Analysis Tools
immune system
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• despite a 20% drop in peripheral B cells, mice show no major defects in B cell migration, as determined by vivo bone marrow egress assays, in vitro chemotaxis assays, and in vivo lymph node and splenic homing indices in adoptive transfer experiments
• naive mice show normal basal antibody titers of IgM, IgG2b, IG2c, IgG3, and IgA in serum
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• B220 staining shows a 20% reduction in the proportion of B cells in the bone marrow and a slight reduction in the number of B cells in spleen, with no significant change in overall bone marrow or spleen cellularity
• however, the proportions of early (B220+ CD43+) and late (B220+ CD43-) B cell progenitors are not altered in the bone marrow; both early B cell progenitor Hardy populations (A, B, and C based on BP-1 and CD24 expression levels) and late B cell progenitor populations (D, E, and F based on IgM and IgD expression levels) are similar to those in wild-type controls
• splenic B cells subpopulations including follicular B cells (CD21+ CD24lo), transitional T1 B cells (CD21loCD24hi), transitional T2/marginal zone B cells (MZB) (CD21hiCD24hi), and MZB cells (CD21hiCD23lo) are similar to those in wild-type controls
• B cell populations in the peritoneal cavity including B2 (CD5-CD11b-), B1b (CD5-CD11b+), and B1a (CD5+ CD11b+) are also similar to those in wild-type controls
• moreover, MZB cell subpopulations (CD21hi CD23-) and B (B220+) cells are normal in inguinal lymph nodes (LNs), consistent with normal B cell maturation
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• mice show a reduction in the absolute numbers of immature B cells in the bone marrow
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• mice show a reduction in the absolute numbers of mature B cells in spleen
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• 7 days after s.c. injection of Vaccinia virus (VACV; Western Reserve) into the footpad, mice show a 35% decrease in the proportion of germinal center CD95+ GL-7+ B cells in the draining popliteal LNs relative to VACV-infected wild-type controls
• however, no significant reduction is noted in the proportion of germinal center B cells in mediastinal LNs 9 days after intranasal injection of Influenza virus (PR8 strain), and the proportion of plasma cells (CD138+ IgDlo) is not significantly changed after VACV or Influenza virus infection
• moreover, the numbers of follicular helper T cells (CXCR5+PD-1+) in the draining LNs remain normal after either virus infection
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• in vitro, isolated primary B cells exhibit a mild defect in response to BCR activation, showing a 35% reduction of early Akt phosphorylation with delayed kinetics and a slight decrease in ERK phosphorylation
• however, intracellular calcium mobilization upon BCR activation is unaffected and B cells spread and gather antigen microclusters normally on antigen-loaded planar lipid bilayers even in the absence of ICAM1
• purified naive B cells show normal BCR internalization and antigen polarization relative to wild-type cells
• naive CFSE-labelled B cells stimulated with anti-IgM or CD40L (a key activating signal from T helper cells) in the presence of IL4 show normal proliferation/division indices
• naive CFSE-labelled B cells stimulated with beads coated with anti-IgM and OVA and cocultured with CTV-labeled OTII T cells show normal B and T cell proliferation/division indices
• naive CTV-labeled naive B cells stimulated with CpG DNA (a TLR9 ligand) or LPS (a TLR4 ligand) show normal proliferation/division indices and plasma cell differentiation; no changes are observed in the secretion of IgM and IL-6 upon stimulation with CpG or in the secretion of IgM upon stimulation with LPS, indicating normal B cell activation by TLR ligands
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• mice exhibit impaired germinal center responses upon viral (VACV) infection
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• mice exhibit impaired early IgM and IgG3 responses upon immunization with NP-KLH in Alum (a classic T-dependent antigen)
• however, defects are overcome by normal generation and affinity maturation of other Ab isotypes in later stages of the immune response
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• mice immunized with NP-KLH precipitated in Alum show a 50% reduction in NP-specific IgG3 titers
