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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

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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 © 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. The paque gradient (Sigma). CpG ODN 2006 binding was performed in live cells for 10 min, costained with anti-TLR9, and fixed/permeabilized. For IC binding, the Alexa Flour Downloaded from ICs and C5b-9 555–labeled ICs were coincubated for 10 min with CpG ODN 2006 Alexa Fluor 488; cells were washed and then fixed for 15 min before image ICs were purified from 50 ml of pooled serum or plasma from 5–10 SLE capture. patients with high levels of complement opsonized ICs. The purification procedures for ICs and C5b-9 have been previously described (23–25). The Western blot analysis nature of the ICs has been characterized for binding to FcgRIIIa in multiple cell types, compared with aggregated human g-globulin and anti-FcgRIIIa Protein lysates were prepared as reported earlier (22). Immunoprecipitates Ab (clone 3G8) (22). In addition, ICs were compared for their potential to (IPs) were generated using 100 mg of protein lysates using 1.0 mg of anti- http://www.jimmunol.org/ activate CD4+ T cells with in vitro formed Ova–anti-Ova ICs (23). FcgRIIIa/b mAb. An appropriate amount of these IPs was run on 4–12% SDS-PAGE gels and after protein transfer, and probed using 1:2000 di- T cell isolation and activation lution of anti-MyD88 (D80F5) or anti-HMGB1 (3935S; Cell Signaling Technologies). The blots were developed using anti-rabbit IgG-HRP at a PBMCs were isolated within 12 h of sample collection, and monocytes were dilution of 1:20,000 and chemiluminescent substrate (Millipore). For removed by overnight plating in a culture dish. The next day, the CD4+ TLR9 Western blotting, we prepared the cell membrane using the Qpro- CD45RA+ cells were purified using naive CD4+ T cell isolation kit II (130- teome plasma membrane kit validated using Cadherin (37601; Qiagen). 094-131; Miltenyi Biotec). CD4+ T cells were isolated using positive The primary monoclonal anti-TLR9 (clone eB72-1665) was used at a selection magnetic beads isolation kit (130-045-101; Miltenyi Biotec). 1:2000 dilution and anti-mouse IgG-HRP secondary at 1:20,000. The IPs Purified naive CD4+ T cells represented a .97% pure population. Purified generated using anti-FcgRIIIa/b mAb were probed with anti-TLR9 sheep by guest on September 23, 2021 cells were maintained in culture with 20 U of IL-2 for 2 d. Thereafter, polyclonal (AF3658; R&D Systems) at 1:5000 dilution overnight at 4˚C these cells were stimulated with plate-bound ICs at 10 mg/ml and purified and a secondary anti-sheep IgG-HRP at a dilution of 1:20,000 for 1 h at soluble C5b-9 at 2.5 mg/ml for each 1 3 106 cells in the presence of plate- RT. We also used a second Ab rabbit anti-TLR9 at 1:2000 to confirm TLR9 bound anti-CD3 at 0.25 mg/ml. Positive control cells were stimulated with protein in the IPs (2254; Cell Signaling Technologies). plate-bound 2 mg/ml anti-CD28 (clone 28.2) and 0.25 mg/ml anti-CD3 (clone OKT3; eBioscience). For inhibition, cells were cultured in 25 nM RNA-seq transcriptome analysis of P505, a Syk inhibitor (PRT06207; Selleckchem), and 50 mM HCQ (263010250; Acros Organics). Cells were cultured for 48 h in the presence Cells were purified and differentiated using a mixture of four cytokines, IL- of IL-2 (20 IU), for each 1 ml of medium (PeproTech). After 48 h, cells 1b, IL-6, IL-23 and TGF-b, as reported earlier (19). RNA was purified were restimulated and processed for staining at 96 h. from naive CD4+ T cells activated with anti-CD3+anti-CD28 or anti-CD3 +ICs+C5b-9 using an RNA purification kit from Clontech Laboratories. A Flow cytometric analysis total of 20 ng of RNA was used to generate the RNA-seq libraries using the Ion AmpliSeq Transcriptome Human Gene Expression Kit as per manu- Cell surface staining was done using anti–CD4-PE-eFluor-610 (61-0049- facturers’ recommendations. The library was sequenced on Ion Torrent 42; eBioscience) for 30 min as per manufacturer’s recommendation. ICs Sequencer by the Genomic Core (Saint Louis University School of Med- were labeled using Alexa Fluor 488 5-TFP ester (A3005) or Alexa Fluor icine). Statistical analysis was performed between two treatments using 555 succinimidyl ester (A3007) and Alexa Fluor 594 succinimidyl ester Fisher exact t test and subjected to gene ontology annotation analysis using (A3008) from Molecular Probes. The IC conjugates were in the range of R. The analysis for differential expression and pathways were performed. 23 to 30 mM fluorochrome/protein ratio. These ICs were used in flow and Heat maps and cluster analysis were performed using CIMminor (http:// cell staining. For staining with CpG ODN 2006-Alexa Fluor 488, we used nci.nih.gov). Reads per million from the RNA-seq data were used as input a 0.3 mM final concentration from a stock solution previously prepared and for the generation of the heat maps in Figs. 9 and 10. Total normalized stored in TE buffer. CpG ODN staining was carried out at room temper- reads obtained from FcgRIIIa or CD28 cosignaling were divided with ature (RT) for 10–15 min in live cells in FACS buffer containing 2% FCS. reads obtained from untreated sample of the same subject. Relative fold TLR9 staining was performed and confirmed using two mAb conjugates, change was used for generating heat maps. Genes upregulated by anti–TLR9-PE (eBioscience) and anti–TLR9-allophycocyanin (clone FcgRIIIa-pSyk signal were subjected to gene ontology analysis, and net- eB72-1665; BD Biosciences). For studying the effect of CpG ODN 2006, work diagram was generated using STRING 10.0 software (28). cells were stimulated and polarized in the presence of IL-1b, IL-6, IL-23, and TGF-b as reported by us earlier (19). Purified naive CD4+ T cells at a density of 0.25 M cells per well were plated in 96-well plates (Nunc) and Results activated as previously reported (19, 22). On day 7, these cells received Activated CD4+ T cells express FcgRIIIa CpG and non-CpG ODN at a final concentration of 5 mM/ml. This concentration was used because human PBMCs and CD4+ T cells respond Immune cells express both membrane and cytosolic Fc receptors, at this concentration for cytokine production (26, 27). ODN 2006 59- which play a central role in the intracellular routing of proteins for TC*GTC*GTTTTGT C*GTTTTGTC*GTT-39 (CpG ODN) and control degradation to the proteasome or Ag presentation pathways (29). ODN 59-TGCTGCTTTTGTGCTTTTTGTGCTT-39 were synthesized by We have shown the expression of FCGR3A RNA transcripts and IDT. Phosphorothioate CpG ODN 2006 was also purchased with 3Alex- + aF488N modification at 39 end for use in flow analysis and microscopy. protein, both in the activated primary human CD4 T cells and Flow staining for cytokine production was performed at 48 h posttreat- T cell lines (19, 21, 22). In SLE patients, pathogenic ICs are The Journal of Immunology 3 opsonized with complement fragments C3b/iC3b, a ligand for surface staining of all three NA-TLRs was observed upon FcgRIIIa- CD11b. However, SLE-ICs bound to CD4+ T cells via FcgRIIIa pSyk cosignaling in primary human CD4+ T cells. A similar staining and not via CD11b (also known as ITGAM or CR3; A.K. Chau- pattern was again observed for these NA-TLRs in P116 cells, a han, unpublished observations). Naive CD4+ T cells from SLE ZAP702/2 mutant cell line, which can only signal using Syk. P116 is patients upon activation on day 4 showed 3.82 and 1.97% of IC a mutant Jurkat cell line, which is the most studied cell type for T cell binding populations, representing the FcgRIIIa-expressing cells. activation and function (32). P116 cells showed marked intense NA- On postactivation day 9, these same cells showed 12.3 and 15.9% TLR staining upon FcgRIIIa-pSyk cosignaling at the cell surface of IC-bound CD4+ T cells, respectively (Fig. 1A). Naive CD4+ (Fig. 3B, 3D, 3F). This was also observed in DIC images for TLR9 T cells isolated from normal subjects also show similar results staining (Fig. 3I). The isotype control for Abs used for NA-TLR (data not shown). Induced expression of FcgRIIIa was observed in staining and monomeric IgG conjugated to Alexa Fluor 488 with cells activated using either anti-CD3+anti-CD28 (CD28 costimu- the same protein/fluorochrome ratio as for the ICs did not show lation) or anti-CD3+ICs+C5b-9 (FcgRIIIa-pSyk costimulation) staining (Fig. 3M–O). Minor peripheral staining for TLR8 was ob- (22). High-resolution stimulated emission depletion (STED) mi- served from CD28 cosignaling compared with TLR3 and TLR9 croscopy of fixed and permeabilized cells show staining for staining in P116 cells (Fig. 3C). Lamp1 staining was observed only in FcgRIIIa, both on the cell surface and in the cytosol (Fig. 1B). the cytosol (Fig. 3G). LysoTracker deep red showed staining for ly- FcgRIIIa receptors in these cells show capping, a phenomenon sosomes, which was also the site for NA-TLR staining in FcgRIIIa- associated with low-affinity Fc receptors. FcgRIIIa staining was pSyk costimulated cells (Fig. 3H). In both human CD4+ T cells and also observed in live cells on the cell surface within 10 min of P116 cells, FcgRIIIa-pSyk cosignaling enhanced NA-TLR staining at incubation with labeled ICs. the cell surface. Untreated cells do not show the staining for these Downloaded from proteins, which is consistent with our published RNA transcript data NA-TLRs on the cell surface (Fig. 3J–L) (19). The enhanced expression of TLR3, TLR8, and + In human CD4 T cells, NA-TLR expression has not been reported TLR9 RNA transcripts was observed using RT2-PCR arrays (19). at the protein level (6, 30). We examined the presence of NA-TLR ThesedatasuggestthattheFcgRIIIa-pSyk cosignaling influences the + proteins in CD4 T cells treated with either anti-CD3+anti-CD28 amount and the subcellular distribution of NA-TLR proteins in ac- + or anti-CD3+ICs+C5b-9. We stained naturally occurring NA-TLR tivated human CD4 T cells. http://www.jimmunol.org/ proteins in human CD4+ T cells and P116, a CD4+ T cell line. This was done to alleviate concerns such as mislocalization of over- TLR9 recognizes CpG ODN on the cell surface expressed fluorescently tagged recombinant proteins (31). Purified To confirm the presence of the TLR9 on the cell surface, we next human CD4+ T cells stimulated via CD28 cosignaling showed less examined the presence of TLR9 protein in purified membranes intense staining for NA-TLRs, which was restricted to the cytosol. isolated from activated CD4+ T cells and Raji cells (Burkitt’s These cells also showed prominent FcgRIIIaexpressiononthecell lymphoma), a cell line that expresses TLR9 protein. Western blot surface and moderately in the cytosol (Fig. 2Aa, 2Ae, 2Ai). However, analysis showed two protein bands in the cell lysates. However, upon FcgRIIIa-pSyk costimulation, CD4+ T cells showed cell surface the membrane preparations showed only an ∼130-kDa protein staining for all three examined NA-TLRs. In addition to the cell (Fig. 4A, left panel). These data suggest that the uncleaved form by guest on September 23, 2021 surface, staining was observed in endosomes (Fig. 2Ab, 2Af, 2Aj). of TLR9 protein accumulates at the cell surface. The examination Differential interference contrast (DIC) confocal images confirmed of IPs generated using anti-FcgRIIIa/b mAb also showed a the cell surface staining of NA-TLRs (Fig. 2Ac, 2Ag, 2Ak). Exam- prominent 130 kDa band and a lower faint band using two anti- ination of Z-stacks of Huygens deconvolution images further con- TLR9 polyclonal Abs; the image was shown with sheep anti- firmed these findings (Fig. 2B). A small amount of TLR8 staining TLR9 (Fig. 4A, right panel). The FcgRIIIa protein in the IPs was observed upon CD28 costimulation. Strong peripheral cell has been characterized and reported previously by us (22).

