FCRL4 Is an Fc Receptor for Systemic IgA, but Not Mucosal Secretory IgA Yanling Liu, Sofiya Goroshko, Leslie Y. T. Leung, Shilan Dong, Srijit Khan, Paolo Campisi, Evan J. Propst, Nikolaus This information is current as E. Wolter, Eyal Grunebaum and Götz R. A. Ehrhardt of September 28, 2021. J Immunol published online 8 June 2020 http://www.jimmunol.org/content/early/2020/06/05/jimmun ol.2000293 Downloaded from
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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 © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 8, 2020, doi:10.4049/jimmunol.2000293 The Journal of Immunology
FCRL4 Is an Fc Receptor for Systemic IgA, but Not Mucosal Secretory IgA
Yanling Liu,* Sofiya Goroshko,* Leslie Y. T. Leung,* Shilan Dong,* Srijit Khan,* Paolo Campisi,† Evan J. Propst,† Nikolaus E. Wolter,† Eyal Grunebaum,‡ and Go¨tz R. A. Ehrhardt*
Fc receptor–like (FCRL) 4 is an immunoregulatory receptor expressed on a subpopulation of human memory B cells of mucosa- associated lymphoid tissue. Fc receptor function of FCRL4 was demonstrated by binding of IgA to FCRL4 following heat aggregation of the Ig. In this study, we demonstrate that FCRL4 recognizes J chain–linked systemic IgA in the absence of heat aggregation. We further demonstrate that mucosal secretory IgA is not recognized by FCRL4 and that systemic IgA binding can be competitively inhibited by recombinant secretory component protein. Finally, we provide evidence that primary FCRL4- bearing human memory B cells are constitutively bound to IgA. Our study provides a mechanism for the negative regulatory Downloaded from activity of FCRL4 on AgR-mediated B cell activation. The Journal of Immunology, 2020, 205: 000–000.
he immunoregulatory receptor, Fc receptor–like (FCRL) composed of three to nine Ig domains with significant sequence ho- 4, is selectively expressed on a morphologically and func- mology to those of classical FcgRandFcεR (3, 13). Key observations tionally distinct subpopulation of human memory B cells from Wilson et al. (14) demonstrated binding of FCRL4 to heat-
T http://www.jimmunol.org/ (Bmem) (1–3). This immunoregulatory activity is mediated via aggregated IgA, but not heat-aggregated IgG. Heat aggregation of phosphorylation of ITIM in the intracellular domain and subsequent Igs as a means of providing avidity and simulating immune complex recruitment of the SHP-1 and SHP-2 tyrosine phosphatases (4–6). formation suggests low-affinity binding of IgA to FCRL4. IgA exists Recent studies showed that the Ab repertoire encoded by FCRL4+ in the two isotypes IgA1 and IgA2, both of which can be detected in Bmem contained reduced levels of somatic mutations and displayed monomeric and polymeric isoforms. In serum, IgA levels are lower increased reactivity to commensal microbiota compared with the than those of IgG, and IgA1 levels exceed those of the IgA2 isotype. FCLR42 counterparts (7). In healthy individuals, FCRL4+ Bmem Although both monomeric and polymeric IgA are present, mono- are restricted to sites of the mucosa (1, 8, 9). However, in the context meric IgA is the prevalent isoform, accounting for ∼90% of total of autoimmune disorders and chronic infectious diseases, FCRL4+ circulating IgA (15–17). In contrast, polymeric IgA is the dominant Ig by guest on September 28, 2021 Bmem were detected in circulation (10–12). isotype in humans at sites of the mucosa where it is produced by In humans, the FCRL family consists of six type I transmembrane lamina propria plasma cells and transported across epithelial barriers receptors, the first five of which are predominantly expressed on to the luminal sites of the gastrointestinal or respiratory tract. On the various B lineage cell populations. The extracellular domains are basolateral side of the epithelium, dimeric J chain–linked IgA binds to the polymeric IgR, the extracellular domain of which is cleaved following transcytosis and remains attached to the Ab as a secretory *Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada; †Department of Otolaryngology – Head and Neck Surgery, Hospital for component (SC). The transported IgA in complex with the SC is Sick Children, University of Toronto, Toronto, Ontario M5G 1X8, Canada; and termed secretory IgA (sIgA) (18, 19). All three forms of IgA, mo- ‡ Division of Immunology and Allergy, Hospital for Sick Children, Toronto, Ontario nomeric IgA, dimeric IgA, and sIgA, are recognized by the classical M5G 1X8, Canada CD89 IgAR, although CD89 binding of dimeric IgA is stronger than ORCIDs: 0000-0001-6793-4498 (S.G.); 0000-0002-4883-582X (L.Y.T.L.); 0000- 0003-0392-4316 (S.D.); 0000-0002-2750-8117 (P.C.); 0000-0002-8013- binding observed for monomeric IgA or sIgA (20). 