100) in complete media with NAD 1 (500 mmol/L) or PBS as a control. Naive CD4 1 T cells were cultured in the presence of 10 mg/mL plate-bound anti-mouse a-CD3 (clone 17A2) and 2 mg/mL soluble a-CD28 (clone 37.51) or complete media only. For CD80 blockade, experiments were performed in cell-cell contact conditions, as described above, with a-CD80 neutralizing antibody (clone 16-10A1; eBioscience). Cells were cultured for 96 hours, and CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17A 1 T-cell frequencies were assessed by using flow cytometry. Isolation and coculture of human naive CD4 1 T cells Human naive CD4 1 T cells were isolated from PBMCs by means of density gradient centrifugation with the SepMate kit (STEMCELLTechnologies), followed by the EasySep Human Na€ ıve CD4 1 T Cell Isolation Kit (STEMCELL Technologies). Blood was obtained from healthy adult volunteers in accordance with guidelines of and approved by the Institutional Review Board of Beth Israel Deaconess Medical Center. Informed consent was obtained from each volunteer in accordance with the Declaration of Helsinki. Human naive CD4 1 CD25 2 CD45RA 1 CD45RO 2 CCR7 1 CD62L 1 T cells were then sorted by means of flow cytometry. Naive CD4 1 T cells were purified to greater than 98% by using cell sorting. Naive human CD4 1 T cells were then plated on the bottom of the 24-well transwell cell culture system (Costar), and LAD-2 cells were cocultured at a ratio of 1:100 in the upper transwell compartment. Cells were stimulated with NAD 1 (500 mmol/L) or PBS as a control. Naive CD4 1 T cells were cultured in complete media only or in the presence of 10 mg/mL soluble anti-mouse a-CD3 (clone 17A2) and 5 mg/mL soluble a-CD28 (clone 37.51). For cell-cell contact experiments, LAD-2 cells and naive CD4 1 CD44 2 CD62L 1 T cells were cocultured (at a ratio of 1:100) in complete media with NAD 1 (500 mmol/L) or PBS as a control. Naive CD4 1 T cells were cultured in the presence of 10 mg/mL soluble anti-mouse a-CD3 (clone 17A2) and 5 mg/mL soluble a-CD28 (clone 37.51) or complete media only. After 96 hours, CD4 1 T-cell IFN-gcytokine production was assessed by using flow cytometry. DC and macrophage depletion For DC depletion, WT, Rag2 2/2 gc 2/2 , and MC 2/2 mice were treated intravenously with 0.5 mg of liposomal clodronate (Encapsula NanoSciences, Nashville, Tenn) at days 8, 5, and 1 before NAD 1 administration. This regimen ensured depletion of greater than 99% CD11c 1 DCs, as described previously. 26,27 As a control group, mice were injected with the same amount of isotype-matched rat IgG as control mice. Flow cytometry Fluorescence-labeled anti-mouse CD4 (clone GK1.5), CD11b (M1/70), CD11c (N418), CD41 (eBioMWReg30), CD61 (2C9. G3), IL-1b(NJTEN3), IL-4 (11B11), IL-6 (MP5-20F3), IL-10 (JES5-16E3), IL-12/IL-23p40 (C 17.8), IL-17 (eBio17B7), IFN-g(XMG 1.2), latency-associated peptide (TW7-16B4), and TNF-a(MP6-XT22) were obtained from eBioscience. All antibodies were used at a concentration of 2 to 5 mg per 1 310 6 cells. To set the gates, flow cytometric dot plots were based on comparison with isotype controls, fluorescence minus one, and permeabilized and unpermeabilized unstained cells. Intracellular staining for IL-1b, IL-4, IL-6, IL-10, IL-12/IL23p40, IL-17, IFN-g, latency-associated peptide, and TNF-awas performed according to the manufacturer’s protocols. Cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences, San Jose, Calif). Flow cytometry was performed on a BD FACSCanto II (BD Biosciences) by using standard procedures, and data were analyzed with FlowJo software (TreeStar, Ashland, Ore). ELISA Mouse IL-4, IL-17A, and IFN-glevels were measured with commercial kits (eBioscience), as described previously. 