FIGURE 1. Human CD4+ T cells express FcgRIIIa. (A) Puriﬁed activated human naive CD4+ T cells on day 4 show 3.82 and 1.97% cells that express FcgRIIIa. These same cells on day 9 show an increased expression for FcgRIIIa to 12.3 and 15.9%. Shown are results from two of four patients analyzed in two separate experiments. (B) STED image shows both membrane (red arrow) and cytosolic FcgRIIIa (yellow arrow). 4FcgRIIIa-pSyk SIGNALING IN NA-TLR RESPONSES

FIGURE 2. FcgRIIIa-pSyk signaling induces NA-TLR protein expression and locates them to the cell surface in human CD4+ T cells. (A) NA-TLRs are observed on the cell surface along with IC binding in cells costimulated by FcgRIIIa-pSyk and not by CD28 cosignaling. Cells costained with Alexa Fluor 488–labeled ICs (green) and NA-TLRs (red), TLR3 (Aa), TLR8 (Ae), and TLR9 (Ai) after anti-CD3+anti-CD28 treatment and TLR3 (Ab), TLR8 (Af), and TLR9 (Aj) upon anti-CD3+ICs+C5b-9 treatment. DIC images for TLR3 (Ac), TLR8 (Ag), and TLR9 (Ak); images are the same as in (Ab), (Af), and (Aj). Nonstimulated cells stained for TLR3 (Ad), TLR8 (Ah), and TLR9 (Al). (B) Huygens Z-stack images show NA-TLRs migrated to the cell surface upon FcgRIIIa-pSyk costimulation (Bb, Bd,andBf) and not from CD28 cosignaling (Ba, Bc,andBe). Data are representative of three independent experiments. Downloaded from

Lysosomal cathepsins and asparagine endoprotease cleaves TLR9 expression of TLR9, which bound to CpG ODN (Supplemental to its mature form that is present in the endosomes (33). The Fig. 1). chimeric TLR9 expressed on the surface of HEK 293 cells recognizes CpG DNA even when endosomal acidiﬁcation is blocked Syk inhibitor P505 and HCQ inhibit NA-TLRs accumulation at (14). Using CpG ODN 2006-Alexa Fluor 488 as a ligand, we the cell surface http://www.jimmunol.org/ observed that TLR9 bound to its ligand on the cell surface in In ﬂow binding analysis, FcgRIIIa-pSyk costimulated P116 cells activated human CD4+ T cells, which was also the site for the show enhanced TLR9 expression when examined at 96 h, which FcgRIIIa protein (Fig. 4B). Binding of CpG ODN 2006 occurred was successfully inhibited by P505 (Fig. 5C). We further exam- within 10 min in live cells, and it localized with TLR9 on the cell ined the effect of P505 and HCQ on FcgRIIIa and NA-TLR lo- surface (Fig. 4C). The average size of the observed ODN 2006 calization using high-resolution STED imaging (Fig. 6A). spot was 350 nm. NA-TLR spots ranged from 200 to 300 nm. Staining and overlay histograms in activated P116 cells showed These studies were performed with STED high-resolution imaging, the FcgRIIIa protein localized together with TLR3, TLR8, and

which can recognize structures as small as 40 nm. The size mea- TLR9 proteins (Fig. 6A). P505 disrupted this localization of NA- by guest on September 23, 2021 sured includes primary and secondary labeled Ab protein, which TLR proteins with FcgRIIIa on the cell surface in activated P116 contributed to the size of the observed spots. Both cell images cells (Fig. 6B). TLR8 showed patchy staining with FcgRIIIa, and histogram overlays suggest that the TLR9 and FcgRIIIa are which was largely restricted to cytoplasm in P505-treated cells present together on the same site at the cell surface (Fig. 4). We (Fig. 6B, middle panels). HCQ inhibits the endosome acidification also confirmed the presence of TLR9 by flow analysis using CpG and blocks NA-TLR signaling (34). Similar to P505, HCQ also ODN 2006 binding to live P116 cells and staining them for TLR9. disrupted the association of NA-TLRs with FcgRIIIa on the cell This analysis showed an increase in double-positive TLR9+ODN+ surface. Both drugs reduced the intensity and size of NA-TLR cell population upon FcgRIIIa-pSyk activation (Fig. 5A). A sim- protein spots, which were comparatively smaller than those ob- ilar enhanced CpG ODN binding from 4.23 to 34.7% was also served in the untreated cells (Fig. 6B, P505 and HCQ panels). observeduponactivationinthehumannaiveCD4+ T cells P505 also reduced the FcgRIIIa expression. Cells treated with isolated from SLE subjects (Fig. 5B). These cells show enhanced HCQ compared with P505 showed higher level of FcgRIIIa