8530 (E.J.P.); 0000-0002-5868-5191 (G.R.A.E.). In an effort to gain deeper understanding of FCRL4 recognition of Received for publication March 16, 2020. Accepted for publication May 15, 2020. IgA, we determined that FCRL4 readily binds systemic IgA (defined in This work was supported by Canadian Institutes of Health Research Grants MOP- this study as serum IgA or mucosal IgA that are not transported across 12614 and PJT-16216 (to G.R.A.E.). epithelial barriers, in contrast to mucosal sIgA that is transported to the Y.L. designed, conducted, and analyzed experiments. S.G. conducted and analyzed luminal side of mucosal membranes), but not mucosal sIgA, in experiments. L.Y.T.L. analyzed experiments and critically appraised the manuscript. complex with the SC. Furthermore, we provide evidence that FCRL4+ S.D. analyzed experiments and critically appraised the manuscript. S.K. analyzed experiments and critically appraised the manuscript. P.C. provided specimens and primary Bmem are constitutively decorated with J chain–linked IgA, a critically appraised the manuscript. E.J.P. provided specimens and critically ap- feature unique to these mucosal lymphocytes. Constitutive occupancy praised the manuscript. N.E.W. provided specimens and critically appraised the manuscript. E.G. provided specimens and critically appraised the manuscript. G.R.A.E. of FCRL4 with J chain–linked IgA provides an explanation for the designed, conducted, and analyzed experiments and wrote the manuscript. nonresponsiveness of FCRL4-bearing Bmem following in vitro AgR Address correspondence and reprint requests to Dr. Go¨tz R. A. Ehrhardt, University ligation and suggests a, to our knowledge, novel regulatory model for of Toronto, Medical Sciences Building, Room 7316, 1 King’s College Circle, B lineage cells preferentially encoding microbiota-reactive AgR. Toronto, ON M5S 1A8, Canada. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: Bmem, memory B cell; FCRL, Fc receptor–like; Materials and Methods HA, hemagglutinin; IgA1-J, J chain–containing dimeric IgA1; SC, secretory compo- Abs and reagents nent; sIgA, secretory IgA. Fluorescently labeled Abs to IgG, IgM, IgD, CD3, CD19, and CD38 were Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 obtained from BD Biosciences (San Jose, CA). Abs to IgA, IgA1, and
www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000293 2 RECOGNITION OF SYSTEMIC IgA BY FCRL4
IgA2 were obtained from SouthernBiotech (Birmingham, AL). Anti–J Statistical analyses chain Abs were purchased from Thermo Fisher Scientific (Waltham, MA), and anti-FCRL4 Abs were purchased from Life Technologies (Carlsbad, Statistical analyses for Ig binding to transfected cells were determined using CA). Colostrum IgA preparations were obtained from Sigma-Aldrich (St. Kruskal–Wallis and Mann–Whitney U tests; tonsillar Ig-binding experi- Louis, MO). Details for all Ab reagents are listed in Supplemental Table I. ments were analyzed using Wilcoxon matched pair tests. Cell lines and primary cells Supplementary material Supplemental Fig. 1 depicts the gating strategy used to assess IgA/IgG HEK293T cells were grown in DMEM, supplemented with 10% FBS and + 2 100 U/ml penicillin/streptomycin. BJAB cells were grown in RPMI 1640 binding to primary human FCRL4 and FCRL4 Bmem. Supplemental supplemented with 10% FBS, and 100 U/ml penicillin/streptomycin. All Fig. 2 illustrates the absence of Ig binding to empty vector control cells. Supplemental Table I lists the Ab reagents used in this study. cells were grown in a