Fc Receptor−like 5 Expression Distinguishes Two Distinct Subsets of Human Circulating Tissue−like Memory B Cells

This information is current as Huifang Li, Francisco Borrego, Satoshi Nagata and Mate of October 2, 2021. Tolnay J Immunol published online 13 April 2016 http://www.jimmunol.org/content/early/2016/04/13/jimmun ol.1501027 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 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 expression profiles, characterized by markedly Downloaded from 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. http://www.jimmunol.org/

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 2, 2021 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, 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 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. , 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 (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 Downloaded from 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 http://www.jimmunol.org/ 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). Other Abs used in rangement were quantified by separate TaqMan PCR reactions using ABI flow cytometry staining were: CD27-allophycocyanin or -BV421 (clone Prism 7900HT. The average number of replications in each cell subset was M-T271), CD22-FITC (clone HIB22), CD21-PE-Cy7 (clone Bu32), LAIR- determined by the ratio of the measured coding joints and the signal joints. 1–PE (clone DX26), CD38-PerCP-Cy5.5 (clone HIT2), and IgG-BV605 (clone G18-145) from BD Biosciences; IgM-FITC (clone SA-DA4) from Cell proliferation assay SouthernBiotech; CD85j-PE (clone 292305) and CXCR5-FITC (clone Freshly sorted cells were plated at 2 3 104 cells per well in a 96-well 51505) from R&D Systems; and IgA-VioBlue (clone IS11-8E10) and

U-bottom plate in cell culture medium (RPMI 1640 with 10% FBS, gluta- by guest on October 2, 2021 TACI-PE (clone 1A1) from Miltenyi Biotec. All other Abs, including mine, nonessential amino acids, sodium pyruvate, penicillin/streptomycin, FCRL4-PE (clone 413D12), CD85d-PE (clone 42D1), IgD-FITC (clone and 0.5 mM 2-ME) in the presence of various combination of the following IA6-2), CD19-allophycocyanin-Cy7 (clone HIB19), CD11c-PE (clone reagents: 10 mg/ml goat anti-IgA/G/M F(ab9) (Jackson ImmunoResearch Bu15), CXCR3-PE (clone G025H7), CXCR4-PE (clone 12G5), PD-1–PE 2 Laboratories), 500 ng/ml CD40L with 1 mg/ml enhancer (Enzo Life Sci- (clone GH12.2H7), CD32-PE (clone FUN-2), CCR6-PE (clone G034E3), ences), 2.5 mg/ml CpG2006 (U.S. Food and Drug Administration core CD95-PE (clone DX2), CCR7-FITC (clone G043H7), CD62L-FITC facility), 10 ng/ml IL-2 (Insight Genomics), and 20 ng/ml IL-4 (Pepro- (clone DREG-56), CD10-PerCP-Cy5.5 (clone HI10a), CD20-PE (clone Tech). Samples were assessed 4 d later by determining relative live cell 2H7), CD40-FITC (clone 5C3), CD80-PE (clone 2D10), CD86-PE (clone numbers using the CellTiter-Glo luminescent cell viability assay (Prom- IT2.2), HLA-DR–PE (clone LN3), HLA-I–FITC (clone W6/32), HLA-II– ega), according to the manufacturer’s instructions. FITC (clone Tu39),€ ICOSL-PE (clone 9F.8A4), and IL4R-PE (clone G077F6) were from BioLegend. Calcium flux assay Reactivity of anti-FCRL5 F99 mAb Freshly isolated B cells (∼10 3 106 cells/ml) were incubated for 30 min in m Reactivity of F99 mAb with FCRL1–5 was assessed by ELISA and flow the presence of 2 M Indo-1/AM (Thermo Fisher Scientific) and 0.045% cytometry as described (26). Briefly, FCRL1–5 produced as fusion proteins Pluronic F-127 (29) in cell culture medium at 37˚C. Cells were subse- with human IgG Fc were coated on ELISA plates via anti-Fc secondary quently stained with CD19, CD21, CD27, and FCRL5 Abs. Cells were Ab. After washing, F99 mAb was added at 50 ng/ml and bound mAb then washed, resuspended in Dulbecco’s PBS with 2% FBS, and warmed was detected with alkaline phosphatase–labeled goat anti-mouse IgG. to 37˚C before flow cytometry. Baseline was collected for 1 min, and then m 9 In flow cytometry analysis, 293T cells transfected with pcDNA3-based 20 g/ml goat anti-IgA/G/M F(ab )2 was added and the response recorded m expression plasmids encoding FCRL1–5 were incubated with 1 mg/ml for 9 min. Response to 1 M ionomycin was recorded during the last 2 min and served as positive control. The relative concentration of intracellular FCRL5 F99 or the following reference anti-FCRL1–5 mouse Abs: 2+ FCRL1 (clone E3, IgG1), FCRL2 (clone B24, IgG2a), FCRL3 (clone free Ca was calculated as the fluorescence ratio of Indo-1 bound (violet)/ Indo-1 unbound (blue) by FlowJo software. Baselines of samples were H5, IgG2b), FCRL4 (clone A1, IgG2a), and FCRL5 (clone F56, IgG1) (30, 31). Cell-bound mAb was detected by PE-conjugated goat anti- aligned prior to comparison. mouse IgG F(ab9)2 (Jackson ImmunoResearch Laboratories) using BD B cell survival assay Accuri C6 (BD Biosciences). Sort-purified B cell subsets were plated at 1 3 105 cells per well in cell B cell isolation and flow cytometry culture medium in a 96-well flat-bottom plate. The proportions of apo- Human blood samples from anonymous healthy donors who fulfilled the ptotic and dead cells were assessed by flow cytometry after staining with eligibility requirements of the American Red Cross, including restrictions annexin V–FITC (eBioscience) and 7-aminoactinomycin D (7-AAD; BD for travel in malaria-risk countries, were obtained from the National In- Biosciences). stitutes of Health Blood Bank following approved protocols. PBMCs were Microarray analysis separated by Ficoll-Hypaque centrifugation, and then B cells were isolated by negative selection using a B cell enrichment mixture (StemCell Tech- RNA was extracted from 0.5–1 million sort-purified B cell subsets nologies). Human tonsils were obtained from the Cooperative Human (.90% pure) using an RNeasy micro kit (Qiagen). RNA quality and Tissue Network. Tonsil B cells were purified similarly to circulating B cells. integrity were determined using an Agilent 2100 bioanalyzer. Amplifi- The Journal of Immunology 3 cation, Cy3 labeling, hybridization, washing, and scanning were per- B cells were the least enriched of FCRL5+ cells, and the frequencies of formed by Phalanx Biotech Group (San Diego, CA). Labeled cDNA FCRL5+ cells were not significantly different among the remaining generated from 100 ng total RNA was hybridized on Agilent SurePrint 3 three populations (Supplemental Fig.2A–C).Inconclusion,whereas G3 Human 8 60K microarrays. Raw data were analyzed by Rosetta + Resolver system (Rosetta Biosoftware), using the Rosetta error model to FCRL5 cells are found among all B cell subsets, FCRL5 is most account for error due to random factors and systematic biases, and the frequently expressed on TLM B cells and least frequently on naive median scaling method without flagged and control data for normaliza- cells. In the remaining part of this study, we focus on FCRL52 and tion. The raw data and normalized results were deposited in Gene Ex- FCRL5+ TLM B cells, and FCRL5+ cells in other B cell subsets are pression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE66470. not discussed. Statistical analysis FCRL5+ and FCRL52 TLM B cells display distinct sets of Data are expressed as mean 6 SEM. Statistical significances were cal- membrane proteins culated using Prism (GraphPad Software). We investigated the possible heterogeneity of TLM B cells based on FCRL5 expression. Using multicolor flow cytometry, Results we assessed whether selected membrane proteins on TLM B cells FCRL5-expressing cells are enriched in the TLM B cell subset were differentially expressed depending on FCRL5 status. A total in healthy donors of 24 cell surface proteins were examined, including 6 inhibitory Prior to assessing FCRL5 expression in circulating B cells, we receptors, 6 homing receptors, and 12 additional proteins (Fig. 2, first confirmed the specificity of the FCRL5 mAbs used in the Table I). Of note, 19 of the 24 examined cell surface proteins were expressed at significantly different levels on FCRL52 + study. FCRL5 mAb clone F99 only bound FCRL5 but not FCRL1–4 and FCRL5 Downloaded from in ELISA and in flow cytometry analysis using transfected 293T cells TLM B cells, including all six inhibitory receptors, CD22, CD32, (Supplemental Fig. 1A, 1B). The specificity of the other FCRL5 mAb CD85d, CD85j, LAIR-1, and PD-1; three homing receptors, CCR6, used, clone 509f6 obtained from a commercial source, was reported CXCR4, and CXCR5; and nine additional proteins, BAFFR, in particular not to bind FCRL4 (22). In an effort to identify cir- CD11c, CD20, CD80, CD86, CD95, HLA-I, HLA-II, and TACI culating B cell subsets enriched for FCRL5+ cells, a number of (Table I). Importantly, neither FCRL52 nor FCRL5+ TLM B cells staining strategies were used. The staining strategy that revealed a expressed cell surface FCRL4 (Supplemental Fig. 1C), reported subset containing a significant portion of FCRL5+ cells was the one on some atypical CD212/lo memory B cells (2, 5). FCRL5+ TLM http://www.jimmunol.org/ used by Moir et al. (2) for HIV-viremic patients. According to this B cells expressed significantly more of five inhibitory receptors, staining protocol, B cells are classified by CD27 and CD21 ex- CD22, CD32, CD85d, CD85j, and PD-1, than did any other B cell pression as naive (CD21+/CD272), classical memory (CD27+), and subset (Fig. 2A, Table I). Alternatively, FCRL52 TLM B cells TLM (CD212/lo/CD272) (Fig. 1A). TLM B cells were mainly CD38lo expressed significantly more surface CD85d, CD85j, and LAIR- (Supplemental Fig. 2D), in contrast to CD38hi/CD212/lo/CD272 1 than did either naive or memory B cells. Regarding homing transitional cells (34). FCRL5 was expressed on average by 5.4% of receptors, FCRL5+ TLM cells expressed the lowest levels of CD19+ B cells (Fig. 1B). FCRL5 expression on B cell subsets from a CXCR4 and CXCR5 among all B cell subsets, whereas FCRL52

