A variant in human AIOLOS impairs adaptive immunity by interfering with IKAROS

Abstract

In the present study, we report a human-inherited, impaired, adaptive immunity disorder, which predominantly manifested as a B cell differentiation defect, caused by a heterozygous IKZF3 missense variant, resulting in a glycine-to-arginine replacement within the DNA-binding domain of the encoded AIOLOS protein. Using mice that bear the corresponding variant and recapitulate the B and T cell phenotypes, we show that the mutant AIOLOS homodimers and AIOLOS-IKAROS heterodimers did not bind the canonical AIOLOS-IKAROS DNA sequence. In addition, homodimers and heterodimers containing one mutant AIOLOS bound to genomic regions lacking both canonical motifs. However, the removal of the dimerization capacity from mutant AIOLOS restored B cell development. Hence, the adaptive immunity defect is caused by the AIOLOS variant hijacking IKAROS function. Heterodimeric interference is a new mechanism of autosomal dominance that causes inborn errors of immunity by impairing protein function via the mutation of its heterodimeric partner.

Extended Data Fig. 1. Lymphocyte and dendritic cell subset analyses of the P1.

(a) Flow cytometric analysis of peripheral blood lymphocytes and monocytes in the P1 and a control subject. Gating strategies are shown above each plot. Numbers within the plots represent percentage of the defined populations. Briefly, the ratio of CD4/CD8 cells was inverted and T cells had an activated phenotype (HLA−DR⁺CD38⁺). CD4⁺ T cells were skewed to memory phenotype (CD45RA⁻CD45RO⁺) and T_H1* cells (CD4⁺CD45RO⁺CD161⁺CXCR3⁺CCR6⁺) were increased. iNKT cells were decreased. NK cells, mDCs, and eosinophils were also decreased. (b) CD3 and TCR expression levels in T cells of the P1 (blue) and control (red). Numbers represent MFI of P1’s T cell subsets relative to a control subject. Surface expression levels of TCRβ, CD3, and CD8 were decreased in T cells of the P1, despite comparable expression of CD4. iNKT: invariant NK T cell, mDC: myeloid dendritic cell, pDC: plasmacytoid dendritic cell, Eo: Eosinophil, Baso: Basophil, MFI: Mean fluorescence intensity

Extended Data Fig. 2. Whole exome analysis of the patients.

(a) Patients and healthy family members who were analyzed by whole-exome sequencing are indicated with an asterisk. (b) Filtering strategy for whole-exome analysis. Patient-specific variants were selected by familial segregation. Variants resulting from sequencing errors were filtered out by ignoring the variants with high in-house frequency. Variants shared by the P1 and the P2 were selected and further narrowed down to rare variants by using cut-off as dbSNP minor allele frequency (MAF) of 0.0001. Candidate variants other than IKZF3 were BCL9 (NM_004326.2:c.3934delG, NP_004317.2:p.Gly1312Alafs, and NM_004326.2:c.3936_3937insTTT, NP_004317.2:p.Gly1311_His1312insGly), BMS1 (NM_014753.3:c.3557C>T, NP_055568.3:p.Ala1186Val), CCDC102A (NM_033212.3:c.1135C>T, NP_149989.2:p.Arg379Trp), DENND4B (NM_014856.2:c.307G>A, NP_055671.2:p.Val103Ile), KIAA1462 (NM_020848.2:c.2215G>A, NP_065899.1:p.Gly739Ser), KRTAP2–2 (NM_033032.2:c.333_334delGA, NP_149021.2:p.Thr112Profs), NCSTN (NM_015331.2:c.464A>G, NP_056146.1:p.Glu155Gly), TCHH (NM_007113.2:c.1072_1074delGAG, NP_009044.2:p.Glu358del), and TMEM129 (NM_138385.3:c.40G>C, NP_612394.1:p.Val14Leu). (c) Alignment of amino acid sequences of the second zinc finger domain of AIOLOS orthologues from several species. Gray-shaded letters indicate identical amino acid in relation to human AIOLOS. Glycine residue at 159 position in human AILOS is well conserved beyond species. (d) Expression pattern of IKZF family genes during human B cell development. CLP/Pre-pro-B cell (CD34⁺CD10⁺CD19⁻), pro-B cells (CD34⁺CD10⁺CD19⁺), large pre-B cells (CD34⁻CD10⁺CD19⁺CD79B⁺IgM⁻) and small pre-B cells (CD34⁻CD10⁺CD19⁺CD79B⁻IgM⁻) were isolated by FACS sorting from the bone marrow aspirate of a healthy donor. RNA was extracted and subjected to RNA-seq analysis. FPKM of IKZF genes in the indicated populations are shown.