FIGURE 3. NA-TLRs locate at the cell surface in P116 cells. P116 cells show enhanced protein staining for NA-TLRs on the cell surface from FcgRIIIa-pSyk cosignaling TLR3 (B), TLR8 (D), TLR9 (F) and not from CD28 cosignaling TLR3 (A), TLR8 (C), and TLR9 (E). LAMP1 (G) and LysoTracker deep red show cytoplasmic stain (H). Stain- ing for TLR9 on DIC background (I). Non- stimulated cells stained for TLR3 (J), TLR8 (K), and TLR9 (L). Isotype controls (M and N) and monomeric IgG labeled with Alexa Fluor 488 (O) do not show staining. Data are representative of three independent experiments. The Journal of Immunology 5

Flow cytometry analysis of the NIH 3T3 cells also did not show any binding of labeled ICs to cells (data not shown), further corroborating the speciﬁcity of FcgRIIIa and MyD88 association in CD4+ T cells upon FcgRIIIa-pSyk costimulation. Next, we examined the subcellular distribution and the association of HMGB1 with FcgRIIIa. Similar to MyD88, in the human CD4+ T cells upon costimulation via FcgRIIIa-pSyk, HMGB1 moved to the cell surface, which did not occur upon CD28 costimulation (Fig. 7A, lower panel). FcgRIIIa and MyD88 or HMGB1 association was further examined in P116 cells to ex- clude the possibility of FcgRIIIa bearing contaminating cells in human preparations. P116 cells gave results similar to human CD4+ T cells (repetitive result not shown). IPs prepared with anti- FcgRIIIa mAb showed the presence of HMGB1 in the activated P116 cells, PBMCs, and THP1 monocytes (Fig. 7Bb). Our results thus suggest that both MyD88 and HMGB1 are upregulated by FcgRIIIa-pSyk costimulation in CD4+ T cells and associate with FcgRIIIa. It will be of interest to further investigate whether

HMGB1 overproduction by FcgRIIIa-pSyk cosignaling results in Downloaded from the secretion of HMGB1. HMGB1 protects extracellular nucleic acids from DNase cleavage.

FIGURE 4. CpG ODN 2006 binds to TLR9 on the cell surface in activated naive CD4+ T cells. (A) Membrane protein fraction shows

uncleaved TLR9 of 130 kDa mass in Western blotting (lanes 5–7). Cell http://www.jimmunol.org/ lysate shows both cleaved and noncleaved protein; lane 1, Raji cells; lane 2, HEK 293 cells; lane 3, Jurkat stimulated; and lane 4, P116 stimulated. Membrane protein fractions: lane 5, Raji cells; lane 6, P116 cells; and lane 7, P116-stimulated cells. Anti-TLR9 sheep polyclonal detects protein in anti-FcgRIIIa/b IPs (right panel). Lane 8, P116 lysate without anti- FcgRIIIa/b Ab; lane 9, P116 cells; lane 10, P116 cell stimulated; and lane 11, P116 cell stimulated with anti-CD28. Shown is one of two experiments. (B) CpG ODN 2006 binds to sites that show TLR9 staining on the cell surface (arrow) (Ba), magnified view (Bb), and histograms (Bc). (C) CpG ODN 2006 (green, Alexa Fluor 488) bound to the sites that also show by guest on September 23, 2021 staining with labeled ICs (arrow) (red, Alexa Fluor 555) (Ca), magnified view (Cb), and histograms (Cc). Data are representative of two independent experiments. expression. These data suggest that both P505 and HCQ influence FcgRIIIa-pSyk signaling in CD4+ T cells. MyD88 and HMGB1 associate with FcgRIIIa TLR8 and TLR9 both signal via MyD88, an adaptor protein, whereas TLR3 uses TRIF. To further confirm the role for FcgRIIIa- pSyk cosignal in NA-TLR responses, we examined the association of MyD88 with FcgRIIIa in human CD4+ T cells costimulated with either CD28 or FcgRIIIa-pSyk cosignaling. Costaining for MyD88 and FcgRIIIa in permeabilized human CD4+ T cells at 96 h after costimulation by FcgRIIIa-pSyk shows both of these proteins to localize at the cell surface. MyD88 remained in the cytosol upon CD28 costimulation, whereas FcgRIIIa-pSyk costimulation moved MyD88 to the cell surface (Fig. 7A). In innate cells, TLR4 signaling triggers MyD88 to form a signaling complex “myddosome” at the cell surface (35). We also examined the association of FcgRIIIa with MyD88 by Western blot analysis of the IPs generated using anti-FcgRIIIa mAb. MyD88 associated FIGURE 5. FcgRIIIa-pSyk cosignaling upregulates TLR9 expression. with FcgRIIIa in P116 cells, PBMCs (SLE), and THP1 monocytes Binding of labeled ODN 2006 in live P116 cells. (A) Flow staining in P116 (Fig. 7Ba). A small increase was also observed upon CD28 co- cells upon FcgRIIIa-pSyk costimulation shows binding of Alexa Fluor 488–CpG ODN 2006 within 10 min in TLR9+ (22%) cells. (B) Human stimulation. A pronounced increase in MyD88 association with naive CD4+ T cells costimulated via FcgRIIIa-pSyk on day 5 show en- FcgRIIIa was observed in the IPs prepared from P116 cells upon hanced binding of labeled CpG ODN (34.7%, right panel) compared with FcgRIIIa-pSyk costimulation. These findings are consistent with untreated control cells (4.23%, left panel). (C) Flow staining of activated the cell staining results and the observed increase in RNA tran- P116 cells for TLR9, untreated control, stimulated twice with ICs+C5b-9 scripts in human CD4+ T cells reported by us earlier (19). NIH (center) and treated with P505 during stimulation. Shown are one of four 3T3 cells, a negative control, did not show MyD88 in the IPs. for (A) and (B) and one of two experiments in (C). 6FcgRIIIa-pSyk SIGNALING IN NA-TLR RESPONSES