24,25 RNA extraction and quantitative PCR BMMCs from C57BL/6 mice were cultured in the presence of NAD 1 (500 mmol/L), LPS, or placebo (PBS). After 24 hours of culture, cells were collected, and mRNA was extracted by using the RNAqueous extraction kit, according to the manufacturer’s protocols (Applied Biosystems, Foster City, Calif). Briefly, cells were homogenized in lysis buffer (total volume of 0.5 mL) and passed through a column. After successive washes, RNA was eluted. For real-time PCR reactions, IL-1a(Mm00439620_m1), IL-b (Mm01336189_m1), IL-4 (Mm00445259_m1), IL-6 (Mm004446190_m1), IL-10 (Mm00439616_m1), IL-12a(Mm00434165_m1), IL-23 (Mm00 518984_m1), TGF-b 1 (Mm01178820_m1), TNF-a(Mm00443260_g1), TLR2 (Mm00442346_m1), TLR4 (Mm00445273_m1), CD86 (Mm00 444543_m1), CD80 (Mm00711660_m1), inducible costimulator ligand (Mm00497237_m1), OX40 ligand (Mm00437214_m1), and IL-33 (Mm00 505403_m1) measurements were performed with TaqMan primers and probes from Applied Biosystems. The housekeeping gene glyceraldehye3-phosphate dehydrogenase (Mm99999915_g1) was used as a control. FIG 1. NAD 1 induces T-cell activation in vivo and T-cell differentiation in vitro after TCR activation. A, C57BL/6 mice were treated daily with intraperitoneal injection of 40 mg of NAD 1 or a placebo solution (PBS). After 7 days, mice were killed, and CD4 1 T cells were isolated from spleens. Frequencies and total numbers of CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17A 1 cells were analyzed by using flow cytometry. B, CD4 1 T cells (3 310 6 ) from 5C.C7 RAG-2–deficient mice were injected into Rag2 2/2 (B10.A background) mice as adoptive transfer recipients. Mice were then treated with NAD 1 or PBS for 7 days and subsequently killed to analyze frequencies and total numbers of CD4 1 IFN-g 1 cells by using flow cytometry. Cand D, Sorted naive CD4 1 CD44 2 CD62L 1 T cells were isolated from spleens of C57BL/6 mice and cultured in complete media alone with a-CD3/a-CD28 with or without IL-2 or in the presence of 50 mmol/L NAD 1 . After 96 hours, frequencies of CD4 1 IFN-g 1 cells and IFN-g, IL-4, and IL-17A cytokine secretion were assessed by using flow cytometry (Fig 1, C) and ELISA (Fig 1, D), respectively. Statistics were as follows: n 515 (Fig 1, Aand B)orn510 (Fig 1, Cand D). Data were derived from 3 independent sets of experiments. The Student ttest and ANOVA were used accordingly to compare groups: **P< .01 and ***P< .001. Data are presented as means 6SDs. FACS, Fluorescein-activated cell sorting; FITC, fluorescein isothiocyanate; PE, phycoerythrin. = J ALLERGY CLIN IMMUNOL VOLUME 142, NUMBER 6 RODRIGUEZ CETINA BIEFER ET AL 1897 RNA sequencing analysis BMMCs from C57BL/6 mice were cultured in the presence of NAD 1 (500 mmol/L), LPS (10 mg/mL; Escherichia coli O127:B8), or placebo (PBS). After 16 hours of culture, cells were collected and RNAwas extracted with the RNAqueous extraction kit, according to the manufacturer’s protocols (Applied Biosystems), as described above. cDNA was obtained by using New England Biolabs kits (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; New England Biolabs, Ipswich, Mass). Briefly, mRNAwas extracted with polyT magnetic beads, and then firstand second-strand syntheses were performed. Once double-strand cDNA was generated, DNA was cleaned up with magnetic beads and then went into library prep. Library preparation was performed by ligating on the P5 and P7 Illumina adaptors along with an index and amplifying the sequencing library by using PCR. The final library was cleaned up by using magnetic beads and made ready for sequencing. FastQ files were aligned against the Ensembl GRCm38.75 genome by using the STAR aligner (version 2.3.1z4) with default parameters. 28 Alignment files (BAM format) were filtered to retain only primary alignments (Samtools viewF 0x0100) and inspected for duplication rate with Picard tools MarkDuplicates; for downstream analyses, duplicate reads were not removed because of the high-quality input RNA. 29 Reads were quantified at the gene level by using featureCounts with annotated exon features in the Ensembl GRCm38.75 GTF file. 30,31 The resulting count matrix was normalized and analyzed for differential expression with DESeq2 software. Ingenuity Pathways Analysis (Ingenuity Systems, Redwood City, Calif) applications were used to generate canonical pathways. Statistical analysis Data are presented as means 6SDs. Statistical analysis was done with the 2-tailed Student ttest (between 2 groups) and 1-way ANOVA (among multiple groups), where appropriate. Survival was compared by using the log-rank test. Pvalues of less than .05 were considered statistically significant. Study approval Animal care and use were in accordance with the National Institutes of Health and Institutional Animal Care and Use Committee guidelines. RESULTS NAD 1 requires an intermediary signal to promote CD4 1 T-cell differentiation We have demonstrated previously that NAD 1 regulated CD4 1 T-cell differentiation independently of the cytokine milieu and well-established transcription factors. 