Published April 13, 2016, doi:10.4049/jimmunol.1501027 The Journal of Immunology Fc Receptor–like 5 Expression Distinguishes Two Distinct Subsets of Human Circulating Tissue–like Memory B Cells Huifang Li,* Francisco Borrego,†,‡ Satoshi Nagata,x and Mate Tolnay* Fc receptor–like (FCRL) 5 is a novel IgG binding protein expressed on B cells, with the capacity to regulate Ag receptor signaling. We assessed FCRL5 expression on circulating B cells from healthy donors and found that FCRL5+ cells are most enriched among atypical CD212/lo/CD272 tissue-like memory (TLM) B cells, which are abnormally expanded in several autoimmune and infec- tious diseases. Using multicolor flow cytometry, FCRL5+ TLM cells were found to express more CD11c and several inhibitory receptors than did the FCRL52 TLM subset. The homing receptor profiles of the two TLM subsets shared features consistent with migration away from lymphoid tissues, but they also displayed distinct differences. Analysis of IgH V regions in single cells indicated that although both subsets are diverse, the FCRL5+ subset accumulated significantly more somatic mutations. Further- more, the FCRL5+ subset had more switched isotype expression and more extensive proliferative history. Microarray analysis and quantitative RT-PCR demonstrated that the two TLM subsets possess distinct gene expression profiles, characterized by markedly different CD11c, SOX5, T-bet, and RTN4R expression, as well as differences in expression of inhibitory receptors. Functional analysis revealed that the FCRL5+ TLM subset responds poorly to multiple stimuli compared with the FCRL52 subset, as reflected by reduced calcium mobilization and blunted cell proliferation. We propose that the FCRL5+ TLM subset, but not the FCRL52 TLM subset, underwent Ag-driven development and is severely dysfunctional. The present study elucidates the heterogeneity of TLM B cells and provides the basis to dissect their roles in the pathogenesis of inflammatory and infectious diseases. The Journal of Immunology, 2016, 196: 000–000. he generation of B cell memory is an important compo- homing receptors consistent with migration away from lymphoid nent of the adaptive immune response. Classical memory organs and toward inflammatory tissues. They also proliferate B cells are defined based on their CD27 expression (1). In poorly in response to B cell stimuli. A similar CD212/lo B cell T 2 humans, recent studies identified atypical CD27 memory B cells, population is expanded in other chronic infections and inflam- indicating heterogeneity within the memory compartment. Tissue- matory conditions where the immune system encounters increased like memory (TLM) B cells, also called atypical memory B cells, Ag load for a prolonged period of time, such as chronic malaria were first defined in 2008 as an abnormally expanded mature (5), systemic lupus erythematosus (6), type Ia common variable by guest on October 1, 2021. Copyright 2016 Pageant Media Ltd. B cell population in the blood of HIV-viremic patients (2). This immunodeficiency (CVID Ia) (7, 8), rheumatoid arthritis (8, 9), 2 2 B cell population is CD20hi/CD21 /lo/CD27 and expresses Fc and Sjo¨gren’s syndrome (10). Additionally, TLM B cells accu- receptor–like (FCRL) 4, a profile similar to that of tonsil tissue mulate in patients with germline heterozygous mutations in memory B cells (3, 4). TLM B cells in HIV-viremic patients ex- CTLA4 (11). TLM B cells are thought to be in a state of ex- press increased levels of inhibitory receptors as well as a profile of haustion, based on enrichment of self-reactive or anti-virus Abs, reduced response to stimuli, and elevated apoptosis (7, 8, 10). A *Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. recent study suggested that TLM B cells produce protective Abs Food and Drug Administration, Silver Spring, MD 20993; †Immunopathology Group, against malaria and therefore may play a functional role in BioCruces Health Research Institute, 48903 Barakaldo, Spain; ‡Ikerbasque, Basque x protecting the host (12). It is possible that TLM B cells con- Foundation for Science, 48013 Bilbao, Spain; and Center for Drug Design Research, http://classic.jimmunol.org National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka stitute a heterogeneous population, and subsets with different 567-0085, Japan functional capacities expand under different conditions such as ORCIDs: 0000-0001-5701-1518 (H.L.); 0000-0001-9156-5215 (S.N.). autoimmune diseases and chronic infections. In the present Received for publication May 4, 2015. Accepted for publication February 29, 2016. study, having established that approximately half of TLM This work was supported by the Intramural Research Program of the Center for Drug B cells from normal donors express FCRL5, we investigated Evaluation and Research/U.S. Food and Drug Administration. H.L. was supported by whetherFCRL5expressionstatuscould discriminate heteroge- the Research Fellowship Program from the Center for Drug Evaluation and Research Downloaded from administered