A forward genetic screen reveals roles for Nfkbid, Zeb1, and Ruvbl2 in humoral immunity

Abstract

Using chemical germ-line mutagenesis, we screened mice for defects in the humoral immune response to a type II T-independent immunogen and an experimental alphavirus vector. A total of 26 mutations that impair humoral immunity were recovered, and 19 of these mutations have been positionally cloned. Among the phenovariants were bumble, cellophane, and Worker ascribed to mutations in Nfkbid, Zeb1, and Ruvbl2, respectively. We show that IκBNS, the nuclear IκB-like protein encoded by Nfkbid, is required for the development of marginal zone and peritoneal B-1 B cells and additionally required for extrafollicular antibody responses to T-independent and -dependent immunogens. Zeb1 is also required for marginal zone and peritoneal B-1 B-cell development as well as T-cell development, germinal center formation, and memory B-cell responses. Finally, Ruvbl2 is required for T-cell development and maximal T-dependent antibody responses. Collectively, the mutations that we identified give us insight into the points at which disruption of an antibody response can occur. All of the mutations identified to date directly affect lymphocyte development or function; none have an exclusive effect on cells of the innate immune system.

Fig. 1.

The antibody response to rSFV-encoded antigen is dependent on T-cell help but not IFN or TLR signaling or MZ B cells. Serum levels of βGal-specific IgG determined by ELISA 14 d after priming with rSFV-βGal in (A) WT mice and mice deficient in CD3ɛ, the IFN-α and -γ receptors, or MyD88 and Ticam1 and (B) control and splenectomized WT mice. Each point represents data from one mouse, and the bar indicates the mean of all values. Background (indicated by the dashed line) was determined by incubating pooled WT sera on uncoated wells. Results are representative of two to three independent experiments.

Fig. 2.

Recovery of the bumble, cellophane, and Worker phenovariants by forward genetic screening. Serum NP-specific IgM (A, C, and E) and βGal-specific IgG (B, D, and F) measured by ELISA in WT and G3 mice immunized 5 or 14 d before with NP-Ficoll and rSFV-βGal. (A and B) Results from 4 wk of screening are pooled with values for bumble index mice in red. (C and D) Results from 4 wk of screening are pooled with values for cellophane index mice in blue. (E and F) Results from 1 wk of screening are shown with values for the Worker index mouse in green. Each point represents data from one mouse. Background (indicated by the dashed line) was determined by incubating pooled WT sera on uncoated wells.

Fig. 3.

Lymphocyte development is impaired in bumble, cellophane, and Worker mice. Single-cell suspensions from WT and mutant mice were analyzed by flow cytometry. (A) IgM⁻ B220^int pro/pre, IgM⁺ B220^int immature, and IgM⁺ B220^high mature B cells as a percent of bone marrow lymphocytes. (B) B220⁺ B cells as a percent of splenocytes and IgD^low IgM^high B220⁺ immature, IgD^high IgM^int B220⁺ mature follicular, and CD21/35^high CD23^low B220⁺ MZ B cells as a percent of splenic B220⁺ B cells. (C) Mean fluorescence intensity of CD44 on CD4⁺ and CD8⁺ T cells and IgM on B220⁺ B cells in spleen. (D) CD19^int B220^high B2 and CD19^high B220^low/int B1 B cells as a percent of peritoneal cavity lymphocytes. (E) CD5^high CD43^high CD19^high B220^low/int B1a and CD5^low CD43^low CD19^high B220^low/int B1b B cells as a percent of peritoneal cavity B1 B cells. (F) CD4⁻ CD8⁻ DN, CD4⁺ CD8⁺ DP, and CD4⁺ or CD8⁺ SP cells as a percent of thymocytes. (G) CD69⁻ and CD69⁺ TCRβ⁺ CD4⁺ or CD8⁺ cells as a percent of CD4⁺ or CD8⁺ thymocytes, respectively. (H) CD4⁺ and CD8⁺ T cells and NK1.1⁺ cells as a percent of splenocytes. Bars indicate the mean (± range) of three to seven mice per genotype analyzed, except for A, in which the mean (± range) is shown for two WT mice.

Fig. 4.

Representative staining for flow cytometric analysis of lymphocyte development in mutant mice. Single-cell suspensions from WT and mutant mice were analyzed by flow cytometry. (A) B220 vs. IgM in bone marrow. (B) IgM vs. B220 in spleen. (C) CD23 vs. CD21/35 on B220⁺ splenocytes. (D) CD19 vs. B220 on peritoneal lymphocytes. (E) CD43 vs. CD5 on CD19^high B220^low/int B1 B cells in the peritoneal cavity. (F) CD4 vs. CD8 in thymus. Flow cytometric plots and the indicated mean percentages of gated populations are representative of three to seven mice per genotype analyzed, except for A, which is representative of two WT mice.

Fig. 5.

The primary T-dependent antibody response is impaired in bumble, cellophane, and Worker mice. WT and mutant mice were immunized with alum-precipitated NP-CGG. Serum levels of NP-specific IgM (A), total NP-specific IgG1 (B), or high-affinity NP-specific IgG1 (C) were measured by ELISA. Each point represents the mean (± range) of greater than or equal to three mice per genotype, except for the day 14 and 28 time points for Worker, with two mice analyzed.

Fig. 6.

cellophane mice have a defect in GC formation. (A) CD38 vs. Peanut Agglutinin (PNA) binding on IgD^low B220^high splenocytes from naïve WT or WT and bumble mice immunized 14 d before (Upper) or WT and cellophane mice immunized 11 d before (Lower) with alum-precipitated NP-CGG. Plots are representative of two to five naïve WT and three to seven immunized mice per genotype. (B) GC B cells as a percent of total B220^high splenocytes in naïve WT or immunized WT and bumble mice. WT vs. bumble, P > 0.05, Mann–Whitney U test. (C) GC B cells as a percent of total B220^high splenocytes in naïve WT or immunized WT and cellophane mice. WT vs. cellophane, P < 0.005, Mann–Whitney U test. In B and C, each point represents data from one mouse, and the bar indicates the mean of all values.

Fig. 7.

The memory B-cell response is reduced in cellophane mice. CD19⁺ B cells were enriched from the spleens of WT or mutant mice immunized 28 d before with alum-precipitated NP-CGG and transferred to CGG-primed WT mice. Immediately after cell transfer, the recipients were injected with NP-CGG in saline; 10 d later, serum levels of NP-specific IgM (A), total NP-specific IgG1 (B), and high-affinity NP-specific IgG1 (C) were measured by ELISA. As controls, NP-specific IgM and IgG1 levels were measured in naïve mice and mice that were immunized with NP-CGG in saline but did not receive memory B cells (boost only). In A and B, all of the recipients made IgM and IgG1 responses equivalent to boost-only controls, indicating that memory B cells were not required for these responses. In C, only mice that received functional memory B cells and were boosted with NP-CGG made high-affinity NP-specific IgG1 responses. Each point represents data from one mouse, and the bar indicates the mean of all values. Background (indicated by the dashed line) was determined by incubating pooled WT sera on uncoated ELISA wells.