• however, mice show a similar increase in NP-specific total IgG titers over time and normal affinity maturation (ratio of NP3 to NP23 titers) of total IgG relative to wild-type controls
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• mice immunized with NP-KLH precipitated in Alum show a 50% reduction in NP-specific IgM titers
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hematopoietic system
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• B220 staining shows a 20% reduction in the proportion of B cells in the bone marrow and a slight reduction in the number of B cells in spleen, with no significant change in overall bone marrow or spleen cellularity
• however, the proportions of early (B220+ CD43+) and late (B220+ CD43-) B cell progenitors are not altered in the bone marrow; both early B cell progenitor Hardy populations (A, B, and C based on BP-1 and CD24 expression levels) and late B cell progenitor populations (D, E, and F based on IgM and IgD expression levels) are similar to those in wild-type controls
• splenic B cells subpopulations including follicular B cells (CD21+ CD24lo), transitional T1 B cells (CD21loCD24hi), transitional T2/marginal zone B cells (MZB) (CD21hiCD24hi), and MZB cells (CD21hiCD23lo) are similar to those in wild-type controls
• B cell populations in the peritoneal cavity including B2 (CD5-CD11b-), B1b (CD5-CD11b+), and B1a (CD5+ CD11b+) are also similar to those in wild-type controls
• moreover, MZB cell subpopulations (CD21hi CD23-) and B (B220+) cells are normal in inguinal lymph nodes (LNs), consistent with normal B cell maturation
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• mice show a reduction in the absolute numbers of immature B cells in the bone marrow
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• mice show a reduction in the absolute numbers of mature B cells in spleen
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• 7 days after s.c. injection of Vaccinia virus (VACV; Western Reserve) into the footpad, mice show a 35% decrease in the proportion of germinal center CD95+ GL-7+ B cells in the draining popliteal LNs relative to VACV-infected wild-type controls
• however, no significant reduction is noted in the proportion of germinal center B cells in mediastinal LNs 9 days after intranasal injection of Influenza virus (PR8 strain), and the proportion of plasma cells (CD138+ IgDlo) is not significantly changed after VACV or Influenza virus infection
• moreover, the numbers of follicular helper T cells (CXCR5+PD-1+) in the draining LNs remain normal after either virus infection
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• in vitro, isolated primary B cells exhibit a mild defect in response to BCR activation, showing a 35% reduction of early Akt phosphorylation with delayed kinetics and a slight decrease in ERK phosphorylation
• however, intracellular calcium mobilization upon BCR activation is unaffected and B cells spread and gather antigen microclusters normally on antigen-loaded planar lipid bilayers even in the absence of ICAM1
• purified naive B cells show normal BCR internalization and antigen polarization relative to wild-type cells
• naive CFSE-labelled B cells stimulated with anti-IgM or CD40L (a key activating signal from T helper cells) in the presence of IL4 show normal proliferation/division indices
• naive CFSE-labelled B cells stimulated with beads coated with anti-IgM and OVA and cocultured with CTV-labeled OTII T cells show normal B and T cell proliferation/division indices
• naive CTV-labeled naive B cells stimulated with CpG DNA (a TLR9 ligand) or LPS (a TLR4 ligand) show normal proliferation/division indices and plasma cell differentiation; no changes are observed in the secretion of IgM and IL-6 upon stimulation with CpG or in the secretion of IgM upon stimulation with LPS, indicating normal B cell activation by TLR ligands
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• mice exhibit impaired germinal center responses upon viral (VACV) infection
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• mice immunized with NP-KLH precipitated in Alum show a 50% reduction in NP-specific IgG3 titers
• however, mice show a similar increase in NP-specific total IgG titers over time and normal affinity maturation (ratio of NP3 to NP23 titers) of total IgG relative to wild-type controls
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• mice immunized with NP-KLH precipitated in Alum show a 50% reduction in NP-specific IgM titers
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