humidified atmosphere at 37˚C and 5% CO2. Serum was obtained from healthy volunteers and tonsillar tissue from pediatric patients undergoing tonsillectomy was obtained from the Hospital for Sick Children (Toronto, ON, Canada) with informed consent according to the Results Declaration of Helsinki and the institute’s Research Ethics Board. FCRL4 recognizes systemic IgA, but not sIgA Generation of FCRL4 and CD89 expression constructs and Binding of heat-aggregated IgA to FCRL4-expressing cells was preparation of recombinant proteins demonstrated in transient transfection studies using 293T cells (14). In experiments aimed at a more detailed understanding Sequences encoding extracellular and transmembrane domains of FCRL4 (nt 61–1222, GenBank accession number BC125173.1; https://www.ncbi. of Ab recognition by FCRL4, we investigated Ig binding to nlm.nih.gov/nuccore/BC125173.1) and CD89 (nt 64–739, GenBank ac- 293T cells transiently transfected with HA- and EGFP-tagged cession number DQ075334.1; https://www.ncbi.nlm.nih.gov/nuccore/ FCRL4 and the classical FcaR CD89 or EGFP control con- Downloaded from DQ075334.1/) were cloned into the pDisplay vector (Invitrogen, Carls- structs lacking the extracellular domain. Unexpectedly, we ob- bad, CA) to provide a N-terminal hemagglutinin (HA)–epitope tag and a signal peptide. The resulting DNA fragments were fused to a C-terminal served robust binding of systemic IgA to FCRL4 expressing cells EGFP marker and cloned into the pLPCX vector for expression in in the absence of heat aggregation, whereas no binding of sys- HEK293T cells. Empty vector control cells contained the signal peptide/ temic IgG or IgM was detectable (Fig. 1A, top). As anticipated, HA-epitope tag/transmembrane domain/EGFP cassette without any addi- the same binding pattern was observed for cells transfected with tional extracellular domain sequences. The extracellular domain of the CD89 but not in negative control EGFP-transfected cells (Fig. 1A, http://www.jimmunol.org/ human polymeric IgR (aa 1–603) was modified by addition of a C-terminal tandem repeat of the 6xHis epitope tag separated by a (4xGly)Ser spacer. bottom, and Supplemental Fig. 2) (22). Conversely, in an inde- The cDNA was custom synthesized, cloned into the pIRESpuro2 vector, pendent series of transfections, we observed mucosal sIgA binding and stably transected into HEK293T cells as described previously (7). to FCRL4-transfected cells only following heat aggregation (Fig. Secreted proteins were purified using nickel affinity chromatography, 1B, top), whereas CD89-transfected cells recognized mucosal eluted with 250 mM imidazole, and extensively dialyzed against PBS. Protein purity was validated using SDS-PAGE followed by Coomassie sIgA with or without heat aggregation with equal efficiency (Fig. Brilliant Blue staining, and protein aliquots were frozen at 280˚C. 1B, bottom). Cell surface expression of the transfected FCRL4 Recombinant IgA or J chain–containing dimeric IgA1 (IgA1-J) proteins and CD89 receptors was verified by detection of the N-terminal were generated by cloning the VH sequences of the anti-DNA Ab 3H9 (21) HA-epitope tag (Fig. 1C). onto an IgA1 backbone, followed by cotransfection of plasmids encoding To more closely examine IgA binding to FCRL4, we by guest on September 28, 2021 H and L chain sequences with or without plasmids encoding the J chain into HEK293T cells. Secreted molecules were purified from culture supernatants expressed the HA-epitope tagged FCRL4 in BJAB B cells. using CaptureSelect IgA affinity resin (Thermo Fisher Scientific), eluted with Using this independent cell line system in which we included 100 mM glycine (pH 2.4) and immediately neutralized in Tris buffer (pH 7. untransfected, EGFP-negative cells as internal controls, we 5). Ab preparations were dialyzed against PBS and tested for dimer for- again observed binding of systemic IgA but not of systemic mation by nonreducing SDS-PAGE and aliquots stored at 280˚C. IgG to FCRL4-expressing cells (Fig. 2A, 2C). Bound IgA Ab binding assays appeared to be predominantly IgA1; although we consistently Serum IgA levels