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Blood, Vol. 111, Issue 3, 1448-1455, February 1, 2008
LMP1 signaling can replace CD40 signaling in B cells in vivo and has unique features of inducing class-switch recombination to IgG1
Blood Rastelli et al.
111: 1448
Supplemental materials for: Rastelli et al
Files in this Data Supplement:
- Figure S1. CD40/LMP1+//CD40+/+ mice show a normal lymphoid compartment in the spleen (JPG, 122 KB)
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(A) Total numbers and percentages of B220+ B and CD5high T cells in the spleen (SP) of wt CD40+/+ control mice (CD40+/+) and CD40/LMP1+ mice. Presented data are means +/− SEM of three mice per group tested in independent experiments. (B) Flow cytometry for B cell subsets in the spleen. Numbers indicate percentages of gated populations of follicular B cells (IgM+IgD+) and marginal zone and transitional B cells (IgM+IgDlow) and follicular B cells (FO) (CD21intCD23+) and marginal zone B cells (MZB) (CD21highCD23low) in the spleen of 7 and 4 mice, respectively. (C) Flow cytometry to identify germinal center (GC) B cells (CD95+PNAhigh) in the spleens of CD40+/+ and CD40/LMP1+//CD40+/+ mice 14 days after immunization with 100 µg NP-CGG. Additionally, the analysis of un-immunized mice is shown (d0) Cells are gated on B220+; numbers indicate the mean percentages +/− SEM of GC B cells of 3 mice.
- Figure S2. Bone marrow-derived dendritic cells (BM-DCs) do not express CD40/LMP1 (JPG, 64.5 KB)
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DCs were enriched from the BM by culturing for 7 days in the presence of GM-CFS. To induce maturation, cells were activated by LPS 12 hours prior to FACS analysis. Cells were gated for living cells (PI negative) and stained for CD11c, CD40 and CD80. (A) Upon LPS stimulation, the endogenous CD40 receptor is up-regulated in wt CD40+/+ BM-DCs, whereas CD40/LMP1 in CD40/LMP1+//CD40−/− BM-DCs is not. As a negative control, BM-DCs of CD40−/− mice were included in the study. (B) CD40 and CD80 staining of CD11c+ BM-DCs of wt CD40+/+ (black line) and CD40/LMP1+//CD40−/− (red line) mice, without (w/o) and after LPS stimulation. LPS induced CD80 expression in both groups, whereas CD40 or CD40/LMP1 expression was induced in wt CD40+/+ but not in CD40/LMP1+//CD40−/− BM-DCs, respectively. (C) CD40, Gr-1 staining of ex vivo isolated B220− BM cells.
- Figure S3. CD40/LMP1+//CD40−/− mice show normal lymphoid compartments (JPG, 89.7 KB)
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(A) Immunohistochemical analyses of the follicular architecture in the spleen. Cryosections were stained with anti-IgM specific for B cells (red) and anti-CD3 specific for T cells (blue). Original magnification, ×50. (B) Flow cytometry for the expression of IgM and IgD on lymphocytes in the spleen (SP). Numbers indicate percentages of gated populations, follicular B cells (IgM+IgD+), marginal zone and transitional B cells (IgM+IgDlow). (C) Flow cytometric analysis of follicular B cells (FO) (CD21intCD23+) and marginal zone B cells (MZB) (CD21highCD23low) in the spleen. Numbers indicate percentages of B220+ B cells displaying a MZB or FO B cell phenotype.
- Figure S4. CD40/LMP1+//CD40−/− B cells do not show higher proliferation rates in the germinal center (JPG, 53.2 KB)
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14 days after immunization with NP-CGG, mice were injected with 150 µl BrdU solution (10 mg/ml) and sacrificed 2 or 6 hours later. Splenocytes were purified and stained using the APC BrdU Flow kit (BD Biosciences). To analyse the incorporation of BrdU into the DNA of GC B cells the stainings were gated on CD95+ and PNA+ cells; numbers indicate the mean percentages +/− SEM of BrdU positive GC B cells of three independent experiments in control wt CD40 +/+ (CD40+/+) and CD40/LMP1+//CD40−/− mice.
- Figure S5. LMP1 signaling induces cytokine-independent class switch recombination to IgG1 (JPG, 133 KB)
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(A) After 5 days of culture in the presence of anti-CD40 antibody, CD40/LMP1+//CD40−/− (red line) but not wt CD40+/+ B cells (black line) showed a certain fraction of IgG1 positive cells, although they had comparable division rates (visualized by CFSE labeling). (B) Quantitative real time RT-PCR for  1 sterile transcript in wt CD40+/+ (black diamonds) and CD40/LMP1+//CD40−/− (red triangles) B cells stimulated with anti-CD40, or anti-CD40 and IL-4, respectively, over 4 days. CD40/LMP1+//CD40−/− B cells showed a more than two fold induction of the 1 sterile transcript after 24 and 48 hours of anti-CD40 stimulation. Upon anti-CD40 and IL4 stimulation the 1 sterile transcript induction was approximately 8 fold in CD40/LMP1+//CD40−/− and 10 fold in wt CD40+/+ B cells, respectively. Mean results from two to three independent cultures are expressed as fold induction relative to resting B cells at day 0. 1 sterile transcript abundance was normalized to GAPDH and was comparable in both wt CD40+/+ and CD40/LMP1+//CD40−/− B cells at day 0. (C) Quantitative real time RT-PCR for IL4, IL13, IL21, BAFF and APRIL in wt CD40+/+ and CD40/LMP1+//CD40−/− B cells stimulated with anti-CD40, or anti-CD40 and IL-4, respectively, over 4 days. No transcript products of any of the analyzed cytokines could be detected in stimulated B cells. As a positive control for IL4, IL13 and IL21 expression, CD4+ T cells were cultured in TH2 conditions (IL4, anti-IL12 and anti-INF-) and re-stimulated for the indicated times with anti-CD3 and anti-CD28. IL4, IL13 and IL21 transcript abundance in T cells was normalized to HPRT. As a positive control for BAFF and APRIL expression, ex vivo isolated splenic B220− cells were used, and the mRNA abundance was normalized to GAPDH. Primer sequences can be provided on request.
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