25 It remains unclear whether NAD 1 promotes CD4 1 T-cell differentiation by acting directly on CD4 1 T cells or through an intermediate cell type. C57BL/6 WT naive mice were treated daily with intraperitoneal injection of NAD 1 or a placebo solution (PBS) and CD4 1 T-cell responses were assessed to characterize the direct effect of NAD 1 on CD4 1 T cells in the absence of antigen challenge. After 7 days, mice were killed, and systemic CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17A 1 T-cell frequencies were evaluated by using flow cytometry. Data indicated that NAD 1 administration was sufficient to promote a significant increase in frequencies of CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17 1 T cells in vivo (Fig 1,A). Of note, NAD 1 administration to Rag2 2/2 mice adoptively transferred with naive CD4 1 T cells from Rag 5C.C7 transgenic mice promoted CD4 1 IFN-g 1 T-cell differentiation in the absence of moth cytochrome C peptide challenge (Fig 1,B). Thus we next assessed in vitro whether NAD 1 directly promotes CD4 1 T-cell differentiation and cytokine production. Splenic naive CD4 1 CD44 2 CD62L 1 T cells from C57BL/6 WT mice were cultured in the presence or absence of a-CD3/aCD28 with or without IL-2 and in the presence of NAD 1 or PBS. After 96 hours of culture, CD4 1 T cells were assessed for IFN-g, IL-4, and IL-17 production by using flow cytometry and ELISA. Consistent with our previous reports, flow cytometry revealed that NAD 1 promotes a robust increase of CD4 1 IFN-g 1 Tcell frequencies after TCR activation, particularly in the presence of IL-2 (Fig 1,C). These findings were confirmed by means of ELISA, indicating increased IFN-g, IL-4, and IL-17 production after CD3/CD28 activation (Fig 1,D). Of note, the highest CD4 1 IL-17 1 T-cell frequencies and IL-17 secretion were observed after TCR activation and in the absence of IL-2, which was consistent with previous reports indicating that IL-2 inhibits T H 17 development. In contrast, no changes in CD4 1 IFN-g 1 T-cell frequencies or IFN-g, IL-4, and IL-17 cytokine production were observed with NAD 1 treatment in the absence of TCR activation (Fig 1,Cand D). Furthermore, increasing NAD 1 concentrations in the absence of TCR activation did not result in a change in CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17 1 T-cell frequencies (see Fig E1,A, in this article’s Online Repository at www.jacionline.org). Collectively, our in vivo data indicate that NAD 1 promotes CD4 1 T-cell differentiation in the absence of antigen challenge, whereas our in vitro data indicate that NAD 1 requires TCR activation, suggesting that NAD 1 -mediated CD4 1 Tcell differentiation observed in C57BL/6 WT naive mice requires an intermediary signal. NAD 1 regulates CD4 1 T-cell fate in the absence of major APCs We next investigated whether other immune cells that are known to activate CD4 1 T cells, in particular APCs, are involved in NAD 1 -mediated CD4 1 T-cell differentiation. It has been shown that ATP, a coenzyme, can promote T H 17 cells through IL-6, IL-23, and TGF-bcytokine production by CD11c 1 cells. 32 Because NAD 1 also acts as a coenzyme, we first assessed in vitro the effects of NAD 1 on CD11c 1 DCs. CD11b 1 CD11c 1 DCs were isolated from spleens of C57BL/6 mice and cultured in the presence of increasing NAD 1 concentrations or PBS. As a positive control, CD11b 1 CD11c 1 DCs were cultured in the presence of 1 mg/mL LPS. After 16 hours, cells were collected and cytokine expression was quantified by using real-time PCR. Consistent with numerous reports, stimulation of CD11b 1 CD11c 1 cells by LPS resulted in increased mRNA expression levels of IL-1a, IL-1b, IL-6, IL-23, and TNF-abut not IL-12 and a downregulation of TLR4. 