Essential requirement for IER3IP1 in B cell development

Abstract

In a forward genetic screen of mice with N-ethyl-N-nitrosourea-induced mutations for aberrant immune function, we identified animals with low percentages of B220⁺ cells in the peripheral blood. The causative mutation was in Ier3ip1, encoding immediate early response 3 interacting protein 1 (IER3IP1), an endoplasmic reticulum membrane protein mutated in an autosomal recessive neurodevelopmental disorder termed Microcephaly with simplified gyration, Epilepsy and permanent neonatal Diabetes Syndrome (MEDS) in humans. However, no immune function for IER3IP1 had previously been reported. The viable hypomorphic Ier3ip1 allele uncovered in this study, identical to a reported IER3IP1 variant in a MEDS patient, reveals an essential hematopoietic-intrinsic role for IER3IP1 in B cell development and function. We show that IER3IP1 forms a complex with the Golgi transmembrane protein 167A and limits activation of the unfolded protein response mediated by inositol-requiring enzyme-1α and X-box binding protein 1 in B cells. Our findings suggest that B cell deficiency may be a feature of MEDS.

Keywords: B cells; ENU; IER3IP1; TMEM167A; unfolded protein response.

Fig. 1.

Identification of mouse Ier3ip1 mutations responsible for B cell deficiency. (A) The frequency of peripheral blood B cells from third-generation (G3) descendants of two independent ENU-mutagenized male mice (G1), with REF (+/+), HET (+/mutant), or VAR (mutant/mutant) genotypes for Ier3ip1 (n = 2 to 43 mice/genotype). Data were normalized to the average B cell frequency of age-matched C57BL/6 mice at the time of the experiment (n = 20 mice/experiment). (B) Manhattan plot showing –log₁₀ P-values (y axis) plotted vs. the chromosomal positions of mutations (x axis) identified in the G1 founders of the affected pedigrees. The alarmist and emergent pedigrees were combined for linkage analysis (i.e., superpedigree mapping) (18). (C) Sequence alignment of human and mouse IER3IP1. Transmembrane domains are highlighted in green. Discordant amino acids between human and mouse IER3IP1 are highlighted in violet. The alarmist (A18V) and emergent (I69K) mutations identified by forward genetic screening in the present study are boxed in orange and blue, respectively. Natural IER3IP1 variants reported to cause MEDS in humans are boxed in violet. (D) Serum glucose and insulin levels in 4-wk-old Ier3ip1^a/a and wild-type littermates (n = 5 to 19 mice/genotype). (E) Representative hematoxylin and eosin (H&E) staining and insulin immunohistochemistry (IHC) of pancreases isolated from 8-wk-old Ier3ip1^a/a and wild-type littermates. (F) A representative photograph and weights of brains isolated from 12-wk-old Ier3ip1^a/a and wild-type littermates (n = 4 to 5 mice/genotype). (G) Immunoblot analysis of IER3IP1 and GAPDH in total cell lysates of splenic pan B cells isolated from Ier3ip1^a/a and wild-type littermates. (H) Representative flow cytometry plots showing B and T cell frequencies in the peripheral blood from 8-wk-old Ier3ip1^a/– and wild-type littermates. Data are representative of one (A) or three independent experiments (D–H). Data points represent individual mice (A, D, and F). Error bars indicate SD. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons (A) or Student’s t test (D and F). **P < 0.01; ***P < 0.001.

Fig. 2.