representative donor is shown on Fig. 1C. Whereas on average 39.3% TLM cells expressed the lowest level of CCR6; these three pro- by guest on October 2, 2021 of TLM B cells expressed FCRL5, only 2.8 and 7.4% of naive and teins were expressed at significantly different levels between classical memory B cells, respectively, were FCRL5+ (Fig. 1B). The FCRL5+ and FCRL52 TLM cells (Fig. 2B, Table I). Of note, frequency of FCRL5+ cells in TLM B cells varied considerably across surface levels of CCR7 and CXCR5, homing receptors for lym- donors, ranging from 5.4 to 86.9%. When B cell subsets were alter- phoid tissues, were similarly reduced on both TLM B cell sub- nativelyclassifiedbyIgDandCD27expression,IgD+/CD272 naive sets, compared with naive and memory B cells. With regard to

FIGURE 1. FCRL5 expression on circulating B cells from healthy donors. (A) B cells were stained for CD19, CD21, CD27, and FCRL5 (mAb clone F99 or 509f6) to identify B cell subsets. (B) Frequencies of FCRL5+ cells on the indicated B cell subsets (n = 31). Horizontal lines indicate mean. ***p , 0.001, using one-way ANOVAwith a Dunnett test, comparing TLM cells to each other subset. (C) Staining of B cell subsets from a representative donor using isotype control (gray shade) or FCRL5 mAb (black line). 4 FCRL5 EXPRESSION DISTINGUISHES TLM B CELL SUBSETS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. The expression of surface proteins on circulating B cell subsets. B cells were stained for CD19, CD27, CD21, FCRL5 (mAb clone F99 or 509f6), and for (A) inhibitory receptors, (B) homing receptors, and (C) other markers and analyzed by flow cytometry. Cell subsets were identified as shown in Fig. 1A. On top of each panel, histograms from a representative donor are shown in the following order (bottom to top): isotype control (black line), naive (grayshade), classical memory (gray shade), FCRL52 TLM (gray shade), and FCRL5+ TLM (black shade). The bottom part of each panel summarizes data from all donors after subtraction of isotype control (n = 6–8); mean fluorescence intensities (MFI) 6 SE are shown. Statistical analyses of the data are shown in Table I. additional surface proteins, FCRL5+ TLM B cells expressed (Fig. 2C, Table I). CD11c was the marker among all evaluated significantly more CD11c and CD20 than did any other B cell surface proteins most differentially expressed between the two subset, as well as the highest levels of CD86, CD95, and HLA-II TLM subsets. The Journal of Immunology 5

Table I. Statistical analyses of surface protein levels among B cell subsets

p Valueb

FCRL52 FCRL5+ Friedman TLM FCRL52 TLM TLM FCRL5+ TLM FCRL5+ TLM Naive versus Surface Test versus versus Classical versus versus Classical versus Classical Markers (p Value)a Naive Memory Naive Memory FCRL52 TLM Memory Inhibitory receptors CD22 ,0.001 0.156 0.844 ,0.05 ,0.05 ,0.05 ,0.05 CD32 ,0.001 0.0625 0.563 ,0.05 0.0625 ,0.05 ,0.05 CD85d ,0.001 ,0.05 0.0938 ,0.05 ,0.05 ,0.05 ,0.05 CD85j ,0.001 ,0.05 0.0938 ,0.05 ,0.05 ,0.05 ,0.05 LAIR-1 ,0.001 0.438 ,0.05 0.0625 ,0.05 ,0.05 ,0.05 PD-1 0.0012 0.0781 0.219 ,0.05 ,0.05 ,0.05 ,0.05 Homing receptors CCR6 0.0251 ,0.05 ,0.05 0.375 0.375 ,0.05 0.467 CCR7 ,0.001 ,0.05 ,0.05 ,0.05 ,0.05 0.3125 0.0781 CD62L 0.0447 ,0.05 0.0547 0.25 0.25 0.7422 0.195 CXCR3 ,0.001 ,0.05 ,0.05 ,0.05 0.8125 0.0781 ,0.05 CXCR4 ,0.001 ,0.05 0.313 ,0.05 ,0.05 ,0.05 ,0.05 CXCR5 ,0.001 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 0.0625

Other Downloaded from BAFFR ,0.001 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 ,0.05 CD11c ,0.001 ,0.05 0.0625 ,0.05 ,0.05 ,0.05 ,0.05 CD20 ,.001 ,0.05 0.0625 ,0.05 ,0.05 ,0.05 ,0.05 CD40 ,0.001 ,0.05 ,0.05 ,0.05 ,0.05 0.844 0.0938 CD80 ,0.001 0.0625 0.0625 ,0.05 0.563 ,0.05 ,0.05 CD86 0.0015 0.0625 0.313 ,0.05 0.156 ,0.05 ,0.05 CD95 ,0.001 ,0.05 0.0781 ,0.05 0.0781 ,0.05 ,0.05 HLA-I ,0.0001 0.313 ,0.05 ,0.05 0.844 ,0.05 ,0.05 http://www.jimmunol.org/ HLA-II ,0.0001 ,0.05 0.156 0.219 ,0.05 ,0.05 ,0.05 ICOSL 0.002 0.219 ,0.05 0.219 ,0.05 0.688 ,0.05 IL4R ,0.0001 ,0.05 ,0.05 ,0.05 ,0.05 0.0625 ,0.05 TACI ,0.001 0.0938 ,0.05 ,0.05 0.156 ,0.05 ,0.05 Data summarized in Fig. 2 were analyzed. aFirst, simultaneous comparison of mean fluorescence intenstiy values from all B cell subsets were performed using a Friedman test. All tested cell surface markers were found significantly different (p , 0.05), prompting further pairwise comparisons. bData from the indicated B cell subsets were compared pairwise by a Wilcoxon signed test. For results considered significantly different, ,0.05 is listed for simplicity; actual p values were 0.01–0.05. by guest on October 2, 2021