33,34 More importantly, in the presence of NAD 1 , CD11c 1 CD11b 1 DCs exhibited increased mRNA expression levels of IL-1a, IL-1b, IL-6, IL10, IL-12, IL-23, TGF-b1, TNF-a, TLR2, and TLR4 in a dosedependent manner (see Fig E1,B), suggesting that NAD 1 alters CD11b 1 CD11c 1 DC activation. Thus we next assessed whether NAD 1 administration can induce CD11c 1 CD11b 1 DC activation in vivo as well. C57BL/ 6 WT mice were treated daily with 40 mg of NAD 1 or PBS by means of intraperitoneal injection. Consistent with our in vitro findings, we found that NAD 1 -treated WT mice showed increased cytokine production, including IL-1b, IL-4, IL-6, IL10, IL-12, TGF-b1, and TNF-a, by CD11b 1 CD11c 1 cells when compared with levels in PBS-treated WT mice (Fig 2). This is consistent with a previous study indicating that J ALLERGY CLIN IMMUNOL DECEMBER 2018 1898 RODRIGUEZ CETINA BIEFER ET AL FIG 2. NAD 1 treatment induces increased cytokine production by CD11b 1 CD11c 1 DCs in vivo. C57BL/6 mice were treated daily with intraperitoneal injection of 40 mg of NAD 1 or a placebo solution (PBS). After 7 days, mice were killed, and CD11b 1 CD11c 1 DCs were isolated from spleens. Frequencies of CD11c 1 IL-1b 1 , CD11c 1 IL-4 1 , CD11c 1 IL-6 1 , CD11c 1 IL-10 1 , CD11c 1 IL-12 1 , CD11c 1 TGF-b 1 , and CD11c 1 TNF-a 1 cells were analyzed by using flow cytometry. Statistics were as follows: n 515. Data were derived from 3 independent sets of experiments. Data are presented as means 6SDs. The Student ttest was used to compare groups: ***P< .001. APC, Allophycocyanin; PE, phycoerythrin. J ALLERGY CLIN IMMUNOL VOLUME 142, NUMBER 6 RODRIGUEZ CETINA BIEFER ET AL 1899 intracellular NAD 1 levels regulate TNF-acytokine production. 35 Taken together, our in vitro and in vivo data indicated that NAD 1 promotes CD11b 1 CD11c 1 DC activation and cytokine production and might play a central role in NAD 1 -mediated CD4 1 Tcell differentiation. It is well established that DCs can promote CD4 1 T-cell differentiation through cytokine and chemokine release. Moreover, like NAD 1 , ATP, a cofactor in energy metabolism, has been shown to enhance IL-6, IL-23, and TGF-bcytokine production by CD11c 1 cells and promote a T H 17 response. 32 Because our results indicated that NAD 1 promotes cytokine expression by CD11b 1 CD11c 1 DCs, we tested whether CD4 1 T-cell differentiation resulted from NAD 1 -mediated DC activation. As reported by us and others, 26,27 more than 99% of professional phagocytes, including both DCs and macrophages, were depleted in WT mice by means of injection of clodronate liposomes (see Fig E2,A,in this article’s Online Repository at www.jacionline.org). As shown in Fig E2,B, DC depletion did not abolish CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17 1 differentiation. These data suggest that NAD 1 regulates CD4 1 T-cell differentiation independently of DCs and macrophages. Although NAD 1 -mediated CD4 1 T-cell differentiation was not abolished after macrophage and DC depletion, we could not rule out compensation by other APCs, such as B cells. To characterize the role of B cells in NAD 1 -mediated CD4 1 T-cell differentiation, we used transgenic Rag2 2/2 gc 2/2 mice, which lack B, natural killer, and gd T cells. In addition, Rag2 2/2 gc 2/2 mice were subjected to depletion of DCs and macrophages. After depletion (see Fig E2,A), Rag2 2/2 gc 2/2 mice received adoptive transfer of naive CD4 1 CD44 2 CD62L 1 T cells and were subjected to treatment with NAD 1 or placebo solution. The results indicated that NAD 1 induced a significant increase in numbers of CD4 1 IFN-g 1 ,CD4 1 IL-4 1 ,CD4 1 IL17 1 ,andCD4 1 IL-10 1 cellswhencomparedwiththecontrol group of mice treated with a placebo solution (see Fig E3,A, in this article’s Online Repository at www.jacionline.org). Moreover, treatment with diphtheria toxin of CD11b-DTR transgenic mice did not abolish CD4 1 IFN-g 1 ,CD4 1 IL-4 1 , and CD4 1 IL-17 1 differentiation (see Fig E3,B). Taken together, our results indicate that NAD 1 promotes CD4 1 T-cell differentiation independently of B cells in addition to macrophages and DCs. NAD 1 administration regulates CD4 1 T-cell fate through MCs exclusively MCs have been mainly described for their role in allergic and autoimmune responses. 