FcγRIIIa Signaling Modulates Endosomal TLR Responses in Human CD4 + T Cells Anil K. Chauhan This information is current as J Immunol published online 12 May 2017 of September 23, 2021. http://www.jimmunol.org/content/early/2017/05/12/jimmun ol.1601954 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/05/12/jimmunol.160195 Material 4.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 23, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 12, 2017, doi:10.4049/jimmunol.1601954 The Journal of Immunology FcgRIIIa Signaling Modulates Endosomal TLR Responses in Human CD4+ T Cells Anil K. Chauhan Recognition of Ab-opsonized pathogens by immune cells triggers both TLR and Fc receptor signaling. Fc receptors endocytose modified nucleic acids bound to Abs and deliver them to endosomes, where they are recognized by nucleic acid–sensing TLRs (NA-TLRs). We show that in CD4+ T cells, NA-TLRs, TLR3, TLR8, and TLR9 are upregulated by FcgRIIIa-pSyk cosignaling and localize with FcgRIIIa on the cell surface. TLR9 accumulates on the cell surface, where it recognizes CpG oligonucleotide 2006. Subcellular location of NA-TLRs is a key determinant in discriminating self versus viral nucleic acid. Hydroxychloroquine used for treating systemic lupus erythematosus and a Syk inhibitor blocked NA-TLR localization with FcgRIIIa. Engaging TLR9 with CpG oligonucleotide contributes to the development of IL17A+ and IL-21+ populations. RNA-sequencing analysis showed upreg- ulation of proinflammatory cytokines, NF-kB signaling, and heat shock protein pathway RNA transcripts. These data suggest a Downloaded from role for FcgRIIIa-pSyk cosignaling in modulating NA-TLR responses in human CD4+ T cells by affecting the amounts and cellular distribution. These events are important for understanding of autoimmune pathology. The Journal of Immunology, 2017, 198: 000–000. oll-like receptors are a therapeutic target, both in auto- in the kidney biopsies of lupus nephritis patients along with IC immunity and cancer. Endosomal TLRs recognize nucleic deposits (12). The glomeruli in the kidney biopsies of proliferative http://www.jimmunol.org/ T acids and trigger innate immune responses. Nucleic acid– lupus nephritis patients show TLR9 deposits along with ICs (13). sensing TLR (NA-TLR) signaling produces type I IFNs in plas- Subcellular compartmentalization of NA-TLRs is a mechanism by macytoid dendritic cells (pDCs) and generates autoreactive B cells, which cells discriminate between modified self and viral nucleic a major driver of systemic autoimmunity (1–3). FcgRIIa receptors acids (14). TLR9, when experimentally forced to mislocalize to in innate cells endocytose DNA/RNA immune complexes (ICs) and the cell surface in HEK 293 cells, recognizes modified self-DNA, deliver them to NA-TLRs in the endosomes. Such events have not but not the viral nucleic acid (14). However, the mechanisms that been studied in CD4+ Tcells.InCD4+ T cells, TLR expression has drive NA-TLRs to the cell surface are unknown (1). Other factors been documented only at mRNA level (4, 5). Expression of TLR that regulate nucleic acid sensing are the quantity and the sig- + proteins in CD4 T cells is debated (6).TLR agonists modulate naling threshold of TLR proteins (1, 15). In SLE, necrotic cells by guest on September 23, 2021 T cell signaling during the autoimmune response (7, 8). TLR4 ac- release high mobility group box 1 (HMGB1) protein, a DNA tivation leads to the generation of IFN-g– and IL-17A–producing chaperone, which binds with DNA-ICs and is necessary to provide cells (9, 10). The nucleic acid sensing is documented in CD4+ stability to these complexes. These HMGB1-bound DNA-ICs T cells, but the receptor that facilitates nucleic acid delivery to NA- stimulate proinflammatory cytokine production via the TLR9- TLRs is unknown (8). The role of endosomal NA-TLRs in human MyD88 pathway (16). CD4+ T cell responses has not been explored. In B cells, TLR signaling coordinated with BCRs and/or Fc NA-TLR signaling contributes to autoantibody production in the receptors signaling drive cellular responses (17). Synergistic sig- mouse models of systemic lupus erythematosus (SLE), Sjo¨gren’s naling by FcgRIIa/TLR9 pathway generates a distinct signal syndrome, and arthritis (11). NA-TLR protein deposits are present compared with the BCR/TLR 9 pathway (18). Enhanced presence of NA-TLRs on the CD4+ T cell surface can establish a cross-talk, Division of Adult and Pediatric Rheumatology, Saint Louis University School of which raises the possibility of synergized signaling with the TCRs Medicine, St. Louis, MO 63104; and Department of Molecular Microbiology and and/or Fc