Differential surrogate light chain expression governs B-cell differentiation by Yui-Hsi Wang, Robert P. Stephan, Alexander Scheffold, Désirée Kunkel, Hajime Karasuyama, Andreas Radbruch, and Max D. Cooper Blood Volume 99(7):2459-2467 April 1, 2002 ©2002 by American Society of Hematology

Analysis of SLC expression on human and mouse cell lines Analysis of SLC expression on human and mouse cell lines.Pro-B and pre-B cell lines were analyzed for cell surface binding of anti-VpreB and anti-λ5 antibodies by conventional indirect immunofluorescence (dashed line) or by an enhanced immunofluorescence me... Analysis of SLC expression on human and mouse cell lines.Pro-B and pre-B cell lines were analyzed for cell surface binding of anti-VpreB and anti-λ5 antibodies by conventional indirect immunofluorescence (dashed line) or by an enhanced immunofluorescence method using fluorochrome-loaded liposomes as a second-step reagent (solid line). Test cells incubated with an excess of unlabeled primary antibodies served as staining controls (shaded histograms). Results illustrated in this and subsequent figures used the VpreB8 anti-human VpreB, R3 anti-mouse Vpre-B, HSL11 anti-human λ5, and LM34 anti-mouse λ5 monoclonal antibodies. Comparable results were obtained with the other anti-VpreB, anti-λ5, and anti-preBCR antibodies used in these studies (see “Materials and methods”). Yui-Hsi Wang et al. Blood 2002;99:2459-2467 ©2002 by American Society of Hematology

Enhanced and conventional indirect immunofluorescence analysis of cell surface SLC expression by B lineage cells.(A) CD19+ cells in human bone marrow and (B) B220+ cells in mouse bone marrow were examined with digoxigenin-labeled anti-VpreB and anti-λ5 mAbs... Enhanced and conventional indirect immunofluorescence analysis of cell surface SLC expression by B lineage cells.(A) CD19+ cells in human bone marrow and (B) B220+ cells in mouse bone marrow were examined with digoxigenin-labeled anti-VpreB and anti-λ5 mAbs and antidigoxigenin-coated fluorochrome-loaded liposomes as a second-step reagent or with biotinylated anti-VpreB mAb and streptavidin-PE. Yui-Hsi Wang et al. Blood 2002;99:2459-2467 ©2002 by American Society of Hematology

Phenotypic characterization of SLC-bearing B lineage cells in bone marrow.(A) Human adult or fetal cells and (B) mouse cells from wild-type or Rag-2−/− juvenile mice were incubated with, respectively, anti-CD19 or anti-B220 antibodies, plus digoxigenin-labe... Phenotypic characterization of SLC-bearing B lineage cells in bone marrow.(A) Human adult or fetal cells and (B) mouse cells from wild-type or Rag-2−/− juvenile mice were incubated with, respectively, anti-CD19 or anti-B220 antibodies, plus digoxigenin-labeled anti-VpreB and antidigoxigenin-coated fluorochrome-loaded liposomes, before counterstaining with fluorochrome-labeled antibodies against CD34, μHC, or κ/λLC (A) or CD19, CD43, BP-1, or κ/λLC (B). Viable CD19+ cells (A) or B220+ cells (B) were electronically gated for this analysis. Coexpression of μHC by VpreB+ cells, indicated by the upward shift observed with conventional immunofluorescence (A, middle panels), was confirmed in parallel experiments in which the liposome-enhanced assay was also used to detect cell surface μHC expression (see text). Yui-Hsi Wang et al. Blood 2002;99:2459-2467 ©2002 by American Society of Hematology

Analysis of intracellular μHC expression by pre-BCR+ and pre-BCR− subpopulations.(A) Human VpreB+ and VpreB−CD34−subpopulations of CD19+κ/λLC− B lineage cells and (B) mouse VpreB+ and VpreB−subpopulations of B-lineage cells from bone marrow samples were pur... Analysis of intracellular μHC expression by pre-BCR+ and pre-BCR− subpopulations.(A) Human VpreB+ and VpreB−CD34−subpopulations of CD19+κ/λLC− B lineage cells and (B) mouse VpreB+ and VpreB−subpopulations of B-lineage cells from bone marrow samples were purified by immunofluorescence cell sorting before cell permeabilization and analysis of intracellular μHC expression. Dashed lines indicate intracellular μHC staining; solid lines indicate background fluorescence. Yui-Hsi Wang et al. Blood 2002;99:2459-2467 ©2002 by American Society of Hematology

Cell size and cell cycle analysis of pre-BCR+ and pre-BCR− subpopulations.(A) Human VpreB+ and VpreB−CD34−subpopulations of CD19+κ/λLC− B lineage cells and (B) mouse CD19+BP-1+κ/λLC− pre-B cells were sorted on the basis of positive or negative cell surface ... Cell size and cell cycle analysis of pre-BCR+ and pre-BCR− subpopulations.(A) Human VpreB+ and VpreB−CD34−subpopulations of CD19+κ/λLC− B lineage cells and (B) mouse CD19+BP-1+κ/λLC− pre-B cells were sorted on the basis of positive or negative cell surface VpreB expression before evaluation of relative cell size by light scatter profile analysis (upper panels) and cell cycle status assessment by Ki-67 expression and DNA content (lower panels). Yui-Hsi Wang et al. Blood 2002;99:2459-2467 ©2002 by American Society of Hematology

Expression profiles for B lineage genes and intracellular proteins in pro-B and pre-B subpopulations.(A) Human CD34+CD19+VpreB−pro-B, CD19+ κ/λLC−VpreB+pre-B, and CD19+κ/λLC−CD34−VpreB−pre-B cells were isolated from fetal bone marrow samples. Expression profiles for B lineage genes and intracellular proteins in pro-B and pre-B subpopulations.(A) Human CD34+CD19+VpreB−pro-B, CD19+ κ/λLC−VpreB+pre-B, and CD19+κ/λLC−CD34−VpreB−pre-B cells were isolated from fetal bone marrow samples. (B) Mouse CD19+ VpreB+ and CD19+VpreB− pro-B cells were isolated from RAG2−/− bone marrow, and VpreB+ and VpreB− subpopulations of pre-B cells (CD19+BP-1+ κ/λLC−) were isolated from bone marrow of wild-type juvenile mice. Subpopulations were purified by 2 rounds of immunofluorescence-based sorting, and the sorted cells were used as templates for RT-PCR assays. PCR products were visualized on agarose gels by ethidium bromide staining. (C) Analysis of cytoplasmic VpreB, Igβ, and conventional LC protein expression within pre-BCR− pre-B cells. Dashed lines indicate staining of pre-BCR− cells. Solid lines represent staining in control pre-BCR+ cells (i) and CD19+κ/λLC+ B cells (ii) and (iii). Background staining with isotype-matched antibodies of irrelevant specificity is indicated by shaded histograms. Yui-Hsi Wang et al. Blood 2002;99:2459-2467 ©2002 by American Society of Hematology

Comparative models of normal B cell differentiation in humans and mice Comparative models of normal B cell differentiation in humans and mice. , μ heavy chains; , Igα/β; , VpreB/λ5; , BILL-cadherin; , calnexin; Tdt, terminal deoxyribonucleotidyl transferase. Comparative models of normal B cell differentiation in humans and mice. , μ heavy chains; , Igα/β; , VpreB/λ5; , BILL-cadherin; , calnexin; Tdt, terminal deoxyribonucleotidyl transferase. Pro-B cells depicted in this scheme express cell surface CD19. Yui-Hsi Wang et al. Blood 2002;99:2459-2467 ©2002 by American Society of Hematology