Figure 1.
ERdj4 deficiency alters hematopoiesis.
(A) Representative contour plots from flow cytometric analyses of myeloid (CD11b+), B lymphoid (B220+) and erythroid (Ter119+) cells in mouse bone marrow. Numbers indicate frequency of the gated population for each genotype. (B) Absolute number of myeloid, B lymphoid and erythroid cells in mouse bone marrow. n = 6–8 mice/genotype. (C) Left panel, representative contour plots from flow cytometric analyses of hematopoietic progenitor cells (LSK, Lin−cKit+Sca1+) in mouse bone marrow. LSK cells were lineage-negative (NK1.1−Gr.1−Ter119−CD11b−B220−CD3−) and further defined by Flt3 expression into hematopoetic stem cells (HSCs, Flt3−), multipotent progenitors (MPPs, Flt3low) and lymphoid-primed MPPs (LMPPs, Flt3high). Right panel, representative contour plots from flow cytometric analyses of common lymphoid progenitors (CLPs, Lin−IL-7Rα+Flt3+cKitlowSca1low) in mouse bone marrow. Numbers indicate frequency of the gated population for each genotype. (D) Absolute number of HSCs, MPPs, LMPPs and CLPs in mouse bone marrow. n = 9–11 mice/genotype.
Figure 2.
The loss of ERdj4 impairs B cell development beginning at the large pre-B cell stage.
(A) Representative contour plots from flow cytometric analyses of B cell development using the Hardy classification scheme. Numbers indicate frequency of the gated population for each genotype. Upper panel, gated on IgM− bone marrow cells. Middle panel, gated on B220+CD43+ bone marrow cells. Lower panel, gated on total bone marrow cells. Fraction A, B220+CD43+BP-1−HSA−; Fraction B, B220+CD43+BP-1−HSA+; Fraction C, B220+CD43+BP-1+HSAlow; Fraction C’, B220+CD43+BP-1+HSAhigh; Fraction D, IgM−B220+CD43−; Fraction E, B220lowIgM+; Fraction F, B220highIgM+. (B–C) Absolute numbers of Hardy fractions. n = 5–8 mice/genotype.
Figure 3.
The defect in B lymphopoiesis is cell autonomous.
(A, D) Schematics of bone marrow transplants. Recipient mice received two doses of lethal irradiation (7+4.75 Gy) followed by intravenous injection of 5×106 bone marrow cells. (B, E) Representative contour plots from flow cytometric analyses of B cells in the bone marrow 16 weeks post-transplant. Numbers indicate frequency of gated population for each genotype. (C, F) Frequency and absolute number of B cells in the bone marrow as determined by flow cytometry. n = 6–8 mice/genotype.
Figure 4.
Deficiency of ERdj4 increases cell death during B lymphopoiesis.
Developing B cells were stained with 7-AAD and analyzed by flow cytometry. (A) Representative histograms from flow cytometric analyses of B cell progenitors positive for 7-AAD. Numbers indicate frequency of gated population for each genotype. (B) Average frequencies of 7-AAD+ B cell progenitors. n = 7–8 mice/genotype. Pro-B, IgM−CD19+B220+CD43+FSClow; Large pre-B, IgM−CD19+B220+CD43+FSChigh; Small pre-B, IgM−B220+CD43−; IMB, B220lowIgM+.
Figure 5.
Follicular B cells are reduced in ERdj4gt/gt mice.
(A) qRT-PCR of ERdj4 mRNA in spleens isolated from mice; samples were normalized to β-actin. RQ, relative quantitation. n = 5 mice/genotype. (B) Representative contour plots from flow cytometric analyses of splenic B cell subsets. Numbers indicate frequency of gated population for each genotype. Transitional B cells (T1, IgM+CD23−; T2, IgM+CD23+; T3, IgM−CD23+) were gated on CD19+CD93+ cells. Follicular (FOB, CD21lowCD23+) and marginal zone (MZB, CD21+CD23low) B cells were gated on CD19+ cells. (C–D) Absolute numbers of transitional and mature splenic B cell subsets. n = 5–6 mice/genotype.
Figure 6.
Basal immunoglobulins are increased in ERdj4gt/gt mice.
Basal serum immunoglobulin levels were quantitated in ERdj4+/+ (open circles) and ERdj4gt/gt (closed circles) mice by isotype-specific ELISA. n = 6–8 mice/genotype.
Figure 7.
ERdj4 deficiency enhances proliferation, survival and immunoglobulin isotype switching of LPS-treated B cells.
(A) CFSE dilution of B220+ cells treated with or without LPS. n = 3 samples/group. (B) Mean fluorescence intensity (MFI) of CFSE in B220+ cells treated with LPS. n = 3 samples/group. (C) Frequency of live B220+ cells treated with LPS. n = 3 samples/group. (D) Left panel, representative contour plots from flow cytometric analyses of B220+IgG3+ cells following treatment with LPS. Numbers indicate frequency of gated population for each genotype. Right panel, absolute numbers of B220+IgG3+ cells. n = 3 samples/group.
Figure 8.
Impaired T cell-dependent antibody responses in ERdj4gt/gt mice.
(A–B) Mice were immunized with either TNP-Ficoll (A, 50 µg/100 µl i.p.) or TNP-CGG (B, 100 µg/200 µl i.p.). Plasma TNP-specific IgM and IgG antibodies were quantitated at baseline and weekly post-immunization. n = 4–5 mice/group.