were determined by ELISA using plates coated with observed weak IgA2 binding, values did not reach statistical monoclonal anti-IgA1/2 Abs and detection with HRP-labeled anti-human significance in six independently performed experiments IgA Abs. A dilution series of purified colostrum IgA served as standard. (Fig. 2A, 2C). No Ig binding to empty vector-transfected cells Transfected cells were incubated with human serum (diluted to IgA con- was observed (Supplemental Fig. 2). In humans, the majority centration of 5 mg/ml), colostrum IgA (5 mg/ml), or recombinant IgA of serum IgA is monomeric, with IgA1 being the predominant (1 mg/ml) for 30 min on ice in PBS containing 0.5% BSA. Heat aggregation of colostrum IgA was performed by incubation at 60˚C for 30 min. Cells subclass (17). To gain further insight into IgA binding to were washed twice with PBS/0.5% BSA and incubated with fluorescently FCRL4, we generated recombinant IgA1 and IgA1-J (Supplemental labeled isotype-specific Abs, followed by two additional wash steps and Fig. 2). We observed no binding of monomeric IgA1 to FCRL4- analysis using a Guava EasyCyte flow cytometer (MilliporeSigma, Bur- expressing BJAB cells, whereas binding of dimeric IgA1-J lington, MA). For SC competition assays, Ab preparations were pre- incubated with various amounts of recombinant SC for 20 min on ice prior was readily detectable (Fig. 2B, 2C). Neither recombinant to addition to transfected cells. Dead cells were excluded by gating on IgA1 nor IgA1-J bound to empty vector-transfected cells propidium iodide–negative cells, and data were analyzed using the FlowJo (Supplemental Fig. 2), and aggregation of IgA1 or IgA1-J by software package (Ashland, OR). preincubation with F(ab9)2 fragments of anti-k LchainAbs Analysis of tonsillar Bmem did not induce binding of IgA1 or enhance binding of IgA1-J to FCRL4-expressing cells (Fig. 2D). These experiments dem- Cell suspensions of tonsil tissue were generated by tissue mincing using a onstrate selective J chain–dependent IgA binding to FCRL4- 70-mm steel mesh. Mononuclear cells were prepared by density gradient centrifugation with lymphocyte separation medium (Sigma-Aldrich). Cells transfected cells. were incubated with anti–J chain Abs, followed by APC-labeled goat anti- SC prevents IgA recognition by FCRL4 mouse Abs, Subsequently, cells were blocked extensively with 5% normal mouse serum then incubated with Abs to the CD3, CD19, IgD, IgA1, IgA2, SC bound to IgA is a key feature distinguishing mucosal sIgA from IgG, CD38, and FCRL4 Ags and fixable Aqua LIVE/DEAD cell exclusion systemic IgA. This prompted us to explore whether the presence or reagent (Thermo Fisher Scientific), followed by data acquisition on a BD LSR II instrument (San Jose, CA) and data analysis using the FlowJo absence of SC could potentially regulate IgA binding to FCRL4. software package. The gating strategy for Bmem (CD32/CD19+/IgD2/ Preincubation of recombinant IgA1-J with various amounts of CD382) analysis is shown in Supplemental Fig. 1. recombinant SC prevented IgA1-J binding to FCRL4-expressing The Journal of Immunology 3 Downloaded from
FIGURE 1. Recognition of systemic IgA by FCRL4. 293T cells were transiently transfected with HA-epitope–tagged FCRL4-EGFP (top row) or CD89- http://www.jimmunol.org/ EGFP (bottom row), followed by incubation with (A) serum from healthy donors or (B) heat-aggregated (aggr.) or nonaggregated (non-aggr.) mucosal sIgA. Bound Ig was detected by isotype-specific, fluorescently labeled Abs. Contour plots show a representative experiment with indicated cell frequencies. Scatter plots indicate relative Ig binding values determined by normalizing median fluorescent intensity (MFI) of the various Ig isotypes to MFI values obtained from corresponding negative control transfected cells. Shown are values from seven individual experiments with horizontal bars delineating mean 6 SEM. (C) Validation of cell surface expression of HA-epitope–tagged proteins. *p , 0.05, **p , 0.01, ***p , 0.001, n.s., p . 0.05, determined using Kruskal–Wallis (systemic Ig) and Mann–Whitney U tests (mucosal sIgA) (n = 7).