In summary, TLM B cells, regardless of FCRL5 expression, had Accumulation of somatic hypermutation (SHM) is another de- comparable general homing receptor expression profiles, reduced fining mark of memory B cell development. We evaluated the SHM levels of CD40, and intermediate levels of IL4R. Overall, however, status of FCRL52 and FCRL5+ TLM B cells and compared them the surface marker profiles of TLM B cells were distinctly differ- to that of naive and IgM+CD27+ memory B cells. Because most ent depending on FCRL5 expression status. In particular, FCRL5+ TLM B cells expressed IgM (Fig. 3A), IgM+ B cells were sorted TLM B cells were characterized among other circulating B cell and used for SHM evaluation. The IgH V region (VH) from two subsets by their high expression of CD11c, CD20, and five inhibi- donors was amplified from single cells and sequenced. VH region tory receptors, CD22, CD32, CD85d, CD85j, and PD-1. sequences are shown in Supplemental Table I and summarized in Fig. 3C. As expected, naive cells lacked SHM, whereas all ana- FCRL5+ TLM B cells include more isotype-switched cells, lyzed IgM+ memory B cells contained SHM. The proportion of have extended replication history, and accumulated more + 2 cells containing SHM was higher in the FCRL5 subset (∼60%) mutations in Ig V regions than FCRL5 TLM B cells than in the FCRL52 subset (∼30%) (Supplemental Table I). The Isotype switch is one hallmark of memory B cell development. mean SHM frequency of cells in the FCRL5+ subset was 2.14% + We found that FCRL5 TLM B cells contained a higher proportion (range, 1.36–2.93%), significantly higher than that of FCRL52 + + + of IgG and IgA cells and fewer IgD cells than did naive B cells, cells (mean, 0.75%; range, 0.18–1.32%), but lower than that of displaying a surface Ig profile comparable to that of classical IgM+ memory B cells (mean, 4.86%; range, 3.77–5.75%). Addi- 2 memory B cells (Fig. 3A). FCRL5 TLM B cells also possessed tionally, no recurrent VDJ combination was found in any B cell + significantly fewer IgD cells than did naive B cells, but very few subset, supporting the notion that the cell populations are poly- + isotype-switched cells were present. Of note, FCRL5 TLM B cells clonal. + 2 contained significantly more IgG cells than did FCRL5 TLM In conclusion, the elevated switched isotype expression, ex- B cells. tended replication history, and accumulation of SHM in most of To compare the in vivo replication histories of circulating B cell the FCRL5+ subset suggest Ag-driven expansion and development. subsets, the average numbers of cell divisions were determined In contrast, the FCRL52 subset was similar to naive B cells in from sorted cells using a k-deleting recombination excision circles these attributes. assay. We found that the average number of cell divisions of 2 + FCRL5+ TLM B cells was significantly higher than that of naive FCRL5 and FCRL5 TLM B cells have distinctly different and FCRL52 TLM B cells, but less than that of classical memory transcriptional profiles B cells (Fig. 3B). In contrast, FCRL52 TLM B cells were similar To further characterize the differences between the FCRL5+ and to naive B cells. FCRL52 TLM subsets, we compared their transcriptional profiles 6 FCRL5 EXPRESSION DISTINGUISHES TLM B CELL SUBSETS

Moreover, microarray analysis identified differentially regu- lated in other functional categories. Genes encoding CDK6 and CDC25B, which play roles in cell cycle progression, were downregulated in the FCRL5+ subset. Notably, genes encoding the Src family kinases FGR and HCK and the protein tyrosine phos- phatases PTPRO, PTPRS, and PTPN2 were all upregulated in the FCRL5+ subset. We also identified several genes involved in regulating apoptosis such as BCL2A1, MCL1, BID, CASP8, and CASP9 to be differentially expressed in the two B cell subsets. Finally, several genes encoding transcription factors were found expressed differently: HES4, AIRE, CREM, TOX, TOX2, TBX21 (T-bet), SOX5, and BCL6 were upregulated, whereas FOXP1 and EGR1 were downregulated in FCRL5+ TLM cells. In conclusion, the transcriptional profiles of FCRL5+ and FCRL52 TLM subsets are markedly different, suggesting they represent separate B cell populations with distinct functional attributes. The transcriptional profile of FCRL5+ TLM B cells is unique among peripheral B cell subsets To identify genes differentially regulated in FCRL5+ TLM B cells Downloaded from compared with other circulating B cell subsets, we selected nine differentially expressed genes identified by microarray analysis for comparative analysis among four B cell subsets. In agreement with microarray results, the expression levels of these genes by quantita- tive PCR were significantly different in FCRL52 and FCRL5+ TLM B cells, with the exception of CD40 and CREM (Fig. 4). IL4R was http://www.jimmunol.org/ found to be expressed at relatively low levels by both classical mem- ory B cells and FCRL5+ TLM B cells. Although NFKBIA (IkBa) mRNA level was the lowest and BCL6 was highest in FCRL5+ TLM B cells, the differences among circulating B cell subsets were mod- erate. Notably, FCRL5+ TLM B cells were markedly different from all other peripheral B cell subsets in their very high expression of ITGAX (CD11c), RTN4R, SOX5,andTBX21 (T-bet).

FCRL5+ and FCRL52 TLM B cells respond differently to by guest on October 2, 2021 FIGURE 3. Surface Ig expression, replication history, and somatic mutation rates of B cell subsets. (A) B cells were stained for CD19, CD27, stimulation and are prone to die in culture CD21, FCRL5, and surface Ig and then analyzed by flow cytometry. To assess the functional consequences of the profound phenotypic Surface Ig expressed by the indicated B cell subsets are shown (n = 6–8). differences, we evaluated the capacity of the FCRL52 and FCRL5+ B ( ) Cells from each subset were sorted from five donors. The average TLM subsets to respond to stimuli, as reflected by intracellular number of replications was evaluated by a k-deleting recombination ex- calcium mobilization and cell proliferation. BCR stimulation cision circles assay. (C) B cells were stained for CD19, IgM, CD21, CD27, + + + 2 triggered only a small elevation of intracellular calcium concen- and FCRL5. CD19 /IgM cells were classified as naive (CD21 /CD27 ), + IgM+ memory (CD27+), and TLM (CD212/lo/CD272), which were tration in the FCRL5 subset that rapidly returned to baseline. + 2 Alternatively, calcium influxes triggered by the same stimulus in further divided into FCRL5 and FCRL5 subsets. Cells from each 2 subset were single sorted from two donors. Somatic mutation rates of naive and FCRL5 TLM B cells were not only stronger but also + 2 + VH genes from 18 naive, 26 IgM memory, 53 FCRL5 ,and40 lasted longer (Fig. 5A). Additionally, the FCRL5 subset showed a FCRL5+ TLM B cells are shown. Statistical analysis using one-way substantially reduced proliferative response to stimulation through ANOVA with a Bonferroni multiple comparison is shown between the BCR, CD40, and/or TLR9 in the presence of IL-4 and IL-2. + 2 FCRL5 or FCRL5 TLM cells and the other subsets. *p , 0.05, **p , BCR and TLR9 costimulation, with or without CD40 engagement, , 0.01, ***p 0.001. induced poor cell proliferation in the FCRL5+ subset compared with naive B cells, whereas the FCRL52 subset underwent an using cells sorted from three healthy donors. A total of 954 unique intermediate level of proliferation (Fig. 5B). A similar trend was genes (515 upregulated, 439 downregulated) were found to be observed when cells were stimulated through the BCR and CD40, more than 2-fold differentially expressed between the two TLM or through CD40 alone, with both TLM B subsets exhibiting subsets at significant levels. Selected differentially expressed significantly reduced proliferation compared with naive cells. genes, grouped according to broad functional categories, are listed We also compared the in vitro survival of B cell subsets. Live cells in Table II. Consistent with flow cytometry results, FCRL5 and were sorted and kept in culture medium in the absence of stimuli. ITGAX (CD11c) were among the most strongly upregulated genes Both TLM B cell subsets contained significantly higher percentages in the FCRL5+ subset. Other differentially regulated genes identi- of dead cells (defined as annexin V+/7-AAD+) after 16 h of culture fied by both flow cytometry and microarray include genes involved than did naive B cells (Fig. 5C). A similar trend was observed after in cell migration and adhesion, such as CXCR4 and CXCR5,aswell 48 h of culture, albeit not significantly different. as genes encoding inhibitory receptors, such as LILRB2 (CD85d) In summary, FCRL5+ TLM B cells exhibit a functional defect in and PDCD1 (PD-1). Of note, microarray analysis revealed two calcium signaling and activation-induced proliferation compared additional inhibitory receptors, SIGLEC6 and LILRB3 (CD85a), to with both naive and FCRL52 TLM B cells, whereas the two TLM be upregulated in the FCRL5+ subset. cell subsets share an equally elevated propensity to die in culture. The Journal of Immunology 7

