Identification of Stage-Specific Surface Markers in Early B Cell Development Provides Novel Tools for Identification of Progenitor Populations This information is current as of September 28, 2021. Christina T. Jensen, Stefan Lang, Rajesh Somasundaram, Shamit Soneji and Mikael Sigvardsson J Immunol published online 25 July 2016 http://www.jimmunol.org/content/early/2016/07/23/jimmun ol.1600297 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/07/23/jimmunol.160029 Material 7.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published July 25, 2016, doi:10.4049/jimmunol.1600297 The Journal of Immunology Identification of Stage-Specific Surface Markers in Early B Cell Development Provides Novel Tools for Identification of Progenitor Populations Christina T. Jensen,* Stefan Lang,* Rajesh Somasundaram,† Shamit Soneji,* and Mikael Sigvardsson*,† Whereas the characterization of B lymphoid progenitors has been facilitated by the identification of lineage- and stage-specific surface markers, the continued identification of differentially expressed proteins increases our capacity to explore normal and malignant B cell development. To identify novel surface markers with stage-specific expression patterns, we explored the reactivity of CD19+ Bcell progenitor cells to Abs targeted to 176 surface proteins. Markers with stage-specific expression were identified using a transgenic 2 reporter gene system subdividing the B cell progenitors into four surface IgM stages. This approach affirmed the utility of known Downloaded from stage-specific markers, as well as identifying additional proteins that selectively marked defined stages of B cell development. Among the stage-specific markers were the cell adhesion proteins CD49E, CD11A, and CD54 that are highly expressed selectively on the most immature progenitors. This work identifies a set of novel stage-specific surface markers that can be used as a complement to the classical staining protocols to explore B lymphocyte development. The Journal of Immunology, 2016, 197: 000–000. he development of mature B lymphocytes from multi- associated with other cell lineages. The earliest progenitors also express http://www.jimmunol.org/ potent progenitors in the bone marrow (BM) represents CD117 (KIT) and CD127 (IL-7R) (1), markers expressed on cells one of the most carefully explored differentiation pathways within the common lymphoid progenitor compartment (6). Whereas T + in the hematopoietic system. This is much due to seminal work in the the most immature CD19 progenitors express CD43 and CD117 early 1990s identifying surface proteins that could be used for the (KIT), expression of CD25 (IL-2Ra) is initiated later in development identification and isolation of defined developmental stages (1). in association with the expression of a functional pre-BCR (1, 7, 8). A Combined expression of the surface markers B220, CD43 (S7), similar expression pattern has been reported for the CD2 protein mainly CD24 (HSA), and BP1 (aminopeptidase A) and IgM (2) allowed for expressedonCD19+ cells displaying cytoplasmatic IgH chains (9). the fractionation of specific progenitor compartments displaying Despite that a set of stage-specific surface markers has been stage-specific features. In vitro differentiation experiments, analysis identified, these may not be sufficient for proper analysis when by guest on September 28, 2021 of the recombination stage of the IgH locus, and expression of exploring the phenotype of transgenic mice or transformed cells. lineage-restricted genes (2, 3) revealed that cells progressed through Furthermore, the use of multiple surface markers in specific development from the B220+CD43+CD242BP12 (fraction A) stage combinations may allow for the identification of additional sub- into B220+CD43+CD24+BP12 (fraction B) cells that subsequently populations in the developmental pathway. In light of this, our aim could mature into a B220+CD43+CD24+BP1+ (fraction C) cell. Frac- was to identify novel markers for defined stages of B cell devel- tion D was defined based on expression of B220 but lack of expres- opment in the mouse BM. First, we sought differentially expressed sion of CD43 or IgM, whereas fractions E and F expressed detectable surface proteins using an Ab library to identify markers expressed levels of surface IgM. The B220+CD192 