BJAB cells, whereas preincubation with an unrelated 6xHis-tagged SC recognize an at least partially overlapping region on J chain–
recombinant control protein (VLR4) did not alter IgA1-J bind- linked IgA. by guest on September 28, 2021 ing (Fig. 3A). Similarly, preincubation of heat-aggregated mucosal + – sIgA with recombinant SC resulted in loss of binding to FCRL4 FCRL4 , but not FCRL4 , Bmem constitutively bind IgA (Fig. 3B). Binding of systemic IgA to FCRL4 was also prevented The robust binding of systemic IgA to FCRL4 transfected cells in by preincubation with recombinant SC and followed by detection the absence of experimental strategies to generate IgA aggregates with either anti-IgA or anti–J chain Abs (Fig. 3C, 3D). These and readily detected cell surface J chain in these experiments observations are in agreement with a model in which FCRL4 and prompted us to investigate the potential of systemic IgA decorating
FIGURE 2. Recognition of J chain–containing IgA by FCRL4. BJAB cells expressing FCRL4 or negative control empty vector EGFP were incubated with (A) serum, followed by detection of bound total IgA, IgA1, IgA2, or IgG or (B) Recombinant IgA1 or recombinant J chain–containing IgA1-J, and detection of bound total IgA. Contour plots show a representative experiment. (C) Scatter plots indicate relative Ig-binding values determined by nor- malizing median fluorescent intensity (MFI) of the various Ig isotypes to MFI values obtained from corresponding negative control transfected cells. Shown are values from six individual experiments with horizontal bars delineating mean 6 SEM. *p , 0.05, **p , 0.01, determined using Kruskal–Wallis (systemic Ig) and Mann–Whitney U tests (recombinant IgA) (n = 6). (D) Recombinant IgA1 or IgA1-J was preincubated with various dilutions of anti- human k (aIgk) L chain (Fab9)2 fragments relative to a constant amount of recombinant IgA starting at equimolar concentrations prior to addition to FCRL4-expressing BJAB cells. Bound IgA1 or IgA1-J MFI signals from FCRL4-expressing, EGFP-positive cells were normalized to MFI values from parental EGFP-negative control cells. Relative MFI values from two independent experiments are depicted by open and closed circles, respectively. 4 RECOGNITION OF SYSTEMIC IgA BY FCRL4 Downloaded from http://www.jimmunol.org/
FIGURE 3. Competition of IgA binding to FCRL4 by recombinant SC. Recombinant IgA1-J (A), heat-aggregated mucosal sIgA (B), or systemic IgA (C and D) was preincubated with recombinant SC in 1:10 dilution steps, starting at 10-fold excess of SC over IgA prior to the addition to FCRL4-expressing BJAB cells and detection of bound IgA (A–C) or J chain (D). Preincubation with 6xHis-tagged VLR4 as a negative control was performed with amounts corresponding to the highest concentration of SC. Bound IgA was detected with isotype-specific Abs, quantitated following gating on EGFP-positive cells and normalized to signal intensities obtained from cells without recombinant, mucosal, or systemic IgA incubation (2). Contour plots depict a repre- sentative experiment shown with the highest concentration of SC during preincubation. Scatter plots indicate relative Ig-binding values determined by normalizing median fluorescent intensity (MFI) of the various IgA Abs to MFI values obtained from corresponding negative control stained cells. Shownare values for six individual experiments with horizontal bars delineating mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, determined using Kruskal–Wallis by guest on September 28, 2021 tests (n =6). primary tonsillar FCRL4+ Bmem. Staining of tonsillar B cells with purified from colostrum or serum sources following heat aggre- anti–J chain Abs consistently allowed the selective detection of J gation of the Igs. Heat aggregation is typically used as a means to chain–reactive cells (3.06%; 61.44 SEM; n = 10) expressing in- simulate immune complexes and to provide avidity in support termediate or high levels of FCRL4 (Fig. 4A). Subsequently, we of low-affinity interactions of the binding partners. Although J aimed to detect