Table II. Selected genes differentially expressed in FCRL5+ and FCRL2 TLM B cells

FCRL5+ versus Average Expression FCRL52 Confirmed by Flow or Functional Gene Fold Fold Quantitative Group Symbol Aliases FCRL52 FCRL5+ Increase Decrease p Value PCR Surface RTN4R Reticulon 4 receptor; Nogo-66 receptor; 130 4,224 32.4 ,0.0001 Yes molecules NgR1 PIGR Polymeric Ig receptor 73 538 7.4 ,0.0001 FCRL5 Fc receptor–like 5 183 1,120 6.1 0.000430 Yes CD68 CD68 Ag 170 843 5.0 0.000122 CD97 CD97 Ag 7,005 34,012 4.9 ,0.0001 FCRL3 Fc receptor–like 3 9,838 43,497 4.4 ,0.0001 HLA-DQA1 MHC class II, DQa1 8,126 28,099 3.5 0.0251 CD63 CD63 Ag 3,854 11,452 3.0 ,0.0001 CD84 CD84 Ag; SLAM family member 5 174 414 2.4 0.00101 CD19 CD19 Ag 55,638 133,997 2.4 0.000252 Yes FCER2 CD23; low-affinity Fc receptor for IgE 642 111 5.8 ,0.0001 CD24 CD24 Ag 7,078 677 10.4 ,0.0001 CD38 CD38 Ag 339 26 13.1 ,0.0001

Activation SLAMF7 CD319; SLAM family member 7 441 3,806 8.6 ,0.0001 Downloaded from TNFSF9 4-1BBL; TNF superfamily 9 132 652 4.9 0.000217 SLAMF1 SLAM family member 1 653 2,077 3.2 ,0.0001 TNFRSF13B TACI; TNF receptor superfamily 13B 103 284 2.8 0.0419 Yes CD40 CD40 Ag, TNF receptor superfamily 5 988 526 1.9 0.0419 CD69 CD69 Ag 22,010 10,526 2.1 0.00661 FCRL1 Fc receptor–like 1 3,068 1,476 2.1 0.00108 CR2 CD21; C3d receptor 1,284 52 24.5 ,0.0001 Yes http://www.jimmunol.org/ Inhibition LILRB2 CD85d; leukocyte Ig-like receptor, 672 7,959 11.9 ,0.0001 Yes subfamily B PDCD1 PD-1; programmed cell death 1 103 1,132 10.9 ,0.0001 Yes SIGLEC6 CD327; sialic acid binding Ig-like lectin 210 909 4.3 0.00658 6; CD33L LILRB3 CD85a; leukocyte Ig-like receptor, 177 583 3.3 0.000114 subfamily B3

Cytokines/ IL2RB IL-2 receptor b; CD122 65 1,220 18.7 0.000140 ,

cytokine TNFRSF1B TNF receptor 2; TNF receptor 234 1,568 6.7 0.0001 by guest on October 2, 2021 receptors superfamily 1B IL28RA IL-28 receptor a; IFN-l receptor 1,372 7,992 5.8 0.000210 EBI3 EBV induced 3; IL-27b; IL-35 subunit 245 965 3.9 0.0111 Yes IL15 IL-15 686 278 2.5 0.0227 IL6 IL-6; IFN-b2 573 176 3.3 0.00149 LTB Lymphotoxin b; TNF superfamily 3 48,601 14,192 3.4 0.000930 IL4R IL-4 receptor 69,133 14,527 4.8 ,0.0001 Yes IL23A IL-23a, subunit p19 1,186 173 6.8 0.000359

Migration/ ITGAX Integrin aX; C3 receptor 4; CD11c 1,129 13,451 11.9 ,0.0001 Yes adhesion ITGB2 Integrin b2; C3 receptor 3, 4; CD18; 11,154 33,335 3.0 ,0.0001 LFA-1 ITGB7 Integrin b7 10,962 29,815 2.7 0.00605 CXCR4 Chemokine (C-X-C motif) receptor 4; 98,487 44,391 2.2 ,0.0001 Yes CD184 ITGAM Integrin aM; C3 receptor 3 subunit; 1,609 618 2.6 0.0149 CD11b SELL L-; CD62L 7,523 2,229 3.4 ,0.0001 CXCR5 Chemokine (C-X-C motif) receptor 5; 5,032 1,302 3.9 0.00800 Yes CD185 CCR7 Chemokine (C-C motif) receptor 7 14,430 2,596 5.6 ,0.0001

Signaling PTPRS Protein tyrosine phosphatase, receptor 1,919 22,562 11.8 ,0.0001 type, S GAS7 Growth arrest–specific 7 78 335 4.3 0.000371 PRKCH Protein kinase Ch 454 1,721 3.8 0.000477 FGR v-fgr Oncogene homolog 31,245 115,898 3.7 0.00900 MAP3K8 MAPK kinase kinase 8 1,435 4,443 3.1 ,0.0001 PTPRO Protein tyrosine phosphatase, receptor 222 604 2.7 0.0290 type, O HCK Hemopoietic cell kinase 702 1,782 2.5 0.01144 PTPN2 Protein tyrosine phosphatase, 138 339 2.4 ,0.0001 non–receptor type 2 PIK3CG PI3K, catalytic g 637 283 2.3 0.000576 NFKBIA IKBA; NF-kB inhibitor a 76,821 30,016 2.6 ,0.0001 Yes (Table continues) 8 FCRL5 EXPRESSION DISTINGUISHES TLM B CELL SUBSETS

Table II. (Continued)

FCRL5+ versus Average Expression FCRL52 Confirmed by Flow or Functional Gene Fold Fold Quantitative Group Symbol Aliases FCRL52 FCRL5+ Increase Decrease p Value PCR CDC25B Cell division cycle 25 homolog B 2,250 862 2.6 ,0.0001 PTPRK Protein tyrosine phosphatase, receptor 1,486 339 4.4 ,0.0001 type, K CDK6 Cyclin-dependent kinase 6 1,295 172 7.5 ,0.0001 TCL1A leukemia/lymphoma 1A 59,526 7,255 8.2 ,0.0001 TCL1B T cell leukemia/lymphoma 1B 5,000 198 25.2 ,0.0001