Extended Data Fig. 3. Wild-type AIOLOS and AIOLOS^G159R ChIP-seq in NALM-6 human pre-B cell line.

(a) Genomic sequence of the IKZF3 knock-out (KO) NALM-6 cell line. Exon 2 of IKZF3 was targeted by CRISPR-Cas9. Each allele of IKZF3 was cloned and sequenced. The knockout clone had an indel in exon 2, resulting in a frameshift and premature termination of the protein. Grey shading indicates inserted nucleotides. Amino acid in red were changed by the frameshift. (b) Western blotting of AIOLOS in wild-type (WT) and IKZF3-KO NALM-6 cell lines. Representative of three independent experiments. (c) Triplicates of ChIP-seq tracks showing five representative loci with unique and common binding by AIOLOS^WT and AIOLOS^G159R in the IKZF3-KO NALM-6 cell line reconstituted with FLAG-tagged AIOLOS^WT or AIOLOS^G159R. Numbers represent the signal values of binding enrichment of the detected peaks. Structure of the genes are shown at the bottom. Locations of binding motifs (GGGAA and GGAGC) within the ChIP-seq track regions are indicated at the bottom. (d) The top significant DNA binding motifs with p-values for AIOLOS^WT and AIOLOS^G159R abstracted from the peaks with all statistically different bindings and non-differential bindings between quadruplicate ChIP-seq samples. The AIOLOS consensus binding sequence (GGGAA) is delineated by the red square and TGGAA motif is delineated by the black square, whereas binding motifs specific to the AIOLOS^G159R peaks (GGAGC, GGAGG, and GCAGG) are delineated by the blue square. GGGAA and TGGAA motifs were consistently associated with AIOLOS^WT, whereas GGAGC, GGAGG, GCAGG, and CCCAGA motifs were repeatedly shown association with AIOLOS^G159R. Peaks with nondifferential binding between AIOLOS^WT and AIOLOS^G159R were enriched with relatively low accumulation of AIOLOS canonical binding motifs. (e) EMSA showing binding of AIOLOS^WT and AIOLOS^G159R binding to AIOLOS consensus sequence (indicated in red font, IK-BS4 probe) or AIOLOS^G159R specific motif (GGAGC, indicated in blue font) containing probe designed from genome regions with high AIOLOS^G159R peaks. Direct binding of AIOLOS^G159R to GGAGC motif was observed. Representative of three independent experiments.

Extended Data Fig. 4. Expression of Aiolos in thymocyte of Ikzf3^+/+, Ikzf3^+/G158R and Ikzf3 ^G158R/G158R mice, and supplementary flowcytometric analysis.

(a) Total cell lysates from the thymus of Ikzf3^+/+ and Ikzf3^−/− mice were subjected for immunoblot using anti-Aiolos antibody. Representative of three independent experiments. (b) Expression levels of IKZF family genes determined by RNA-seq in pre-B cells of mice with the indicated genotype (n = 3 for each genotypes). Bar graphs show mean with SD. * p <0.0094, ** p <0.0054, determined by one-way ANOVA. (c, d) Total cell lysates from the thymus of Ikzf3^+/+, Ikzf3^+/G158R and Ikzf3^G158R/G158R mice were subjected for immunoblot using anti-Aiolos antibody. Expression levels of Aiolos were normalized by Gapdh protein. Numbers indicate relative intensity of Aiolos of indicated genotypes to Ikzf3^+/+ sample(c). Graphs showing summary of relative quantity (RQ) of three independent experiments (d). The expression levels of Aiolos were comparable between the genotypes (n = 3 for each genotypes). Bar graphs show mean with SD. (e) Relative expression of wild-type and mutant Ikzf3 alleles in pre-B cells calculated from RNA-seq data (n = 3 for each genotypes). Bar graphs show mean with SD. (f) Flowcytometric analysis of CD19 and B220 staining in bone marrow and splenic cells in Ikzf3^+/+, Ikzf3^+/G158R and Ikzf3^G158R/G158R mice. (g) Flowcytometric analysis of follicular B cell and marginal zone B cells in indicated cell subsets. IgM and IgD expression in B220⁺CD19⁺ cells were also shown.

Extended Data Fig. 5. T cell phenotypes in Ikzf3^+/G158R and Ikzf3^G158R/G158R mice.