systemic IgA on Bmem distinguished by presence chain–linked IgA1 dimer formation supports binding to FCRL4, or absence of FCRL4. Consistent with the detection of J chain on we observed that preincubation of systemic IgA, recombinant J FCRL4+ Bmem, we observed a large proportion of IgG/IgA1 chain–linked IgA1, and heat-aggregated mucosal sIgA with double-positive cells among ex vivo FCRL4+ Bmem, represent- recombinant SC completely abrogated FCRL4 binding. Combined ing transmembrane IgG AgR and systemic IgA1 bound to FCRL4 with the readily observed binding of systemic IgA to FCRL4- (Fig. 4B, top row). In contrast, IgG- and IgA1-double-positive expressing cells in the absence of heat aggregation, these obser- cells were absent in the FCRL42 Bmem compartment (Fig. 4B, vations indicate that potential denaturation and/or loss of SC on top row). Similarly, IgG/IgA2-double-positive cells were detected mucosal sIgA during heat aggregation, but not the avidity created only among FCRL4+ Bmem, albeit at slightly lower levels than during heat aggregation, results in FCRL4 recognition of mucosal IgG/IgA1-positive cells (Fig. 4B, bottom row). As expected, sIgA. Specific recognition of systemic IgA, and not systemic the appearance of IgG/IgA1- or IgG/IgA2-double-positive cells IgM, further suggests that IgA and J chain, but not solely the J among FCRL4-bearing Bmem resulted in decreased frequencies chain, may form the protein interface recognized by FCRL4. of cell surface IgG+/IgA2 Bmem. These ex vivo studies are Although systemic IgA bound strongly to FCRL4-expressing cells, consistent with the in vitro Ig-binding experiments that demon- recombinant IgA1-J exhibited weaker binding. These reduced strated J chain–linked IgA binding to FCRL4-transfected cells. median fluorescent intensities could be inherent to IgA generated using a cell system different from plasma cells for protein gen- Discussion eration, or it could reflect the presence of copurified monomeric IgA is the most prevalent Ig of mucosal tissue and can interact with IgA secreted by IgA and J chain–coexpressing cells. We further multiple cell surface localized receptors, including the FcaRI observed that IgA1 binding to FCRL4-expressing cells was more (CD89) (23), Fca/mR (24), transferrin receptor (25), DC-SIGN/ pronounced than the signals observed for IgA2 both for serum SIGNR (26, 27), asyaloglycoprotein receptor (ASGR) (28), IgA1/2 as well as for the IgA1/2 isotypes that decorate primary ex polymeric IgR (29), and FCRL4 (14). Initial experiments dem- vivo tonsillar Bmem. IgA1 is the predominant isotype in serum as onstrating IgA binding to FCRL4-transfected cells used IgA well as in the orogastric and respiratory tracts, whereas IgA2 is the The Journal of Immunology 5
FIGURE 4. Constitutive binding of J chain–linked IgA to FCRL4+ Bmem. (A) Tonsillar B cells were stained with Abs to J chain, CD19, IgD, CD38, and FCRL4, followed by analysis for J chain–reactive cells (top) or isotype control reactive cells (bottom). Shown is a representative of eight independent tonsil samples. (B) Analysis of IgA binding to FCRL4+ and FCRL42 Bmem. Shown is a representative of CD19+/CD382/IgD2/FCRL4+ or CD19+/CD382/IgD2/ FCRL42 Bmem of 10 independent tonsil samples analyzed for IgG and IgA1 (top panel) or IgG and IgA2 (bottom panel) reactivity. Scatter plots indicate Downloaded from observed frequencies of the analyzed tonsil samples. **p , 0.01, determined using Wilcoxon matched pairs tests (n = 10). major isotype in the lower gastrointestinal tract (30, 31). Affinity IgA on FCRL4-bearing Bmem may also contribute to proin- measurements of the interactions of the IgA isotypes to FCRL4 flammatory processes in autoimmune and chronic infectious dis- will determine whether the observed stronger binding of IgA1 to orders in which dysregulated FCRL4+ Bmem were reported in