Apoptosis/ BCL2A1 BCL2-related protein A1 1,303 4,208 3.2 0.0114 survival CASP9 Caspase 9 649 1,974 3.0 0.0153 MCL1 Myeloid cell leukemia sequence 1 2,336 6,080 2.6 0.00283 CASP8 Caspase 8 612 1,434 2.3 0.00694 BID BH3 interacting domain death agonist 581 1,340 2.3 0.00154 TNFRSF10A TNF receptor superfamily 10A; death 339 151 2.2 0.00989 receptor 4 TNFSF10 TRAIL; TNF superfamily 10 225 91 2.5 ,0.0001

BNIP3 BCL2/adenovirus E1B interacting 476 178 2.7 0.000885 Downloaded from protein 3 BIRC3 cIAP2; baculoviral IAP repeat 7,182 2,327 3.1 ,0.0001 containing 3 PAWR PRKC apoptosis WT1 regulator 1,016 282 3.6 ,0.0001

Transcription HES4 Hairy and enhancer of split 4 334 5,953 17.8 ,0.0001 factor BCL11B B cell CLL/lymphoma 11B 43 550 12.9 0.000522 TOX2 TOX HMG-box family 2 745 9,445 12.7 ,0.0001 http://www.jimmunol.org/ AIRE Autoimmune regulator 134 1,050 7.9 ,0.0001 CREM cAMP-responsive element modulator 245 1,813 7.4 ,0.0001 E2F7 E2F transcription factor 7 151 1,051 7.0 0.0119 TOX Thymocyte selection–associated 200 1,177 5.9 0.000868 HMG-box TBX21 T-bet; T-box 21 971 3,971 4.1 ,0.0001 Yes SOX5 SRY-box 5 111 373 3.4 ,0.0001 Yes BCL6 B cell CLL/lymphoma 6 1,435 4,375 3.0 0.0221 Yes NFATC2 NFAT, cytoplasmic 2 169 385 2.3 0.0117

EGR1 Early growth response 1 5,150 2,355 2.2 0.0144 by guest on October 2, 2021 SMAD3 SMAD family 3 2,528 1,042 2.4 ,0.0001 FOXP1 Forkhead box P1 1,165 220 5.3 0.0253 LEF1 Lymphoid enhancer-binding factor 1 585 31 18.7 ,0.0001