progenitors can be further on subfractions of CD19+ BM cells. The utility of these marker to enriched based on their expression of CD93 (AA4.1) (4) as well as identify specific B cell stages was tested using reporter transgenic by exclusion of cells expressing Ly6C, NK1.1, or Dx5 (5), markers mice carrying a l5 promoter–regulated hCD25 gene (10) and a Rag-1–regulated GFP gene (11) because the combined expression of these allowed us to identify subpopulations of Ig2 cells without *Department of Molecular Hematology, Lund University, 22184 Lund, Sweden; and †Department of Clinical and Experimental Medicine, Linko¨ping University, 58185 the use of any classical stage-specific surface markers. Using this Linko¨ping, Sweden general approach, we were able to identify a number of stage- ORCIDs: 0000-0002-6753-7983 (C.T.J.); 0000-0002-0854-2328 (S.L.); 0000-0003- specific markers, notably the cell adhesion molecules CD49E, 0669-9644 (R.S.). CD11A,andCD54allexpressedathighlevelsonpro–Bcells.These Received for publication February 18, 2016. Accepted for publication June 22, 2016. and other markers such as CD98 could be used in combination with This work was supported by grants from the Swedish Cancer Society, the Swedish more classical B linage markers to develop new protocols to isolate Research Council, the Knut and Alice Wallenberg Foundation, the Swedish Child- hood Cancer Foundation, and by a donation from Henry Hallberg. cells from specific B cell differentiation stages. The sequences presented in this article have been submitted to Gene Expression Omnibus under accession number GSE74290. Materials and Methods Address correspondence and reprint requests to Prof. Mikael Sigvardsson, Lund University, Department of Molecular Hematology, BMC B12, 22184 Lund, Sweden. Animal models E-mail address: [email protected] Wild-type mice and the reporter mice, carrying an hCD25 reporter under The online version of this article contains supplemental material. the regulatory elements of the Lambda5 (l5, Igll1) promoter (10), Rag1- 2/2 Abbreviations used in this article: BM, bone marrow; FDR, false discovery rate; GFP knock-in mice (11, 12), and Rag1 mice (13) were on a C57BL/6 FMO, fluorescence minus one. background. BM was harvested from mice aged 6–10 wk. Animal pro- cedures were performed with consent from the local Ethics Committee at Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 Lund University (Lund, Sweden). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600297 2 IDENTIFICATION OF STAGE-SPECIFIC B CELL MARKERS information). Because the mouse Abs in the library generated a degree of background staining, the data obtained were verified using directly con- jugated Abs (see Supplemental Table I for Ab information). Next generation sequencing Three thousand sorted pro–B1, pro–B2, pre–B1, pre–B2, and pre–B3 cells, immature B cells, and mature B cells were sorted from individual mice into 1 ml of RNAlater (Ambion/Life Technologies). Total RNA was isolated using RNAeasy micro kit (Qiagen) according to the manufacturer’s recommenda- tions. cDNA was generated using the Ovation RNA sequencing system V2 (NuGEN), and libraries were constructed with Ovation ultralow system V2 1– 16 (NuGEN) according to the manufacturer’s instructions. The libraries were subjected to 50 cycles of HiSeq 2000 and NextSeq 500 sequencing from two to four samples per cell type. FASTQ files were mapped against the mouse FIGURE 1. Ab screen indicates extensive heterogeneity of cell surface genome mm10 using the HISAT version 0.1.5-beta with default settings. Gene proteins in early B lymphoid progenitor compartments. (A)FACSprofiles expression levels were quantified using the R package Rsubread against displaying the gating strategy used to identify differentially expressed sur- RefSeq and HAVANA annotations for Ig segment analysis, and differentially face markers on CD19+ BM cells using an Ab library. ABx indicates any of expressed genes were identified using ANOVA on DESeq normalized data using the cell types as factors. The p values were corrected using Benjamini– the explored Abs in the library. Expression patterns of one of the library Abs + Hochberg correction. All Ig variable and joining segments were combined (CD24) in the CD19 cells are shown as representative sample (lower panel). into a single expression