FCRL4 is a reflection of the relative abundance of the isotype or if nonmucosal locations (10–12). In our analysis of ex vivo tonsillar http://www.jimmunol.org/ FCRL4 displays preferential IgA1 recognition. Bmem, we were able to detect J chain and IgA binding to Bmem FCRL4 is unique among IgA-binding receptors, as it is a potent expressing high and intermediate levels of FCRL4, but not on Bmem negative regulator of B cell Ag receptor signaling (4–6). Coen- with low-level FCRL4 expression. The regulatory mechanisms gagement of FCRL4 with the B cell Ag receptor results in phos- governing FCRL4 expression remain largely unexplored, although phorylation of tyrosine residues located within ITIM consensus induction of FCRL4 expression was reported in response to CpG sequences and inhibition of B cell Ag receptor signal transduction and anti-IgM treatment as well as following HIV gp120 treatment pathways via subsequent recruitment of the SHP1 and SHP2 ty- (34). It will be important to define mechanisms regulating FCRL4 rosine phosphatases. Conversely, cells expressing FCRL4 display expression and to investigate whether different FCRL4 expression enhanced responses to TLR stimulation, indicating that FCRL4 levels represent functional heterogeneity within this cell population. by guest on September 28, 2021 has context-dependent activating or inhibitory functions (5, 6). FCRL4+ Bmem are found in the interfollicular regions of ton- Our demonstration of constitutive occupation of FCRL4 on pri- sillar tissue and Peyer’s patches (8, 9), anatomical locations that mary Bmem by IgA provides a mechanistic explanation for permit potential contact with retrograde transported mucosal sIgA/ studies that showed an inability of FCRL4+ Bmem to proliferate Ag complexes. Receptor-mediated uptake of mucosal sIgA-coated and differentiate following anti-Ig stimulation simulating T cell– Ag by dendritic cells permits sampling of luminal Ags and the independent stimulation, but not in response to cytokine mixtures regulation of tolerogenic or proinflammatory immune responses simulating T cell–dependent stimulation (1). Recognition of J (26, 35). Similar to dendritic cells, B cells are efficient APC; chain–linked IgA as natural ligand for FCRL4 will also permit however, Ag uptake is initiated following Ag recognition by the studies using this reagent to investigate potential activating or B cell Ag receptor (36). Although the biological function of inhibitory function of FCRL4 upon ligand binding. In this context, FCRL4 on Bmem remains to be elucidated, the selective recog- it is interesting that a recent study on FCRL3 determined that this nition of systemic IgA, but not secretory mucosal sIgA, by FCRL4 FCRL family member recognized mucosal sIgA, but not serum may represent a mechanism that prevents AgR-independent up- IgA, and that engagement of FCRL3 inhibited regulatory T cell take and presentation of mucosal sIgA-coated Ag. function and promoted a proinflammatory Th17-like phenotype (32). Structural analysis of FCRL4 and FCRL3 in complex with Acknowledgments their respective IgA ligands will resolve the molecular basis for We are grateful to Dr. Nathalie Simard for help with fluorescent-activated the opposing IgA-binding characteristics. It will also permit a cell sorting and Laura Ernst for technical assistance. comparison with CD89 recognition of IgA unique in that each IgA molecule is recognized by two CD89 receptors (20). Disclosures Although the source of systemic IgA captured by FCRL4 on The authors have no financial conflicts of interest. Bmem remains to be investigated, it is likely secreted by local lamina propria plasma cells, a source of Abs enriched in clonotypes with microbiome- and polyreactive-binding characteristics (33). References The observed preferential microbiome reactivity of AgR encoded 1. Ehrhardt, G. R., J. T. Hsu, L. Gartland, C. M. Leu, S. Zhang, R. S. Davis, and by FCRL4+ Bmem (7) and the accessibility of luminal Ag in M. D. Cooper. 2005. Expression of the immunoregulatory molecule FcRH4 subepithelial dome locations of mucosal tissue indicates a defines a distinctive tissue-based population of memory B cells. J. Exp. Med. 202: 783–791. regulatory mechanism wherein microbial Ag from the micro- 2. Ehrhardt, G. R., A. Hijikata, H. Kitamura, O. Ohara, J. Y. Wang, and environmentcapturedbyIgAboundtoFCRL4willresultin M. D. Cooper. 2008. Discriminating gene expression profiles of memory B cell subpopulations. J. Exp. Med. 205: 1807–1817. coligation of the inhibitory FCRL4 with the AgR to prevent T cell– 3. Li, F. J., W. J. Won, E. J. Becker, Jr., J. L. Easlick, E. M. Tabengwa, R. Li, independent activation. Binding and transport of microbial Ag to M. Shakhmatov, K. Honjo, P. D. Burrows, and R. S. Davis. 2014. Emerging roles 6 RECOGNITION OF SYSTEMIC IgA BY FCRL4