Discussion expressed gene between TLM B cells and classical memory We identified in healthy donors two distinct subsets of TLM B cells, B cells obtained from children exposed to malaria (36). RTN4R discriminated by FCRL5 expression, illuminating the heteroge- protein plays critical roles in the CNS and has multiple binding neity of this pathologically important subset of cells and providing partners, including BAFF (37), and was shown to be expressed clues to understand their development and function in healthy by activated human B cells (38). Of note, FCRL3 mRNA levels individuals. were found elevated in the FCRL5+ subset, whereas FCRL1 was The proposed FCRL5+ and FCRL52 TLM subsets, which on expressed at a reduced level, suggesting coordinated changes in average are comparable in numbers, differ from each other in the expression of FCRL family members. Importantly, however, numerous characteristics. High expression of CD11c, T-bet, SOX5, FCRL4 surface protein was not present on circulating B cells from RTN4R, and a number of inhibitory receptors distinguishes the healthy donors (Supplemental Fig. 1C), as reported (4, 10). The FCRL5+ subset not only from the FCRL52 subset, but also from FCRL5+ TLM B cells described in this study are distinct from the 2 other circulating B cell populations. Although CD11c, T-bet, and FCRL5+ cells identified in HCV-MC patients, which are CD21 /lo/ SOX5 were identified in other studies to mark TLM B cells (2, 3, CD27+ (28). Additionally, the Ig repertoire of FCRL5+ TLM B cells 7, 8, 10, 29), RTN4R to our knowledge was first suggested to be a from healthy donors was highly diverse, unlike the clonally ex- useful marker in the present study. CD11c forms a dimer with panded FCRL5+ cells described in HCV-MC patients (7, 39). CD18, serving as both complement receptor and adhesion mole- Consistent with the idea that TLM B cells migrate away from cule, possibly facilitating Ag uptake and presentation as well as lymphoid tissues and accumulate in nonlymphoid tissues, we contributing to tissue localization (3, 35). Elevated expression of found that compared with naive and classical memory B cells, 2 HLA-II, CD80, and CD86 on the FCRL5+ TLM subset is further both FCRL5 and FCRL5+ TLM B cells expressed substantially indication that the cells might possess a higher capacity to present lower levels of CCR7 and CXCR5, homing receptors for lymphoid Ag to T cells. However, the possible active functional capacities of tissues. Nevertheless, FCRL5+ TLM B cells, compared with 2 TLM B cells remain to be studied, as the cells may play yet un- FCRL5 TLM B cells, expressed higher levels of CCR6 and known roles. RTN4R was the most differentially expressed gene CXCR3, homing receptors for inflammatory sites, and therefore between the two TLM B cell subsets. Notably, RTN4R was also they might preferentially accumulate in stressed or injured tis- found in a recent microarray study to be the most differentially sues with ongoing inflammation. Indeed, CD212/lo B cells over- The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Genes differentially expressed by FCRL5+ TLM B cells compared with other subsets. Cells from each subset were sorted from four to six donors. Cell subsets were identified as shown in Fig. 1A. mRNA levels relative to FCRL52 TLM B cells were determined by quantitative PCR. Statistical analysis using one-way ANOVA with a Dunnett test is shown between FCRL5+ TLM cells and the other subsets. *p , 0.05, **p , 0.01, ***p , 0.001. FIGURE 5. Differential responses to stimulation and survival of FCRL52 and FCRL5+ TLM B cells. (A) Changes in intracellular cal- expressing FCRL5 by microarray analysis were found to be the cium concentrations upon BCR and subsequent ionomycin stimulation predominant B cell population in both the bronchoalveolar lavage are shown in the indicated B cell subsets. Cell subsets were identified fluid of CVID Ia patients and synovial fluid of rheumatoid arthritis as shown in Fig. 1A. A representative of three independent experiments 2 patients (8, 29). Regarding their functional properties, FCRL5 is shown. Arrows indicate the addition of anti-Ig F(ab9)2 Ab and ion- TLM B cells proliferated less upon BCR, CD40, and/or TLR9 omycin. (B) Sorted B cell subsets were cultured with the indicated stimulation than did naive cells, and the proliferative capacity of stimuli. Live cell numbers were estimated 4 d later by measuring ATP the FCRL5+ subset was even lower. Moreover, the FCRL5+ subset levels based on luminescence signal. Results from three independent showed significantly decreased calcium influx upon BCR stimu- experiments are shown using one-way ANOVA with a Bonferroni C lation than did either the FCRL52 subset or naive B cells. The multiple comparison. ( ) Sorted live B cells subsets were cultured in medium without stimulus for 16 (n =4)or48h(n = 5) and then stained reduced responses to stimuli were consistent with elevated levels with annexin V and 7-AAD. Data from each time point were analyzed of multiple inhibitory receptors such as CD22, CD32, CD85d, p , + by one-way ANOVA with a Bonferroni multiple comparison. * 0.05, CD85j, and LAIR1 on the FCRL5 subset, suggesting that the **p , 0.01, ***p , 0.001. cells are functionally exhausted. Several receptor-type protein tyrosine phosphatases, including PTPRO and PTPRS, were up- 10 FCRL5 EXPRESSION DISTINGUISHES TLM B CELL SUBSETS regulated in the FCRL5+ subset, similarly as reported for CD212 autoreactive B cells that become anergic. The poor in vitro re- cells in CVID Ia patients (8), which could also contribute to their sponse of the FCRL5+ TLM subset to a number of stimuli together reduced response to stimulation. We also found clear indications, with prominent expression of multiple inhibitory receptors sug- based on analysis of replication history, switched isotype ex- gests exhaustion, possibly in response to chronic Ag stimulus. pression, and SHM rate, that the FCRL5+ TLM subset, but not the FCRL5 expression itself could be a mark of previous BCR stim- FCRL52 TLM subset, underwent Ag-driven expansion and de- ulation, as FCRL5 is specifically induced upon BCR stimulation velopment, indicating that the cells are not anergic (7, 28, 39). (23). The type of Ag that drives TLM B cells in healthy indi- While this manuscript was under review, two groups reported viduals remains to be defined. In contrast, the FCRL52 TLM that TLM B cells from individuals living in malaria endemic subset is akin to naive B cells in terms of its isotype expression, areas express FCRL5 but are FCRL42 (36, 40), in contrast to short replication history, and low levels of SHM. The FCRL52 previous reports (5), underscoring the importance of FCRL5 as subset proliferates more robustly than does the FCRL5+ subset in a key marker of TLM B cells in malaria. Notably, both new stud- response to stimuli and expresses lower levels of inhibitory re- ies found that TLM B cells are heterogeneous in regard to ceptors. Intriguingly, we found that FCRL5+ TLM B cells from FCRL5 expression and suggested that FCRL5 expression is a healthy adults displayed significantly lower SHM rate and shorter useful marker to discriminate phenotypic and functional subsets. replication history than did classical memory B cells, similarly to FCRL5+ TLM B cells were reported to express elevated levels of what was reported in children exposed to malaria (45). However, CD19, CD20, CD32, and FCRL3 but reduced levels of CD21, adults exposed to malaria, who presumably encountered more compared with FCRL52 TLM B cells (36, 40), in agreement with rounds of malaria infection than did children, display similar SHM our study. Importantly, FCRL5 expression was found associated rates and replication histories in their TLM and classical memory with markedly compromised BCR signaling and Ab production B cell subsets (12, 40). These observations together suggest that Downloaded from and it was suggested that FCRL5 marks dysfunctional cells (36, replication and SHM in TLM B cells is related to the extent of Ag 40). Most of the genes that we identified differentially expressed exposure. Further studies are needed to shed on the origin between the two subsets of TLM B cells were also detected in the and functional roles of the two TLM B cell subsets during an malaria studies, which compared TLM B cells to classical mem- immune response, and in particular on the role of FCRL5 itself. ory B cells. Specifically, 50% of the genes listed in Table II were identified in both malaria studies, whereas 38% of the genes were Acknowledgments http://www.jimmunol.org/ identified in one of the studies. Notably, the direction of changes We thank Mark Kukuruga for technical help with flow cytometry, Shirin in expression show strong correlation; genes that we found ele- Treadwell and Adovi Akue for cell sorting, Antonina Aydanian for VH 2 vated in FCRL5+ TLM B cells compared with FCRL5 TLM sequence analysis, Michael DeSalvo (Phalanx Biotech Group) for micro- B cells were found in the malaria studies to be elevated in TLM array analysis, and Bazarragchaa Damdinsuren, Andrea Franco, Zachary B cells compared with classical memory B cells. Therefore, the Kraus, Venkateswara Simhadri, and two anonymous reviewers for sug- gene expression signature of TLM B cells is dominantly driven gestions. Human tonsils were provided by the Cooperative Human by the unique signature of the FCRL5+ subset. The new malaria Tissue Network, which is funded by the National Cancer Institute. studies strongly support the conclusion that FCRL5 expression by guest on October 2, 2021 discriminates two phenotypically and functionally distinct subsets Disclosures of TLM B cells. The authors have no financial conflicts of interest. A CD212/CD11c+ B cell subset, called age-associated B cells, which has increased capacity to respond to TLR stimulation, pre- References sent Ag, and secrete autoantibodies, accumulates in aged mice 1. Kurosaki, T., K. Kometani, and W. Ise. 2015. Memory B cells. Nat. Rev. and also appears in mice with certain viral infections and autoim- Immunol. 15: 149–159. mune conditions (41–43). Several features of murine age-associated 2. Moir, S., J. Ho, A. Malaspina, W. Wang, A. C. DiPoto, M. A. O’Shea, G. Roby, + S. Kottilil, J. Arthos, M. A. Proschan, et al. 2008. 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Med. combined TLR and BCR stimulation (41), elevated CCR7 expres- 202: 783–791. sion that was proposed to lead to localization of the cells at the 5. Weiss, G. E., P. D. Crompton, S. Li, L. A. Walsh, S. Moir, B. Traore, K. Kayentao, A. Ongoiba, O. K. Doumbo, and S. K. Pierce. 2009. Atypical splenic T/B border (43), and uncompromised survival in culture memory B cells are greatly expanded in individuals living in a malaria-endemic independent of BAFF (41). It is not known whether TLM B cells area. J. Immunol. 183: 2176–2182. accumulate with aging in humans. FCRL4+ tissue memory B cells 6. Wehr, C., H. Eibel, M. Masilamani, H. Illges, M. Schlesier, H. H. Peter, and K. Warnatz. 2004. A new CD21low B cell population in the peripheral blood of described in tonsils constitute another B cell population that shares patients with SLE. Clin. Immunol. 113: 161–171. some of the features of the circulating FCRL5+ TLM B cell subset 7. Charles, E. D., C. Brunetti, S. Marukian, K. D. Ritola, A. H. Talal, K. Marks, I. M. Jacobson, C. M. Rice, and L. B. Dustin. 2011. Clonal B cells in patients (3, 4). The phenotypic similarities of these FCRL4- and FCRL5- with hepatitis C virus-associated mixed cryoglobulinemia contain an expanded expressing cells include their shared CD21, CD32, CD11c, and anergic CD21low B-cell subset. Blood 117: 5425–5437. SOX5 expression profiles, as well as low responsiveness to BCR 8. Isnardi, I., Y. S. Ng, L. Menard, G. Meyers, D. Saadoun, I. Srdanovic, J. Samuels, J. Berman, J. H. Buckner, C. Cunningham-Rundles, and E. Meffre. stimulation. However, distinct differences exist beyond divergent 2010. /CD212 human naive B cells contain mostly au- FCRL expression, including the robust response of FCRL4+ tissue toreactive unresponsive clones. Blood 115: 5026–5036. memory B cells to CD40 stimulation combined with IL-2 and IL-10. 9.Yeo,L.,H.Lom,M.Juarez,M.Snow,C.D.Buckley,A.Filer,K.Raza,and D. Scheel-Toellner. 2015. Expression of FcRL4 defines a pro-inflammatory, RANKL- In this study, we defined two subsets of TLM B cells. The producing B cell subset in rheumatoid arthritis. Ann. Rheum. Dis. 74: 928–935. significant replication history, switched isotype expression, and 10. Saadoun, D., B. Terrier, J. Bannock, T. Vazquez, C. Massad, I. Kang, F. Joly, + M. Rosenzwajg, D. Sene, P. Benech, et al. 2013. Expansion of autoreactive 2 accumulation of SHM in the FCRL5 TLM subset suggest Ag- unresponsive CD21 /low B cells in Sjo¨gren’s syndrome-associated lymphopro- driven expansion. Therefore, this subset is unlikely to represent liferation. Arthritis Rheum. 65: 1085–1096. The Journal of Immunology 11

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2.5 - - - - - F99 Anti Anti Anti Anti Anti

2.0 FCRL1

1.5 FCRL2

1.0 FCRL3 0.5 FCRL4 ELISA absorbance ELISA 0.0 FCRL5

Antibody binding

C Classical FCRL5- FCRL5+ CD19+/IgD-/CD38- Naive memory TLM TLM Tonsil cell

FCRL4

Supplemental Figure 1. Specificity of FCRL5 antibody (clone F99) and assessment of FCRL4 expression on circulating B cell subsets. (A) FCRL5 (F99) mAb binding to recombinant FCRL1- 5/human IgG-Fc fusion proteins were assessed using ELISA. Means ± SD of four replicate wells are shown. (B) FCRL5 (F99) mAb binding to 293T cells transfected with FCRL1-5 using flow cytometry. (C) Freshly isolated circulating B cells were labeled with CD19, CD21, CD27, FCRL5, FCRL4 (black line) or isotype control mAb (grey shadow). B cell subsets were gated as shown in Figure 1 A. As positive control, staining of tonsil memory B cells is shown. A representative of three independent experiments is shown. A B CD19+ 50 G3 G2 40