by the Oak Ridge Associated Universities. neous TLM B cell subsets. Eight members of the FCRL family with sequence similarity to The raw data and normalized results presented in this article have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession Fc receptors were identified in humans, including six cell surface number GSE66470. proteins, FCRL1–6, and two intracellular proteins, FCRLA and Address correspondence and reprint requests to Dr. Mate Tolnay, Office of Biotech- FCRLB (13–15). FCRL1–5 are exclusively expressed on B cells, nology Products, Center for Drug Evaluation and Research, U.S. Food and Drug except for FCRL3, which is also expressed by a subset of regu- Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993. E-mail address: [email protected] latory T cells and NK cells (16–19). FCRL5, the largest protein in The online version of this article contains supplemental material. the family, contains nine extracellular Ig domains as well as two Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; CVID Ia, type Ia ITIM motifs and one predicted ITAM motif in its cytoplasmic tail. common variable immunodeficiency; FCRL, Fc receptor–like; HCV-MC, hepatitis C Using a chimeric receptor containing the cytoplasmic tail of virus–related mixed cyoglobulinemia vasculitis; SHM, somatic hypermutation; TLM, FCRL5, crosslinking FCRL5 and the BCR was shown to recruit tissue-like memory; V , Ig H chain V region. H SHP-1 to the two ITIM motifs of FCRL5, resulting in reduced Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 BCR-induced calcium mobilization and protein tyrosine phos- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501027 2 FCRL5 EXPRESSION DISTINGUISHES TLM B CELL SUBSETS phorylation (20). We and others reported that FCRL5 binds intact Flow cytometry was performed on an LSR II (BD Biosciences), and data IgG via a complex mechanism (21, 22), and therefore immune were analyzed using FlowJo (Tree Star). complexes may link FCRL5 to the BCR, potentially blocking B cell sorting B cell activation similarly to the inhibitory FcgRIIB. Potential activating functions were also proposed for FCRL5 (23), similarly Freshly isolated B cells were stained with CD19-allophycocyanin-Cy7, CD27-allophycocyanin, CD21-PE-Cy7, and FCRL5-PE (clone 509f6) mAbs. to FCRL3 (24) and FCRL4 (25), suggesting dual regulatory ca- Single cells were gated using forward scatter/side scatter plot following pacities. The expression level of FCRL5 is low in peripheral doublet discrimination and then gated on CD19+ B cells. B cell subsets B cells, but it is increased in lymphoid tissues (14, 16). FCRL5 were discriminated based on CD21, CD27, and FCRL5 staining. In another expression is upregulated in multiple B cells malignancies, in- gating strategy, B cells were also stained with IgM-FITC, in addition to the above panel. B cell subsets were sorted using FACSAria (BD Biosciences). cluding hairy cell leukemia, chronic lymphocytic leukemia, man- tle cell lymphoma, and multiple myeloma, but the contribution Quantitative PCR of FCRL5 to disease manifestation and progression is unclear + 2/lo Total RNA was extracted from an equal number of sorted B cells using a (16, 26, 27). FCRL5 cells were found enriched on CD21 / microRNeasy kit (Qiagen), and then random-primed cDNA was synthesized CD27+/IgM+ marginal zone–like B cells in hepatitis C virus–re- using SuperScript III (Invitrogen). All quantitative PCR was performed fol- lated mixed cyoglobulinemia vasculitis (HCV-MC) patients, but lowing protocols defined for RT2 quantitative PCR primer assays, with primers not in healthy donors (28). Intriguingly, in two studies FCRL5 was from Qiagen, using SYBR Green (Applied Biosystems) and ABI Prism 7900HT (Applied Biosystems). All data were normalized to cyclophilin H. found overexpressed in TLM B cells based on microarray analysis (8, 29). Somatic mutation analysis We report that FCRL5 is a marker that distinguishes two subsets Single B cells were sorted into plates or tubes containing 10 ml13 RT of TLM B cells with distinct differences in surface molecules, buffer for SuperScript III and then snap frozen on dry ice and stored at replication histories, somatic mutation rates, gene expression 280˚C. cDNA synthesis and PCR were conducted following the published profiles, and responses to stimuli. method (32). PCR products were purified and sequenced bidirectionally. Sequences were analyzed using the international ImMunoGeneTics in- formation system (http://imgt.org/). Materials and Methods Abs k-Deleting recombination excision circles assay Two FCRL5 mAbs were used, clone 509f6 labeled with PE or eFluor 660 The assay was performed as published (33). Briefly, genomic DNA from (BioLegend; mouse IgG2a) and clone F99 (mouse IgG2b) (26), producing 3 3 105 sort-purified B cells was isolated using a QIAamp genomic DNA similar results. FCRL5-F99 was biotin labeled using a kit from Pierce, mini kit (Qiagen). The coding and signal joints of the IGK-deleting rear- and was used with streptavidin-BV421 (BioLegend).