Severe B cell deficiency caused by an Ier3ip1 mutation in mice. (A) Representative photographs of thymi and spleens isolated from 8-wk-old Ier3ip1^a/a and wild-type littermates. (B) Thymocyte subpopulations in 8-wk-old Ier3ip1^a/a and wild-type littermates. (n = 4 to 6 mice/genotype). (C and D) CD44 mean fluorescence intensity (MFI) on CD4⁺ and CD8⁺ T cells (C) as well as the ratio of B cells to T cells (D) in the peripheral blood from 8-wk-old Ier3ip1^a/a mice and wild-type littermates (n = 9 to 11 mice/genotype). (E) Quantitative analysis of the HSC and progenitor populations in the BM of 8- to 10-wk-old Ier3ip1^a/a and wild-type littermates (n = 3 to 7 mice/genotype). Each cell subset was gated as described previously (15). LT-HSC: long-term hematopoietic stem cells, ST-HSC: short-term hematopoietic stem cells, MPP: multipotent progenitors, LMPP: lymphoid-primed multipotent progenitors, MEP: megakaryocyte–erythroid progenitors, CMP: common myeloid progenitors, and GMP: granulocyte-macrophage progenitors. (F) Numbers of B cell subpopulations in the BM, spleen, and peritoneal cavity of 8- to 10-wk-old Ier3ip1^a/a and wild-type littermates (n = 6 mice/genotype). (G–N) Representative flow cytometry plots showing B cell development in the BM (G and H), spleen (I–L), and peritoneal cavity (M and N) from 8- to 10-wk-old Ier3ip1^a/a and wild-type littermates (n = 6 mice/genotype). Each B cell subset was gated as follows: pre-pro-B: B220^lowLy51⁻CD24^low, pro-B: B220^lowLy51⁻CD24⁺, pre-B: B220^lowLy51⁺CD24⁺, immature B: B220⁺IgM⁺IgD⁻, transitional B: B220⁺IgM^highIgD^low, mature recirculating B: B220⁺IgD⁺IgM⁺, T1: B220⁺CD93⁺IgM^highCD23⁻, T2: B220⁺CD93⁺IgM^highCD23⁺, T3: B220⁺CD93⁺IgM^lowCD23⁺, follicular B (FOB): B220⁺CD93⁻IgM⁺CD21⁺CD23^high, marginal zone precursor (MZP): B220⁺CD93⁻IgM⁺CD21⁺CD2a3^high, marginal zone B (MZ): B220⁺CD93⁻IgM⁺CD21⁺CD23^low, B2: B220⁺CD19⁺, B1: B220^lowCD19⁺, B1a: B220^lowCD19⁺CD43⁺CD5⁺, and B1b: B220^lowCD19⁺ CD43⁺CD5^-. Numbers adjacent or inside outlined regions represent percent cells in each. (O) Total serum IgG1, IgG2a, IgG2b, IgA, IgE, and IgM were detected in Ier3ip1^a/aand wild-type littermates by ELISA (n = 23 mice/genotype). Data are representative of two (B, E, F, and G–O) or three independent experiments (A, C, and D). Data points represent individual mice (B–F and O). Error bars indicate SD. P values were determined by Student’s t test (B–O). *P < 0.5; **P < 0.01; ***P < 0.001; NS, not significant.

Fig. 3.

A cell-intrinsic effect of an Ier3ip1 mutation on B cell function and UPR. (A and B) Repopulation of donor-derived B cells (A) and T cells (B) in the peripheral blood of recipients 12 wk after reconstitution with BM isolated from mice with the indicated genotypes (n = 6 mice/group). (C and D) T cell–dependent (C) and T cell–independent antibody responses (D) after immunization with aluminum hydroxide-precipitated OVA and NP-Ficoll, respectively, in the BM chimeras at 10 wk after reconstitution (n = 6 mice/group). Data are represented as absorbance at 450 nm. (E–I) Flow cytometry analysis of immunoglobulin class switch recombination (E and F), proliferative response (E and G), and cell cycle (H and I) of splenic naive B cells. Splenic naive B cells isolated from Ier3ip1^a/a and wild-type littermates were stimulated with LPS and IL-4 for 72 h at 37 °C (n = 4 mice/genotype). Numbers adjacent to outlined areas indicate percent cells in each. (J) Immunoblot analysis of BiP, IRE1α, XBP1s (spliced), XBP1u (unspliced), p-PERK, PERK, p-eIF2α, ATF4, CHOP, ATF6, IER3IP1, and GAPDH in total cell lysates of splenic pan B cells from Ier3ip1^a/a and wild-type littermates before and after stimulation with tunicamycin for 4 h at 37 °C. Data are representative of two independent experiments. Data points represent individual mice (A–D, F, G, and I). Error bars indicate SD. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons (A–D) or Student’s t test (F, G, and I). ***P < 0.001; NS, not significant.