8,15,16 Although MCs express costimulatory molecules and secrete a myriad of chemokines and proinflammatory cytokines and have been shown to influence T-cell polarization, the mechanisms by which MCs regulate T-cell response remain unclear. Indeed, previous studies have reported that MC-deficient mice display defective CD4 1 but also CD8 1 T-cell responses after Leishmania major infection 36 and in the setting of EAE. 37 Furthermore, we have shown previously that NAD 1 protects against EAE. 25 Thus we next investigated the role of MCs in NAD 1 -mediated CD4 1 T-cell differentiation. WT and MC-deficient mice (Kit W /Kit W-v )were treated daily with intraperitoneal injection of NAD 1 . Treatment with NAD 1 in MC-deficient mice was not able to promote CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17 1 T cells, indicating that MCs were required for NAD 1 -mediated CD4 1 T-cell differentiation (Fig 3). Therefore we next sought to dissect the role of MCs in NAD 1 - mediated T-cell differentiation in vitro. BMMCs were generated, as described in the Methods section. The average yield of MCs (FcεRI 1 c-Kit 1 double-positive cells) increased with time and reached greater than 95% on day 90, as shown by using flow cytometry (see Fig E3,C). BMMCs were cultured over 6 weeks to express homogenous levels of KIT and FcεRI. 8 BMMCs were then directly cocultured with naive CD4 1 CD44 2 CD62L 1 T cells in the presence of NAD 1 or PBS. Moreover, MCs and T cells were cocultured in separate compartments by using a transwell system to determine whether these cells require cell-cell contact. As an additional control, naive CD4 1 CD44 2 CD62L 1 T cells were activated with a-CD3/a-CD28. The results indicated that in the presence of NAD 1 , MCs promoted CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17 1 T-cell differentiation in the absence of TCR activation (media plus NAD 1 ) and independently of cell-cell contact (see Fig E4 in this article’s Online Repository at www.jacionline.org). Moreover, when MCs and naive CD4 1 CD44 2 CD62L 1 T cells were cocultured in the presence of NAD 1 and T cells were activated with a-CD3/a-CD28, frequencies of CD4 1 IFN-g 1 and CD4 1 IL-17 1 T cells increased further (see Fig E4). These results were consistent with our initial findings indicating that cultured naive CD4 1 CD44 2 CD62L 1 T cells in the presence of NAD 1 and a-CD3/a-CD28 promoted CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17 1 (Fig 1,Cand D). Like DCs, MCs express costimulatory molecules and secrete a myriad of cytokines that are known to regulate innate and adaptive immune responses. Thus to determine whether MCs mediate CD4 1 T-cell differentiation through costimulatory molecules, cytokines, or both, MCs were treated with NAD 1 in vitro, and mRNA levels of OX40 ligand, inducible costimulator ligand, CD80, CD86, TNF-a, IL-4, IL-6, and IL-33 were measured by using real-time PCR. NAD 1 induced a modest increase in CD80 and IL-33 and a decrease in IL-4 mRNA expression levels by MCs (see Fig E5,A, in this article’s Online Repository at www.jacionline.org). It is well established that CD80 can either promote or inhibit activation of naive T cells by binding to CD28 or cytotoxic T lymphocyte–associated antigen 4, respectively. Therefore to assess the role of CD80 in NAD 1 -MC–mediated CD4 1 T-cell differentiation, blockade of CD80 with a neutralizing antibody was performed in vitro by using our cell-cell contact coculture system. Our findings indicated that CD80 blockade did not reduce CD4 1 IFN-g 1 or CD4 1 IL17 1 T-cell frequencies when compared with an isotype control (see Fig E5,Band C). Of note, CD80 blockade resulted in increased CD4 1 IL-4 1 T-cell frequencies (see Fig E5,D), suggesting that CD80 can play an inhibitory effect on MCmediated IL-4 cytokine production. Collectively, our findings suggest that MC-mediated CD4 1 