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FCRL4- FCRL4+ memory B
CD38 FCRL4
Supplemental Figure 1: Gating strategy for the analysis of FCRL4+ and FCRL4- tonsilar memory B cells. Tonsilar mononuclear cells were prepared by tissue mincing using a 70μm steel mesh followed by density gradient centrifugation using lymphocyte separation medium. The resuting cell population was incubated with antibodies to the CD3, CD19, IgD, IgA, IgG, CD38, and FCRL4 antigens. Dead cells were excluded using the fixable Aqua Live/Dead reagent. (Left panel) CD19+/CD3- B cells were separated into IgD+/CD38- naive B cells, IgD-/CD38+ germinal center B cells, IgD-/CD38++ plasma cells and IgD-/CD38- memory B cells (red gate). (Right panel) Memory B cells were analyzed according to CD19 and FCRL4 expression and separated into FCRL4+ and FCRL4- cell populations (blue gates). A Systemic Ig Mucosal sIgA
IgA IgG IgM aggr. non-aggr. 0.56 0.13 0 0 0.04 0.05 0.35 0.03 0.6 0.18
103 103 103 103 103
102 102 102 102 102
101 101 101 101 101
100 100 100 100 100 0 0 0 0 0
0 100 101 102 103 0 100 101 102 103 0 100 101 102 103 0 100 101 102 103 0 100 101 102 103
B C Systemic Ig recombinant IgA recombinant IgA IgA IgG IgA1 IgA1 IgA1-J 0.35 0.25 0.25 0.05 0.16 0.04 103 103 103 235 102 102 102 235 180 180 α-IgA 101 101 101 135 135 100 100 100 0 0 0
103 103 103
102 102 102
101 101 101
100 100 100 0 0 0
0 100 101 102 103 0 100 101 102 103 0 100 101 102 103
Supplemental Figure 2: No binding of immunoglobulins to EGFP-transfected negative control cells. (A) 293T cells were transiently transfected with empty vector GFP plasmids, followed by incubation with serum from healthy donors or heat-aggregated (aggr.) or non-aggregated (non-aggr.) mucosal sIgA. Bound Ig was detected by isotype specific fluorescently labeled monoclonal antibodies. Contour plots show a representative of 7 experiments. (B) BJAB cells transduced with negative control empty vector EGFP were incubated with serum, followed by detection of bound total IgA, IgA1, IgA2 or IgG or recombinant monoclonal IgA1 or recombinant J-chain containing IgA1-J, followed by detection of bound total IgA. Contour plots show a representative of 6 experiments. (C) Western blot analysis of recombinant IgA1 and IgA1-J. Recombinant proteins were separated by SDS-PAGE under non-reducing conditions and detected by HRP-labeled anti-human IgA antibodies. Supplemental Table 1: Antibody reagents used in the study.
Antibody Clone ID Source Catalogue # Anti-human CD3 (Pe-Cy7) SK7 BD Biosciences 557851 Anti-human CD19 (APC-Cy7) SJ25C1 BD Biosciences 557791 Anti-human CD38 (V450) HIT2 BD Biosciences 561378 Anti-human IgD (BUV395) IA6-2 BD Biosciences 563813 Anti-human IgG (BV605) G18-145 BD Biosciences 564229 Anti-human IgM (PE) MHM-88 Biolegend 314508 Anti-human IgA (PE) polyclonal Southern Biotech 2050-09 Anti-human IgA (HRP) polyclonal Southern Biotech 2050-05 Anti-human IgA1 (FITC) B3506B4 Southern Biotech 9130-02 Anti-human IgA1 (PE) B3506B4 Southern Biotech 9130-09 Anti-human IgA2 (PE) A9604D2 Southern Biotech 9140-09 Anti-human J chain OTI3B3 ThermoFisher MA5-25840 Anti-mouse Ig (PE) polyclonal Southern Biotech 10-10-09 Anti-mouse Ig (APC) polyclonal Southern Biotech 10-12-11 Anti-human FCRL4 413D12413D12 ThermoFisherThermoFisher 4646-3079-3079-42-42 (PerCP-eFluror710) Anti-HA epitope tag 12CA5 ThermoFisher MA1-12429 Anti-human IgG/A/M polyclonal Jackson 109-006-064 ImmunoResearch Anti-human IgA1/A2 G20-359 BD Biosciences 555883 IgA (colostrum) polyclonal Sigma-Millipore I-1010
Anti-human kappa F(ab’)2 biot. polyclonal Southern Biotech 2062-08