+

30

20 % FCRL5

10

0

+

G4 G1 CD27

CD19 IgD negative G1: Naive memory

G4: Double memory G3: Switched G2: Unswitched

C G2: G3: G4: G1: CD19+ Unswitched Switched Double Naïve memory memory negative

FCRL5

D CD19+ Naive Classical memory TLM

CD21

CD38

Supplemental Figure 2. FCRL5 expression on circulating B cells; transitional B cells. (A, B, C) Freshly isolated B cells were labeled with CD19, CD21, CD27, IgD and FCRL5 or isotype control mAbs. (A) B cell subsets are gated as naïve (IgD+/CD27-), unswitched memory (IgD+/CD27+), switched memory (IgD-/CD27+) and double negative cells. (B) Frequencies of FCRL5+ cells on the indicated B cell subsets (n = 9). (C) FCRL5 staining of different B cell populations (black line), compared to isotype control (gray shaded) in a representative donor. Numbers indicate percentages of FCRL5+ cells. (D) B cells were labeled with CD19, CD27, CD21 and CD38 mAbs. Cell subsets were identified as shown in Figure1A. CD21lowCD38high transitional B cells present in the indicated B cell subsets are gated. A representative of six independent experiments is shown. Supplemental Table I. VH sequencing results from two donors Donor #101 Sample Mutation CDR3 V D J CDR3 sequence number rate (%) length Naïve 3-48*02 5-18*01 4*02 0 CARSRNTAMVFDYW 12 L2 4-59*01 6-13*01 4*02 0 CAHIAAAEVEW 9 L11 3-30*03 7-27*01 3*02 0 CVSGDPHGGGPLSGAFDIW 17 L13 3-53*01 5-18*01 3*02 0.70 CARGLGQLYAFDIW 12 L14 3-23*01 / 1*01 0 CAKAPWRW 6 L15 1-69*01 3-22*01 5*02 0 CARDRAMIVGGGNWFDPW 16 L16 4-59*01 1-26*01 2*01 0 CARCPLVGATTPYWYFDLW 17 L17 1-46*01 5-18*01 4*02 0 CAREDLAPIQLWSW 12 L21 3-7*01 3*9-*01 5*02 0 CARDLPYYDILTGYHNWFDPW 19 L22 3-48*02 33-22*01 4*02 0 CARDQGGLYYYDSSGLPYYFDYW 21 L23 4-39*01 6-13*01 5*02 0 CAEMYSSSWAERW 11 L24 4-61*01 6-13*01 4*02 0 CARGYSSSWVFDYW 12 IgM+ R2 3-48*02 3-10*01 4*02 4.48 CARGRWGEYFDCW 11 memory R12 4-39*01 3-10*01 4*02 5.54 CAKSPYVSGSRIDYW 13 R13 3-73*01 2-2*01 5*01 8.39 CASTNQLSSW 8 R14 3-53*01 6-19*01 4*02 0.38 CARGGQWLTPDYW 11 R15 6-01*01 4-23*01 6*02 8.66 CTGEPTTAGRPTHGMDVW 16 R16 3-33*03 2-15*01 4*02 4.48 CARGCCSGASLAFPINYW 16 R17 3-23*01 5-12*01 4*02 8.21 CAKDLRRADDYW 10 R18 3-33*01 2-15*01 6*02 6.72 CARVSRSSTWNYYYYGMDVW 18 R21 3-30*03 4-23*01 4*02 2.61 CAKGYGGYLDYW 10 R22 3-23*01 6-13*01 4*02 2.99 CAKDQGEDSSRWYVTLPWFW 18 R25 4-39*01 1-1*01 6*02 2.95 CARVLWNNVHHYYYGMDVW 17 R27 3-23*01 3-22*01 4*02 4.12 CAKDSAVGYDSSGYYFYW 16 R28 4-4*07 4-17*01 5*01 9.81 CARHYGDSYW 8 FCRL5- B1 1-2*04 6-13*01 6*02 0 CARARRIADLYYYYGMDVW 17 TLM C1 1-18*01 6-13*01 6*02 1.12 CARDLLQMARYYYGMDVW 16 F1 3-7*01 2-21*02 3*02 0 CARTAADLDAFDIW 12 G1 4-39*01 4-17*01 4*02 0 CARHVGDSSWGYW 11 A2 4-39*01 6-13*01 4*02 4.8 CARLSSSWYSPLPIDYW 15 B2 1-2*02 6-6*01 4*02 0.75 CERDYSSSSGIYYFDYW 15 C2 3-64D*06 3-3*01 4*02 0.37 CVKDSFGSTAYW 10 D2 3-30*04 5-12*01 6*02 0.37 CARDPAHYSGYVHYYYYGMDVW 20 E2 1-69*01 2-2*01 5*02 0 CARGVVPADVNWFDPW 14 F2 3-53*01 3-10*01 4*02 0 CATMTYGSGSYSDW 12 A3 3-30*03 5-18*01 5*02 0 CAREGLQLWVFDPW 12 B3 5-51*01 2-15*01 3*02 0 CASTLTPLRYCSGGSCYPDAFDIW 22 C3 4-39*01 3-22*01 2*01 0 CARHAYYDSSGYTFDLW 15 D3 3-48*02 6-19*01 5*02 0 CARDLYGGYSSAGFDPW 15 F3 1-46*01 3-22*01 3*02 0 CASLGLGEDSSGTDAFDIW 17 H3 1-2*02 6-13*01 4*02 0 CATRSLYYSSSSLHSFDYW 17 A4 1-3*01 3-10*01 4*02 0 CARGGRVDYW 8 H4 3-30*03 5-24*01 6*02 0 CAKDLGRWVENGYGMDVW 16 A5 3-23*01 4-23*01 3*02 0 CAKPGRPYTVDDAFDIW 15 C5 5-51*01 3-16*02 3*02 0 CARRELWSLDAFDIW 13 D5 3-11-*03 3-10*01 4*02 0.37 CRGYYYGSGSYYNNLYFDYW 18 E5 3-53*01 6-19*01 3*02 0 CARDLGYSSGWYFGAFDIW 17 G5 4-31*03 2-21*02 4*02 0.37 CARTAYCGGDCYVYFDYW 16 H5 3-53*01 3-10*01 4*02 0.38 CARNGFGELFFDYW 12 A6 1-3*01 6-19*01 6*02 0 CARDQGMYSSGKYGMDVW 16 D6 3-74*01 3-22*01 4*02 0 CARGGGYYDSSPDYW 13 H6 5-51*01 5-18*01 3*02 0 CARQLWLPGDVSAAFDIW 16 FCRL5+ B7 3-7*01 3-22*01 4*02 1.49 CARVGGMYYYDSSGYYSDYW 18 TLM E7 3-23*01 6-13*01 4*02 0 CAKEGRKGSIAAAGFDYW 16 G7 3-30*04 5-12*01 4*02 1.12 CARDSLTYYFDYW 11 H7 1-8*01 3-10*01 4*02 7.43 CARVGNSGNHFNRRFEYW 16 A8 4-31*03 1-26*01 5*02 3.69 CARGRGTSLNWFDPW 