FIGURE 6. P505 and HCQ prevent localization of NA-TLRs with FcgRIIIa. (A) In activated P116 cells, FcgRIIIa (green, IC binding) coloc- alize with NA-TLRs (red) after 96 h of anti-CD3 +ICs+C5b-9 treatment. Histogram shows TLR stain (upper panel) and IC stain (lower panel) in regions marked with arrow. TLR3 (a and b), TLR8 (c and d), and TLR9 (e and f). (B) STED images of P116 cells, as in (A) treated with P505 and HCQ. FcgRIIIa was more impaired by P505 treatment compared with HCQ. Data are representative of two independent experiments.

Cosynergistic signaling by FcgRIIIa-pSyk and TLR9 regulation of I-kB kinase/NF-kB signaling, were differentially upregulates IL-17A and IL-21 population expressed at p = 2.7e6. Syk augments type 1 IFN signaling via To test whether these two immune receptors functionally cooperate STAT1 and IRF9 in macrophages (36). Cluster analysis showed in the CD4+ T cells, we examined the production of two key increased expression of proinflammatory cytokines that contribute Downloaded from cytokines. IL-17A drives inflammation and IL-21 supports auto- to the various autoimmune disease pathologies. We observed a antibody production by B cells. Both of these cytokines are ele- vated in SLE patients. We examined naive CD4+ T cells isolated from both SLE and normal subjects. Both ODN (p ,0.0181) and CpG ODN 2006 (p ,0.0005) showed a statistically significant increase in the IL-17A–producing cells, compared with cells that http://www.jimmunol.org/ received CpG ODN alone (Fig. 8A, 8D). Even though the CpG ODN showed an increase in the IL-17A–producing cells over the ODN-treated group, it was not statistically significant. The IL-21– producing cells also showed a statistically significant increase in both ODN at p ,0.0016 and the ODN CpG-treated (p , 0.0004) group compared with cells that received CpG ODN alone (Fig. 8B, 8D). The IL-21–producing cells also showed a statistically significant increase upon CpG ODN treatment compared with ODN treatment (p , 0.003). This increase was also seen in by guest on September 23, 2021 CD4+IL-17A gated cells analyzed for IL-21 production at p , 0.0009 (Fig. 8C, 8D). These increases in cytokine-producing cells were observed in both naive CD4+ T cells obtained from SLE patients and normal subjects. These results suggest that both FcgRIIIa-pSyk and TLR9 cosignaling together contributed to the IL-17A and IL-21 production. Cosignaling by FcgRIIIa-pSyk upregulates inflammatory pathway genes To ascertain whether FcgRIIIa-pSyk is a distinct cosignal from CD28, we performed RNA-seq transcriptome analysis of naive human CD4+ T cells activated from two costimulatory pathways in paired samples. RNA-seq analysis showed statistically significant upregulation of several key inflammatory pathways from FcgRIIIa-pSyk cosignaling compared with CD28 cosignaling. Several categories such as cellular response to DNA damage (GO:0006874, 76 genes at p = 6.04e9), dsRNA binding (GO:0003723, 19 genes at p = 1.309e9), and RNA binding (GO:0003723, 260 genes at p = 2.2e16) were upregulated. These data suggest a nucleic acid–sensing activity from FcgRIIIa-pSyk cosignaling. RNA-seq analysis also showed an increase in reads per million for IL-27, IFN-l2, IFN-l3, IFN-l, 1IFN-v1, and IFN-b1 from the FIGURE 7. FcgRIIIa-pSyk cosignaling recruits MyD88 and HMGB1 to the cell surface. (A) Huygens Z-stack images for MyD88, arrow (red) and FcgRIIIa-pSyk costimulation (Fig. 9A). Several type 1 IFNs + showed marked increases, that is, IFN-a14, IFN-a1, IFN-a22P, HMGB1, arrow (red) with labeled ICs (green) in human CD4 T cells. Both MyD88 and HMGB1 moved to the cell surface and localized with IFN-1a, and IFN-a13 (Fig. 9B). NF-kB transcription factor tar- FcgRIIIa in cells costimulated by FcgRIIIa-pSyk and not with CD28. (B) gets showed increased RNA transcription upon FcgRIIIa-pSyk IPs prepared with anti-FcgRIIIa mAb and probed with anti-MyD88 (Ba) cosignaling (Fig. 9C). FcgRIIIa-pSyk cosignaling differentially and anti-HMGB1 (Bb). Both proteins were observed in activated P116 expressed 44 genes in the category GO:0051092, representing cells and positive controls, PBMCs, and THP1 monocytes and not in positive regulation of NF-kB transcription factor activity NIH3T3 (negative control) cells. Data are representative of three inde- (p = 2.5612). In addition, 55 genes in GO:0043123, a positive pendent experiments. The Journal of Immunology 7