Fig. 4.

The IER3IP1 interactome. (A) IER3IP1 interactors identified by coimmunoprecipitation combined with liquid Chromatography with tandem mass spectrometry (LC–MS/MS) using mouse B cell lines (A20) stably expressing FLAG-tagged mouse IER3IP1 or empty-FLAG. Relative protein abundance calculated using precursor ion intensities (abundance in IER3IP1 IP ÷ sum of abundances in IER3IP1 and control IP) is plotted on the y axis. y = 0.5 indicates equivalent abundance in the IER3IP1 IP and control IP. y > 0.5 indicates enrichment in the IER3IP1 IP, with y = 1 indicating that the protein was exclusively detected in the IER3IP1 IP. X axis values represent the precursor ion intensity detected by MS. Data shown are combined from three independent MS analyses (n = 5 IP/cell line). (B) Immunoblot analysis of HUWE1, MCL1, c-Myc, BCL2, IER3IP1, and GAPDH in total cell lysates of splenic pan B cells from Ier3ip1^a/a and wild-type littermates following LPS and IL-4 stimulation for 48 h or left untreated. (C) Flow cytometry analysis of CD72 expression on peripheral blood B cells from 8-wk-old Ier3ip1^a/a and wild-type littermates (n = 7 to 14 mice/genotype). (D) Splenic pan B cells from Ier3ip1^a/a and wild-type littermates were labeled with Indo-1/AM ester. Fluorescence was measured for 60 s to establish a baseline, and then, cells were stimulated with anti-IgM (blue arrowhead). Cytosolic Ca²⁺ flux was monitored with flow cytometry by measuring the ratio of Indo-1 violet and blue fluorescence emission. Kinetic traces are displayed and were normalized to baseline (n = 4 mice/genotype). (E and F) The frequency of peripheral blood B cells from third-generation (G3) descendants of three (E) or two (F) independent ENU-mutagenized male mice (G1), with REF (+/+), HET (+/mutant), or VAR (mutant/mutant) genotypes for nonsense mutations in Mtmr14 (E, n = 3 to 23 mice/genotype) or Rnf219 (F, n = 5 to 21 mice/genotype), respectively. (G) HEK293T cells were transfected with either FLAG-tagged TMEM167A or empty-FLAG, and HA-tagged wild-type IER3IP1 (WT) or mutant IER3IP1 (A18V). Cell lysates were immunoprecipitated using anti-FLAG M2 agarose beads and immunoprecipitates were subsequently analyzed with antibodies against FLAG or HA. (H) HEK293T cells were transfected with HA-tagged TMEM167A or empty-HA, and either FLAG-tagged WT or mutant IER3IP1. Fourteen hours after transfection, cells were treated with cycloheximide (CHX) and harvested at indicated times posttreatment. Cell lysates were immunoblotted with indicated antibodies. (I) TMEM167A expression in IER3IP1 knockout human cerebrum organoids. Raw MS data were obtained from the ProteomeXchange dataset with accession number PDX019350 (7). (J) Quantitative real-time PCR analysis of Tmem167 transcript levels in splenic B cells isolated from Ier3ip1^a/a and wild-type littermates (n = 4 to 6 mice/genotype). Data are representative of one (E and F), two (B–D, G, H, and J), or three (A) independent experiments. Data points represent individual mice (C, E, F, and J) or organoids with indicated genotype (I). Error bars indicate SD. P values were determined by Student’s t test (C and J) or one-way ANOVA with Dunnett’s multiple comparisons (E and F). ***P < 0.001.