13 B8 1-3*01 3-0*01 5*02 3.36 CARGPPIYHSGRYVGNWFDPW 19 C8 4-39*01 3-6*01 3*02 2.21 CARFGADDGFDIW 11 E8 3-9*01 5-4*01 3*02 0 CAKEGDGTSGAFDIW 13 G8 4-4*07 6-9*01 4*02 0 CARGEFAVAPPPTPLYYFDYW 19 A9 5-51*01 4-1*01 6*02 2.61 CARHRGPTGTDYYGMDVW 16 B9 1-46*01 3-16*01 *01 0 CARDAGYDDDFQHW 12 C9 1-18*01 3-10*01 6*02 0 CARDELLWFGESIYYYYGMDVW 20 D9 3-23*04 3-10*01 4*02 0 CAKDRNFYEYYYGSGSYSPYYFDYW 23 F9 4-31*03 6-13*01 4*02 0 CAREAIAAAGLDYW 12 E10 4-31*03 3-22*01 4*02 0 CARVSRVPYYYDSSGYGTDYW 19 G10 3-11*03 6-19*01 6*02 0 CARDREVAGTSDYYYGMDVW 18 A11 1-69*01 6-13*01 6*02 0 CARGIAAAGNYYYYGMDVW 17 B11 4-61*01 2-8*01 1*01 0 CARMGVYDSPSFQHW 13 F11 4-59*01 5-24*01 2*01 0 CARGGDGYNVGDWYFDLW 16 H11 5-51*01 3-16*02 4*02 3.36 CARVRGATERLFDYW 13 B12 3-9*01 5-12*01 6*02 7.46 CARGGDGYNVGDWYFDLW 16 D12 1-18*01 3-16*01 3*02 0 CAREWRSWGAFDIW 12 F12 3-23*01 3-22*01 4*02 2.43 CAKPLDSSGYSDHW 12 Donor #107 Sample Mutation CDR3 V D J CDR3 sequence number rate (%) length Naïve L5 4-4*02 2-21*02 4*02 0 CARSLVFGGDYFFDYW 14 L6 4-39*01 3-22*01 3*02 0 CARGAPFYYDSSGDAFDIW 17 L7 3-21*01 5-18*01 5*02 0 CARGRRYSYGPGGW 12 3-30- L8 3-22*01 4*02 0 CAREVAVITAFDYW 12 3*01 L11 5-51*01 3-16*01 4*02 0 CARLGGATYYFDYW 12 5-10- L12 4-23*01 5*02 0 CASQYGGTKNNWFDPW 14 1*03 IgM+ R1 6-1*01 3-9*01 4*02 1.81 CARRAVTEYGVFDFW 13 memory R2 3-23*01 5-18*01 4*02 8.96 CASRFGM 5 R5 5-51*01 1-14*01 5*01 5.60 CARHPLFGTNSW 10 R6 3-30*03 3-22*01 4*02 3.73 CAKEYDSGGYGAFADYW 15 R7 4-39*01 2-2*01 4*02 3.32 CTRLMGSAWAQSEYW 13 R8 3-48*02 2-8*01 5*02 5.97 CARDSAFGQKNWFDPW 14 R11 3-7*01 6-19*01 4*02 2.61 CARQWPFDYW 8 R12 3-11*06 3-22*01 4*02 0.75 CARALGYSSAYYYYPYW 15 R14 3-7*01 3-3*01 6*03 4.10 CARDLGFHIMDVW 11 R15 2-70*01 6-13*01 2*01 2.21 CARTGGIEGAGVGNFDLW 16 R16 4-59*09 2-15*01 4*02 9.39 CVRRRGGGWADYFDYW 14 R17 4-59*03 2-2*01 4*03 3.77 CARDRSSTTGYMDVL 13 R18 2-5*02 2-2*01 4*02 4.80 CAQRRYHFDYW 9 - FCRL5 1B 2-5*01 6-13*01 5*02 0 CAHRLRYSSSWYGGWFDPW 17 TLM 1C 5-51*01 5-24*01 4*02 0 CARHGDGYNPPSYW 12 1D 3-23*01 3-22*01 4*02 0 CAKGCLWYDSSGYYYDYYFDYW 20 1E 1-69*01 2-2*01 6*02 0 CARWGRGYCSSTSCYYYYGMDVW 21 1F 3-13*01 3-3*01 6*03 0 CARGSWSGLWYYYYYMDVW 19 2A 1-2*02 6-6*01 3*02 0 CARGHPYSSYAFDIW 13 2B 1-2*02 2-8*01 4*02 1.49 CARPGFCTNGVCLWAFDYW 17 2C 1-69*01 3-3*01 5*02 2.24 CAKDRTVWSGTGWFDPW 15 2D 3-49*05 2-2*01 6*02 0 CTRDGEGSSTSSYYGMDVW 17 2F 2-70*01 3-9*01 6*03 1.85 CARMAITYYDLLTGYLGDYYYYMDVW 24 2G 3-30*03 1-26*01 6*03 7.09 CARDSEELLSGSYHYYYMDVW 19 2H 3-23*01 4-17*01 4*02 0 CATQASYGDYVYW 11 3-30- 3A 6-13*01 5*02 0 CARDRTAFGQQLFTGNWFDPW 19 3*01 3B 4-31*03 3-10*01 4*02 0 CARGFGFGEPWGFPRYW 15 3C 3-48*02 3-22*01 4*02 0 CARDDEYYYDSSGSIPFDYW 18 3E 1-2*02 3-10*01 6*03 0 CARGPKSFTYGSGSLNYYYYYMDVW 23 4A 3-30*03 6-13*01 4*02 5.22 CAKEASAGTIDYW 11 4B 1-18*01 4-17*01 4*02 0 CARGSTVTPLDYW 11 4C 1-3*01 6-13*01 4*02 0.37 CAREDSSWYFEDFDYW 14 4D 3-23*01 3-3*02 4*02 11.57 CAKDYISGDGFWDFDWW 15 3-30- 4H 6-19*01 6*03 0 CAREYSSGWYRDDLYYMDVW 18 3*01 5C 1-46*01 5-18*01 4*02 1.49 CARGSYGPDRCDYW 12 5D 3-23*01 6-19*01 5*02 0 CAKDPYSSGWLGWFDPW 15 5H 5-51*01 6-13*01 3*02 0 CAVWPGSSWEDAFDIW 14 6A 4-59*01 2-2*01 5*02 0 CARVGRYCSSTSCYYWFDPW 18 6C 3-9*01 5-12*01 1*01 0 CAKGAYGGYVNW 10 + FCRL5 7A 1-18*01 4-17*01 6*02 0.75 CARNKSVTTDGMDVW 13 TLM 7C 3-30*03 4-17*01 4*02 6.37 CVRPPRFFGDYDTTYW 14 8A 3-30*03 3-9*01 5*02 0.75 CARLRLIRPWFDPW 12 8B 3-64*05 2-21*01 5*02 0.37 CVRDIVVVRWGWFDPW 14 8C 3-53*01 3-10*01 6*02 4.53 CARVGSSNQYYGMDVW 14 8D 1-2*02 3-3*01 6*04 0.75 CARSAGGGSSTSQGYYDFGGAWDVW 23 8E 3-73*02 6-6*01 4*02 0 CTRRGSSAEFDYW 11 9C 3-53*01 3-10*01 6*02 4.53 CARVGSSNQYYGMDVW 14 9F 4-31*03 3-22*01 4*02 4.43 CAGNYYDRSGYYPLSW 14 10A 5-51*01 3-9*01 1*01 1.12 CATPGFDDFQHW 10 10B 1-46*01 3-3*01 4*02 4.48 CARSIPYYDFWSGFYW 14 10F 3-30*03 3-10*01 6*02 5.97 CARDLIYDFPRAYFYGMDVW 18 11A 4-31*03 3-9*01 4*02 0 CARANTYYDILTGYYTPIDYW 19 11C 1-3*01 3-10*01 6*02 5.22 CARDRGDRGVMAYYYGMDVW 18 11D 3-30*03 3-10*01 6*03 0 CAKTGRYYYGSGWVYYYYYMDVW 21 12C 3-30*03 3-10*01 3*02 5.97 CAMFPGLRPFNDAFDLW 15 12D 3-23*01 3-3*01 4*02 5.22 CAKGSGYHFWSGYWGLDYW 17