reports, where we show increase in proinflammatory cytokines in the culture supernatants from FcgRIIIa-pSyk cosignaling (19, 22). These patients showed enhanced expression of IFN-inducible genes, IFIT1, IFITM1, IFITM10, IFI6, IFIH1, and IFITM5 RNA transcripts (Fig. 10A). HSPs, which are grouped as danger signal proteins with HMGB1 termed as “alarmins,” are induced in innate cells during inflammation. HSPs genes were upregulated by FcgRIIIa-pSyk cosignaling (Fig. 10B). Comparable with the cytokine profile, patients 2, 3 and 4 showed a strong signature for the HSP expression upon FcgRIIIa- pSyk cosignaling compared with CD28 cosignaling (Fig. 10B). HSP transcripts for DNAJP5, DNAJB6, DNAJC2, DNAJC5, DNAJC5B, HSF2, HSFY2, HSP90AB3P, HSPB7, HSPE1, S100A7A, S100A7A, S100A7L2, and S100G showed statically significant increase at p , 0.005. Ontogeny analysis of 31 selected HSP genes that were highly upregulated by FcgRIIIa-pSyk cosignaling represent response Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 8. CpG ODN 2006 enhances cytokine production. Naive CD4+ T cells from SLE patients (shown are one in four) and normal subjects (shown are one in four) show enhanced IL-17A production from ODN and CpG ODN treatment upon FcgRIIIa-pSyk cosignaling compared with cells treated with CpG ODN alone (A). CpG ODN-treated cells show enhanced IL-21+–producing cells compared with cells treated with CpG ODN alone (B). Percentage of double-positive IL-17A+IL-21+ cells was enhanced from CpG ODN treatment (C). Statistically signiﬁcant increase in IL-17A+, IL-21+, and IL17A+IL21+ cells was observed from ODN and CpG ODN treatment compared with cells treated with CpG ODN alone (n =8, combined four normal and four SLE) (D). IL-21+ and IL17A+IL-21+ showed statistically signiﬁcant increases from CpG ODN treatment over ODN-treated cells at p , 0.003 and p , 0.0009, respectively. Combined analysis, SLE (n = 4) and normal (n = 4). Data are representative of two independent experiments. strong expression of RNA transcripts for IL-1a, IL-1b,IL-2, IL-17A, IL-22, IL-25, IL-31RA, and IL-34 in two patients, mod- FIGURE 9. FcgRIIIa-pSyk cosignaling upregulates IFN and NF-kB erate response in one patient, and mild response in another of the pathway RNA transcripts. Genes upregulated in human naive CD4+ T cells total four analyzed (Fig. 10A). A total of 24 genes from the from FcgRIIIa-pSyk cosignaling compared with CD28 cosignaling. (A) GO:0019221 (cytokine-mediated signaling pathway) category were Relative expression of IL-27, IFN-l, and IFN-v1. (B) Type 1 IFNs. (C) upregulated (p = 2.2e16). These data are supported by our previous NF-kB transcription factors. Data-normalized read per million (n = 4). 8FcgRIIIa-pSyk SIGNALING IN NA-TLR RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 10. (A)FcgRIIIa-pSyk cosignaling upregulates proinflammatory cytokine RNA transcripts and IFN-induced genes. Heat map shows clustering analysis for relative fold increase in cytokines RNA transcripts in four patients from FcgRIIIa-pSyk cosignaling compared with CD28 cosignaling. Color scale: blue = 0 to red = 38.4. (B)FcgRIIIa-pSyk costimulation upregulates HSP pathway RNA transcripts. Heat map showing cluster analysis of relative fold increase in RNA transcripts from the heat shock pathway observed in RNA-seq experiments. Color scale: blue = 0 to red = 151.003. to unfolded proteins, protein folding, refolding, heat response, and upregulation of several key proinflammatory and innate signaling negative regulation of inclusion body assembly (Supplemental Fig. 2, pathways transcripts from FcgRIIIa-pSyk signaling. Supplemental Table I). In total, 80 genes from protein folding GO: 0006457 were upregulated (p = 2.035e11). These results suggest Discussion that the FcgRIIIa-pSyk pathway triggers stress signal. Also, genes for In this report, we demonstrate that the FcgRIIIa-pSyk cosignaling both ubiquitination and proteasome assembly were differentially participates in upregulating and the subcellular redistribution of regulated by FcgRIIIa-pSyk signaling (A.K. Chauhan, unpublished NA-TLRs. Upon FcgRIIIa cosignaling, TLR9 is expressed on the observations). This is consistent with a role of Fc receptors in the cell surface, which is biologically active and contributes to the intracellular routing of proteins to proteasome assembly (29). CD4+ T cell differentiation. A single report using human CD4+ In conclusion, results show that the FcgRIIIa-pSyk cosignaling effector T cells has shown the enhanced expression of T cell ac- in CD4+ T cells accumulate NA-TLRs on the cell surface. Two tivation markers and cell cycle entry upon TLR9 activation (37). drugs, P505 and HCQ, prevent NA-TLRs association with The responses that drive NA-TLRs to the cell surface and whether FcgRIIIa on the cell surface. Both MyD88 and HMGB1 proteins they retain their function on the cell surface are debated (15). associate with FcgRIIIa and localize on the cell surface, possibly Aberrant oversupply of nucleic acids or its defective degradation forming a signaling complex. CpG ODN treatment of naive CD4+ leads to NA-TLR activation (1). Furthermore, increased expres- T cells in the presence of FcgRIIIa-pSyk cosignaling enhances IL- sion and/or the presence of NA-TLRs at sites that are accessible to 17A and IL-21 production. RNA transcriptome analysis showed modified self nucleic acids are a prerequisite for autoimmunity. The Journal of Immunology 9