T-cell differentiation does not require cell-cell contact or involvement of conventional costimulatory molecules, such as CD80. MC-mediated CD4 1 T-cell differentiation is conserved in the human MC line LAD-2 We next investigated whether this novel pathway is conserved in human subjects and whether human MCs could regulate human CD4 1 T-cell differentiation as well. Naive CD4 1 T cells were isolated from healthy donors and cocultured in direct contact or in J ALLERGY CLIN IMMUNOL DECEMBER 2018 1900 RODRIGUEZ CETINA BIEFER ET AL our transwell system with LAD-2 cells, a well-established human MC line. Similar to murine BMMCs, cocultures were performed in the presence of NAD 1 or PBS and with or without a-CD3/aCD28. Flow cytometry indicated that in the presence of NAD 1 , human MCs promoted CD4 1 IFN-g 1 T-cell differentiation (see Fig E6,A, in this article’s Online Repository at www.jacionline. org). Consistent with our murine BMMC data, human MCs were able to promote T H 1 polarization in the absence of cellcell contact. Taken together, these results suggest that the MCmediated CD4 1 T-cell differentiation pathway through MCs is conserved in human subjects as well. Unique gene expression profile by MCs after NAD 1 activation To unravel a potential signaling pathway involved in MCmediated CD4 1 T-cell differentiation, we next performed an RNA-sequencing analysis on BMMCs that were cultured for 16 hours in the presence of NAD 1 , LPS, or PBS. As shown in Fig 4,A, the results indicated that NAD 1 significantly upregulated 603 genes and downregulated 753 genes (with a Pvalue of less than .05) when compared with MCs treated with PBS. Moreover, when compared with LPS conditions, changes in the number of genes marked as differentially expressed were dramatic. When comparing LPS versus NAD 1 conditions, 6053 genes were significantly upregulated, and 5845 were found to be downregulated, suggesting that NAD 1 signaling machinery is distinct from LPS stimulation. As expected, dramatic changes in gene expression were observed when comparing PBS with LPS treatment (Fig 4,A). To elucidate potential genes involved in MC-mediated CD4 1 T-cell differentiation, we investigated the role of the most upregulated genes in NAD 1 versus PBS conditions and compared them with gene expression characteristics in presence of LPS (Fig 4,B). FIG 3. MCs play a central role in NAD 1 -mediated CD4 1 T-cell differentiation. C57BL/6 WT and WBB6F1/ JKit w /Kit w-v (MC-deficient) mice were treated daily with intraperitoneal injection of 40 mg of NAD 1 or a placebo solution (PBS), as indicated. After 7 days, mice were killed, and systemic levels of CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17A 1 cells were assessed by using flow cytometry. Data were derived from 2 independent experiments (n 55-10). Data represent means 6SDs. ANOVA was used to compare groups: ***P< .001. APC, Allophycocyanin; PE, phycoerythrin. J ALLERGY CLIN IMMUNOL VOLUME 142, NUMBER 6 RODRIGUEZ CETINA BIEFER ET AL 1901 FIG 4. Unique MC gene expression profile after NAD 1 activation. BMMCs from C57BL/6 mice were cultured in the presence of NAD 1 (500 mmol/L), LPS (10 mg/mL; Escherichia coli O127:B8), or placebo (PBS). After 16 hours of culture, cells were collected and RNA was extracted for RNA-sequencing analysis. A, Differential gene expression. B, Gene heat map expression profile. C, Ingenuity Pathway Analysis. J ALLERGY CLIN IMMUNOL DECEMBER 2018 1902 RODRIGUEZ CETINA BIEFER ET AL FIG E1. High doses of NAD 1 do not promote naive CD4 1 T-cell differentiation in vitro, and NAD 1 regulates CD11b 1 CD11c 1 DC cytokine production in vitro in a dose-dependent manner. A, Sorted naive CD4 1 CD44 2 CD62L 1 T cells were isolated from spleens of C57BL/6 mice and cultured in complete media with increasing concentrations of NAD 1 (500 mmol/L and 1 mmol/L) or PBS. After 96 hours, frequencies of CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17A 1 cells were assessed by using flow cytometry (n 510). Data were derived from 3 independent experiments. B, Sorted CD11b 1 CD11c 1 DCs were isolated from spleens of C57BL/6 mice and cultured (1 310 6 