receptors. We have earlier shown that the phosphory- Immunology, Saint Louis University School of Medicine, St. Louis, MO 63104 lation of spleen tyrosine kinase (Syk) by FcgRIIIa triggers the ORCID: 0000-0002-9487-5283 (A.K.C.). differentiation of human naive CD4+ T cells into proinflammatory Received for publication November 16, 2016. Accepted for publication April 13, IFN-g and IL-17A–producing effector T cells (19–22). FcgRIIIa- 2017. pSyk cosignaling also induces expression of NA-TLRs and type 1 This work was supported by National Institutes of Health Grant R01 A1098114 (to IFN RNA transcripts (19). The role for FcgRIIIa-pSyk as a co- A.K.C.). stimulatory signal in NA-TLR responses that contribute to the Address correspondence and reprint requests to Dr. Anil K. Chauhan, Division of + Adult and Pediatric Rheumatology, Saint Louis University School of Medicine, 1402 CD4 T cell differentiation has never been investigated. South Grand Boulevard, St. Louis, MO 63104. E-mail address: [email protected] In this study, we demonstrate that the FcgRIIIa cosignaling ac- The online version of this article contains supplemental material. cumulates functional TLR9 on the cell surface. Purified membrane Abbreviations used in this article: ATCC, American Type Culture Collection; DIC, preparations from cells activated via FcgRIIIa cosignaling show full- differential interference contrast; HCQ, hydroxychloroquine; HMGB1, high mobility length noncleaved 130 kDa TLR9 protein, whereas the cell lysates group box 1; HSP, heat shock protein; IC, immune complex; IP, immunoprecipitate; NA-TLR, nucleic acid–sensing TLR; ODN, oligodeoxynucleotide; pDC, plasmacy- show both noncleaved and cleaved forms of TLR9. TLR9 protein on toid dendritic cell; RNA-seq, RNA-sequencing; RT, room temperature; SLE, sys- the cell surface binds to CpG oligodeoxynucleotide (ODN) and en- temic lupus erythematosus; STED, stimulated emission depletion; Syk, spleen hances the differentiation of naive CD4+ T cells. NA-TLR upregu- tyrosine kinase. lation was observed in both primary human CD4+ T cells and P116 2/2 Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 cells, a ZAP-70 mutant of Jurkat T cells, which can signal only www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601954 2FcgRIIIa-pSyk SIGNALING IN NA-TLR RESPONSES via Syk. A Syk inhibitor, P505, and hydroxychloroquine (HCQ), ment. Cells were analyzed for IL-17A and IL-21 production in flow drugs used for the treatment of SLE, inhibit the cell surface local- analysis using anti–IL-17A-PE-R700 and anti–IL-21-BV421 (BD Biosci- ization of NA-TLRs with FcgRIIIa. HMGB1 and MyD88 proteins ences). A two-tailed paired nonparametric t test was performed using Prism software to analyze the statistical difference in cytokine-producing that participate in nucleic acid sensing and TLR signaling associate populations. Conjugates were titrated and flow compensation was done with FcgRIIIa protein on the cell surface poststimulation via FcgRIIIa using PE- or allophycocyanin-conjugated isotype controls. receptor signaling. RNA-sequencing (RNA-seq) analysis shows the Cell staining for confocal imaging upregulation of IFNs, IFN-inducible genes, NF-kB signaling–driven proinflammatory cytokines, and heat shock protein (HSP) RNA Postactivation cells were harvested and washed with PBS and fixed in 4% transcripts. These results provide new insight into the role of FcgRIIIa formaldehyde for 15 min at RT. Cells were then permeabilized using cold methanol at 220˚C for 10 min. Cells were kept for 1 h in 1% BSA/PBS signaling in T cell biology. and stained using Ag-specific primary Abs at a dilution of 1:50 in BSA/ PBS for 1 h and developed using anti-species isotype-specific Alexa Fluor Materials and Methods fluorochrome conjugate (Life Technologies) at appropriate dilutions. Anti- Cells and cell lines TLR3, -TLR8, and -TLR9 Abs were purchased from R&D Systems. Anti- MyD88 and anti-HMGB1 were obtained from Cell Signaling Technologies Blood was collected with informed consent in the Saint Louis University (rabbit monoclonal). As a control for labeled ICs, we used human IgG- Rheumatology clinic. P116 cells (CRL-2676; American Type Culture conjugated with Alexa Fluor 488. Isotype controls for mouse monoclonal Collection [ATCC]), an acute T cell leukemia ZAP-70 mutant line, was and purified rabbit IgG fraction were used as negative controls (Sigma grown as per guideline from ATCC. Jurkat, HEK 293T, THP1, and Raji Chemicals). For CpG ODN 2006–Alexa Fluor 488 staining, 1:200 dilution were also obtained from ATCC. The PBMCs were isolated using Histo- of primary stock was used to give a final concentration of 0.3 mM.