Data presented in this article suggest that the FcgRIIIa-pSyk Our results demonstrate a role for FcgRIIIa-pSyk in TLR9 re- cosignaling contributes to these events in CD4+ T cells. These sponses. Syk inhibition by P505 blocked both expression of TLR9 observations at protein level are supported by our previous ob- and its subcellular localization with FcgRIIIa. It has been proposed servation of the increased expression of NA-TLR RNA transcripts that the abnormal signaling in SLE CD4+ T cells occurs from the upon FcgRIIIa-pSyk cosignaling (19). The engagement of rewiring of the TCR complex by Syk from the CD3 ligation (55, FcgRIIIa by ICs was necessary for the upregulation of NA-TLRs 56).TheFcreceptorg-chain forms a heterodimer with the TCR and their accumulation at the cell surface (Figs. 2, 3) (19, 22). z-chain in the CD3 complex and supports TCR signaling. Activa- TCR signaling was also required for NA-TLR expression (8, 38, tion of CD4+ TcellsviaFcgRIIIa results in the colocalization of the 39). CpG ODNs 2006 interacted with TLR9 on the cell surface Fc receptor g-chain with the TCR z-chain (24). In human CD4+ within minutes in live CD4+ T cells, suggesting the presence of T cells, the ligation of FcgRIIIa by ICs recruits Syk to the TCR functional TLR9. MyD88, a signaling scaffold protein, is essential complex and not the CD3 ligation by anti-CD3 as reported in mouse for TLR7, TLR8, and TLR9 signaling associated with FcgRIIIa SLE models (19, 22, 56). Phosphorylation of serine/threonine res- (40). In addition, the danger signal protein HMGB1, which is a idues in Syk regulate receptor trafficking from lysosomes to endo- DNA chaperone, is also associated with FcgRIIIa on the cell somes (57). Syk also promotes lysosomal fusion, which then surface (Fig. 7A). Engaging TLR9 with CpG ODN 2006 in cells facilitates delivery of processed Ag to MHC class II (50). Fur- costimulated via FcgRIIIa-pSyk enhanced IL-17A and IL-21 thermore, Syk interact with TRAF3, TRAF6, TAK1, and TBK1 in production, two key cytokines that participate in autoimmune murine macrophages. These genes were upregulated by the pathology. RNA-seq analysis showed upregulation of a number of FcgRIIIa-pSyk cosignaling in CD4+ T cells (51). FcgRIIIa-pSyk key inflammatory pathway genes that are observed during innate cosignal also upregulated lymphocyte phosphatase, encoded by Downloaded from immune responses and inflammation (41). These observations PTPN22. PTPN22 is a known risk factor for SLE pathology, which suggest a novel role for FcgRIIIa-pSyk cosignaling in NA-TLR– also regulates Fc receptor signaling and contributes to the type I mediated inflammatory responses in human CD4+ T cells. IFN production in pDCs (35). These studies strongly support a role The current understanding of NA-TLR mechanisms is based for FcgRIIIa-pSyk signal in abnormal CD4+ Tcellresponses. largely on studying fluorescently tagged recombinant proteins HCQ, which is used to treat SLE, disrupted the presence of NA-

expressed in innate cells and mouse models. Concerns have been TLRs with FcgRIIIa. The mechanism of action by which HCQ http://www.jimmunol.org/ raised about the conclusions drawn from these models, and a provides benefit to SLE patients is not fully understood. It is similar analysis using primary human cells is suggested (15, 17, proposed that HCQ antagonizes immune stimulation by CpG-DNA 42). This study addresses these concerns. Costimulation from (58). HCQ affects TLR7 signaling and production of IFN-a (59). TLR9 acts as a surrogate for CD28 cosignal in mouse T cells (7, HCQ is a weak base that neutralizes the acidic compartment in the 43). CpG ODNs activate complement pathway (44). This is im- endosomes (15). We observed disruption of FcgRIIIa protein lo- portant because ICs and pathogens both activate complement. calization with NA-TLRs upon HCQ treatment. The effect of HCQ Complement activation directly enhances T cell activation (45). on Syk signaling has not been examined. In activated CD4+ T cells, NA-TLRs are observed on the cell HMGB1 is a critical protein that links innate and adaptive im- surface with FcgRIIIa. Signaling by cell surface TLRs, in par- munity (60). Overexpression of HMGB1 from FcgRIIa-pSyk sig- by guest on September 23, 2021 ticular TLR2 and TLR4, triggers proliferation and enhances the naling suggests a role for this signal in coupling of innate and differentiation of mouse CD4+ T cells (30, 46). The location of adaptive responses (19). HMGB1 also promotes DNA delivery to TLR9 rather than nucleotide sequence of the ligand, its species of the endosomes (16, 61). HMGB1 released from necrotic cells origin, covalent modification, or double verses single strandedness complexes with DNA-ICs and stimulates IFN production via the of DNA determines its activation (47). Immune receptors largely TLR9-MyD88 pathway (16). HMGB1 bound to DNA-ICs protects signal from the cell surface, but whether NA-TLRs are able to the DNA from degradation by DNases (15). Class A CpG com- cooperate with FcgRIIIa and signal from the cell surface in CD4+ plexed to HMGB1 induces the association of MyD88 with TLR9 T cells has never been explored (15). Understanding of the (16). MyD88 forms dimers with TIRAP under the cell surface and cosynergistic functional responses by immune receptors from cell acts as an adaptor for TLR4 signaling (40). Recruitment of MyD88 surface and their impact on tolerance breakdown is of paramount to the cell surface is functionally important because an allele of importance. CpG ODN 2006 is a TLR9 ligand that enhances MyD88 directed to the membrane restores LPS responsiveness in B cell proliferation, IL-6, and Ab production. A single report TIRAP-deficient macrophages and in fibroblasts that are deficient in previously demonstrated functional TLR9 on the cell surface in both TIRAP and MyD88 (62). B cell–intrinsic MyD88 signaling primary neutrophils (48). In B cells, BCR, which signals via Syk, drives Ig class switching and GC differentiation (63). MyD88 also delivers CpG DNA to TLR9 (49). Syk kinase partners with Fc forms a signaling complex at the cell surface (40). FcgRIIIa-pSyk receptor g-chain and plays a key role in the maturation of late cosignaling induces overexpression and association of MyD88 with endosomes (50). A report from mouse macrophages has suggested FcgRIIIa on the cell surface, suggesting a role for this signaling in a role for Syk in the endocytosis of surface TLR4 (51). TLR4 TLR-mediated immune responses (17, 19). endocytosis from the cell surface requires Syk-PLCg2 signaling TLR9 signaling results in robust production of type I IFNs, (52). Another report suggested a role for the C330b-DAP12- which promote or inhibit T cell activation, survival, and differ- TLR4-Syk-PI3K signaling complex in amplifying inflammatory entiation (64, 65). FcgRIIIa-pSyk cosignaling induced expression responses in macrophages (53). A role for Syk in the regulation of of all three subsets of IFN RNA transcripts in naive human CD4+ TLR signaling in pDCs is also observed (54). These studies and T cells. This was accompanied by an increase in NF-kB tran- our results show that the FcgRIIIa-pSyk signaling-induced NA- scripts. NF-kB signaling contributes to both IFN and cytokine TLRs are critical for CD4+ T cell responses. We propose that the production. In addition, genes that respond to IFN signaling were accumulation of TLR9 on the cell surface results from the over- upregulated. Of particular interest was IFIH1, an innate sensor that expression of protein and thereafter insufficient cleavage by Ca- senses cytoplasmic DNA and plays a role in RIG-1/MDA5- thepsin, which is required for their relocation to endosomes (47). mediated induction of IFN-a/b (Fig. 10A). The upregulation of This is a critical event that could lead to generation of proin- these pathways supports the hypothesis that FcgRIIIa ligation by flammatory CD4+ T cells. ICs triggers innate signaling pathways in human CD4+ T cells. 10 FcgRIIIa-pSyk SIGNALING IN NA-TLR RESPONSES