cells/well) in complete media and in the presence of increasing concentrations of NAD 1 (100 mmol/L and 500 mmol/L). As a positive control, CD11b 1 CD11c 1 DCs were cultured in the presence of LPS (1 mg/mL). After 16 hours of culture, cells were collected, and mRNA expression levels of IL-1a, IL-1b, IL-6, IL-10, IL-12, IL-23, TGF-b, TNF-a, TLR2, and TLR4 were determined by using real-time PCR. Values are expressed as fold expression relative to the housekeeping gene glyceraldehye3-phosphate dehydrogenase (GAPDH;n55). Data were derived from 2 different experiments. ns, Not significant. *P< .05, **P< .01, and ***P< .001, as determined by means of ANOVA, comparing the indicated groups. Data represent means 6SDs. APC, Allophycocyanin; PE, phycoerythrin. J ALLERGY CLIN IMMUNOL VOLUME 142, NUMBER 6 RODRIGUEZ CETINA BIEFER ET AL 1908.e1 FIG E2. In vivo depletion of DCs by means of liposomal clodronate administration does not alter NAD 1 - mediated CD4 1 T-cell differentiation. A, C57BL/6 WT, MC 2/2, and Rag2 2/2 gc 2/2 mice were treated intravenously with liposomal clodronate at 28 days, 25 days, and 21 day before NAD 1 treatment. Data derived from 2 independent experiments (n 55 per group). B, C57BL/6 WT mice were treated intravenously with liposomal clodronate at 28 days, 25 days, and 21 day before NAD 1 treatment. Mice were then treated with daily intraperitoneal injections of 40 mg of NAD 1 or a placebo solution (PBS). After 7 days, mice were killed, and frequencies of CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17A 1 cells were analyzed by using flow cytometry. Data were derived from 2 independent experiments (n 510). Data represent means 6SDs. ns, Not significant. The Student ttest and ANOVA were used to compare between groups: **P< .01 and ***P< .001. APC, Allophycocyanin; PE, phycoerythrin; SSC, side scatter. J ALLERGY CLIN IMMUNOL DECEMBER 2018 1908.e2 RODRIGUEZ CETINA BIEFER ET AL FIG E3. NAD 1 promotes T-cell differentiation in Rag2 2/2 gc 2/2 mice and flow cytometry of in vitro differentiation of BMMCs. A, Rag2 2/2 gc 2/2 mice were treated intravenously with liposomal clodronate at 28 days, 25 days, and 21 day before NAD1treatment. After depletion, sorted naive CD4 1 CD44 2 CD62L 1 T cells from C57BL/6 WT mice sorted by means of fluorescence-activated cell sorting were adoptively transferred (3 310 6 cells per adoptive transfer). Animals were then treated with daily intraperitoneal injections of 40 mg of NAD 1 or a placebo solution (PBS). After 7 days, mice were killed, spleens were collected, and frequencies of CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17A 1 cells were assessed by using flow cytometry. Data were derived from 2 independent experiments (n 510). B, CD11b-DTR transgenic mice weighing 25 to 30 g were injected with diphtheria toxin (DT; 25 ng/g body weight) 24 hours before and 72 hours after beginning NAD 1 or PBS administration for depletion of CD11b 1 cells. After 7 days of treatment with PBS or NAD 1 , frequencies of CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17A 1 cells were assessed by using flow cytometry. Data were derived from 2 independent experiments (n 510). C, BMMCs were obtained from femurs and tibias of 6to 8-week-old C57BL/6 WT mice. BMMCs were cultured in WEHI-3–conditioned medium over 90 days. Purities of c-Kit 1 FcεRI 1 MCs were then assessed by using flow cytometry. Data represent means 6SDs. The Student ttest was used to compare between groups: *P< .05 and **P< .01. APC, Allophycocyanin; PE, phycoerythrin; SSC, side scatter. J ALLERGY CLIN IMMUNOL VOLUME 142, NUMBER 6 RODRIGUEZ CETINA BIEFER ET AL 1908.e3 FIG E4. Murine MCs promote CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17A 1 T-cell differentiation in the presence of NAD 1 both with and without cell-cell contact. BMMCs were cocultured with isolated naive CD4 1 CD44 2 CD62L 1 T cells from C57BL/6 mice (1:100 ratio) either in cell-cell contact or in separate compartments by using a transwell system. Cells were then treated with NAD 1 (500 mmol/L) or PBS. After 96 hours, frequencies of CD4 1 IFN-g 1 (A), CD4 1 IL-4 1 (B), and CD4 1 IL-17A 1 (C) cells were