Further support for altered signaling in T cells in vivo via FcgRIII- 6. Kabelitz, D. 2007. Expression and function of Toll-like receptors in T 2/2 lymphocytes. Curr. Opin. Immunol. 19: 39–45. pSyk also comes from the ZAP70 mutation in humans. In these 7. Mills, K. H. 2011. TLR-dependent T cell activation in autoimmunity. Nat. Rev. patients, T cells signal via Syk, which results in the development of Immunol. 11: 807–822. autoimmune inflammatory syndrome (66). It is also noteworthy that 8. Landrigan, A., M. T. Wong, and P. J. Utz. 2011. CpG and non-CpG oligodeoxynucleotides directly costimulate mouse and human CD4+ T cells through a IL-27, a cytokine that regulates the differentiation of TH17 and TFH TLR9- and MyD88-independent mechanism. J. Immunol. 187: 3033–3043. cells, was also upregulated (67). We have previously shown potent 9. Reynolds, J. M., G. J. Martinez, Y. Chung, and C. Dong. 2012. Toll-like receptor 4 signaling in T cells promotes autoimmune inflammation. Proc. Natl. 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Human CD4(+) T-cells: a role for low-affinity Fc recep- reported in this article describe a novel mechanism by which tors. Front. Immunol. 7: 215. PAMPs can contribute to adaptive immune responses in autoim- 22. Chauhan, A. K., C. Chen, T. L. Moore, and R. J. DiPaolo. 2015. Induced ex- by guest on September 23, 2021 munity. Further elucidation of these mechanisms is central to pression of FcgRIIIa (CD16a) on CD4+ T cells triggers generation of IFN-ghigh subset. J. Biol. Chem. 290: 5127–5140. understanding of autoimmune pathology and developing novel 23. Chauhan, A. K., and T. L. Moore. 2011. T cell activation by terminal complex of therapies for autoimmunity, particularly for SLE. complement and immune complexes. J. Biol. Chem. 286: 38627–38637. 24. Chauhan, A. K., and T. L. Moore. 2012. Immune complexes and late complement proteins trigger activation of Syk tyrosine kinase in human CD4(+) T cells. Acknowledgments Clin. Exp. Immunol. 167: 235–245. 25. Low, J. M., A. K. Chauhan, D. S. Gibson, M. Zhu, S. Chen, M. E. Rooney, I thank Dr. Grant Kolar from the Department of Pathology, Saint Louis M. J. Ombrello, and T. L. Moore. 2009. Proteomic analysis of circulating im- University School of Medicine for providing support with confocal micros- mune complexes in juvenile idiopathic arthritis reveals disease-associated pro- copy. I thank Dr. Joel Eisenberg from the Department of Biochemistry and teins. Proteomics Clin. Appl. 3: 829–840. Molecular Biology, Saint Louis University School of Medicine for care- 26. Pohar, J., A. Kuznik Krajnik, R. Jerala, and M. Bencina. 2015. Minimal se- ful review of the manuscript and editing. I would also like to thank quence requirements for oligodeoxyribonucleotides activating human TLR9. J. Immunol. 194: 3901–3908. Kathie Mihindukulasuriya and Dr. Dale Dorsett from the Genomic Core at 27. Yasuda, K., M. Rutz, B. Schlatter, J. Metzger, P. B. Luppa, F. Schmitz, T. Haas, Saint Louis University. 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