assessed by using flow cytometry (n 56). Data were derived from 2 independent experiments. *P< .05, **P< .01, and ***P< .001, as determined by means of ANOVA, comparing the indicated groups. Data represent means 6SDs. APC, Allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin. J ALLERGY CLIN IMMUNOL DECEMBER 2018 1908.e4 RODRIGUEZ CETINA BIEFER ET AL FIG E5. MCs do not regulate CD4 1 T-cell differentiation in the presence of NAD 1 through CD80. A, BMMCs from C57BL/6 mice were cultured in the presence of NAD 1 (500 mmol/L) or placebo (PBS). After 24 hours of culture, cells were collected, and mRNA was extracted. mRNA levels of CD86, CD80, TNF-a, IL-4, inducible costimulator ligand (ICOS-L), OX40 ligand (OX40-L), IL-6, and IL-33 were determined by using real-time PCR. Values are expressed as fold expression relative to the housekeeping gene glyceraldehye-3-phosphate dehydrogenase (GAPDH).Band C, BMMCs were cocultured with isolated naive CD4 1 CD44 2 CD62L 1 T cells from C57BL/6 mice (1:100 ratio) in cell-cell contact conditions in the presence of a-CD80, NAD 1 (500 mmol/L), or placebo (PBS), as indicated. B-D, After 96 hours, frequencies of CD4 1 IFN-g 1 (Fig E5, B), CD4 1 IL-17A 1 (Fig E5, C), and CD4 1 IL-4 1 (Fig E5, D) cells were assessed by using flow cytometry (n 56). Data were derived from 2 independent experiments. ns, Not significant. *P< .05, **P< .01, and ***P< .001, as determined by using the Student ttest and ANOVA, comparing the indicated groups. Data represent means 6SDs. APC, Allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin. J ALLERGY CLIN IMMUNOL VOLUME 142, NUMBER 6 RODRIGUEZ CETINA BIEFER ET AL 1908.e5 FIG E6. Conserved MC-mediated CD4 1 T-cell differentiation in the human MC line LAD-2 in the presence of NAD 1 . NAD 1 induces T-cell differentiation in MHC class II 2/2 and WASP 2/2 mice. A, Human MC line LAD2 cells were cocultured with isolated human naive CD4 1 T cells from healthy donors (1:100 ratio) either in cell-cell contact or in separate compartments by using a transwell system. Cells were then treated with NAD 1 (500 mmol/L) or PBS. After 96 hours, frequencies of CD4 1 IFN-g 1 cells were assessed by using flow cytometry (n 56). Data were derived from 2 independent experiments. B, MHC class II 2/2 (B6.129SH2 dlAb1-Ea ) mice were treated daily with intraperitoneal injections of 40 mg of NAD 1 or a placebo solution (PBS). After 7 days, mice were killed, and splenocytes were harvested. Systemic frequencies of CD4 1 IFNg 1 , CD4 1 IL-4 1 , and CD4 1 IL-17A 1 cells were analyzed by means of flow cytometry. Data were derived from 2 independent experiments (n 55). C, WASP 2/2 (B6.129S6-Was tm1Sbs /J) mice were treated daily with intraperitoneal injections of 40 mg of NAD 1 or a placebo solution (PBS). After 7 days, mice were killed, and frequencies of CD4 1 IFN-g 1 , CD4 1 IL-4 1 , and CD4 1 IL-17A 1 cells were assessed by means of flow cytometry. Data were derived from 2 independent experiments (n 55). ns, Not significant. *P< .05, **P< .01, and ***P< .001, as determined by using the Student ttest and ANOVA, comparing the indicated groups. Data represent means 6SDs. APC, Allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; SSC, side scatter. J ALLERGY CLIN IMMUNOL DECEMBER 2018 1908.e6 RODRIGUEZ CETINA BIEFER ET AL FIG E7. NAD 1 alters systemic frequencies of CD4 1 T-bet 1 IFN-g 1 cells and CD11c 1 MHC class II 1 DCs after L monocytogenes infection. C57BL/6 mice were treated for 5 days with daily intraperitoneal injections of NAD 1 (40 mg) or placebo solution (PBS). After 5 days, mice were infected with a nonlethal dose of L monocytogenes (1 310 7 colony-forming units) and killed 3 days later. Spleens were collected, and frequencies of CD4 1 T-bet 1 IFN-g1(A), CD4 1 IFN-g 1 (B), and CD4 2 IFN-g 1 (C) T cells and CD11b 1 CD11c 1 MHC class II 1 DCs (D) were assessed by using flow cytometry. Data were derived from 2 independent experiments (n 55). Data represent means 6SDs. ns, Not significant. The Student ttest was used to compare between groups: *P< .05 and ***P< .001. J ALLERGY CLIN IMMUNOL VOLUME 142, NUMBER 6 RODRIGUEZ CETINA BIEFER ET AL 1908.e7