(a) Flow cytometric analysis of thymocyte and lymph node T cells in Ikzf3^+/+, Ikzf3^+/G158R, and Ikzf3^G158R/G158R mice. Expression of indicated surface markers in total thymocytes, lymphocyte gated lymph node cells and CD3e⁺ lymph node cells are shown. Numbers indicate the percentage of cells in each gate or each quadrant. Mature T cells in lymph node of Ikzf3^G158R/G158R mice showed decrease of CD8⁺ T cells and increase of CD4⁻CD8⁻ T cells. Similar but milder phenotypes were observed in Ikzf3^+/G158R mice. CD4⁺ T cells in lymph node of Ikzf3^G158R/G158R mice showed skewing to CD44⁺ memory phenotype, which also recapitulated the patient’s phenotype. (b) TCRβ, CD3ε, CD4, and CD8a expression levels in thymocyte and lymph node T cell subsets of Ikzf3^+/+, Ikzf3^+/G158R, and Ikzf3^G158R/G158R mice. Numbers represent relative MFI against Ikzf3^+/+ mice. Similar to the human patients, Ikzf3^+/G158R and Ikzf3^G158R/G158R mice showed decreased expression of TCRβ and CD3ε expressions in thymocytes and lymph node T cells, respectively. (c) Emergence of CD4^loCD8⁺ cells in thymus of Ikzf3^G158R/G158R mice. CD4 expression in CD8α⁺ thymocytes (delineated by red line) is shown in the histogram. Numbers represent relative MFI against Ikzf3^+/+ mice.

Extended Data Fig. 6. Accumulation of Wild-type AIOLOS in pericentromeres.

FLAG-ChIP-seq was performed in IKZF3-KO NALM-6 cell line reconstituted with FLAGtagged AIOLOS^WT or AIOLOS^G159R. Quadruplicates of ChIP-seq tracks showing the pericentromeric regions with TGGAA repeats, and a non-pericentromeric region containing TGGAA repeats. The locations of TGGAA motif are indicated at the bottom. As indicated by the binding motif analyses, AIOLOS^WT predominantly bound to TGGAA-rich regions.

Extended Data Fig. 7. C-terminal sequence of Ikzf3^G158R:Δc-ZF (Ikzf3^G158R:D461fs) allele and T cell phenotypes of Ikzf3^{+/G158R:Δc-ZF} mouse.

(a) Direct sequencing of Ikzf3 mRNA by Ikzf3^G158R allele-specific PCR amplification of cDNA amplified using an Ikzf3^G158R mutant allele-specific 5' primer and universal C-terminal 3' primer. Complementary DNA was synthesized from total RNA extracted from peripheral blood of F0 founder mice generated by CRISPR-Cas9 genome editing. The C-terminal sequence of the Ikzf3^G158R mutant allele confirmed the single nucleotide insertion (indicated as a purple letter) which results in the frame-shift and disruption of ZF5–6 structure. (b) Expressions of CD4 and CD8 in lymph node T cells of Ikzf3^+/G158R, Ikzf3^+/+, and Ikzf3^{+/G158R:Δc-ZF} mice. Numbers represent relative MFI against Ikzf3^+/+ mice.

Figure 1.. A heterozygous IKZF3 variant associated with B cell deficiency.

(a) The pedigree of patients with a heterozygous IKZF3 variant. Squares and circles represent males and females, respectively. Black symbols represent family members with B cell deficiency and harboring a heterozygous IKZF3 variant. The right panel shows the IKZF3 sequences of the patients (P1–P3) and a control subject. The arrow indicates the heterozygous IKZF3 c.475G>C variant. (b) Flow cytometric analysis of peripheral blood and BM of P1 and a healthy control. Numbers indicate the percent of cells in each quadrant. (c) A schematic of AIOLOS, which is encoded by IKZF3. The blue and yellow boxes represent the N-terminal ZFs that mediate DNA binding and the C-terminal ZFs that mediate dimerization, respectively. The missense variant G159R is located at the second ZF.

Figure 2.. Impaired binding of AIOLOS^G159R to the AIOLOS consensus sequence.

(a) The structure of human AIOLOS with four N-terminal ZFs in a complex with DNA generated by homology modeling using human PR/SET domain 9 (PRDM9) as a template. The N-terminal four ZFs (ZF1–4) of AIOLOS are shown in cyan, red, blue, and purple, respectively. Zinc ions bound to the four ZFs are shown as gray spheres. (b) Homology modeled structure of the DNA binding domain of the second ZF of AIOLOS showing that G159 is in contact with DNA and the side chain of R159 in the AIOLOS^G159R mutant creates steric clashes with DNA. G159 is shown in yellow and R159 in gray. (c) A line plot showing the distance between Cα-G159 with N1-guanine 28 and Cα-R159 with N1-guanine 28 of DNA in WT AIOLOS (black) and the AIOLOS^G159R mutant (red), respectively, indicating that the G159 to R159 substitution pushed the DNA away from the second ZF, thereby reducing the binding capacity of AIOLOS^G159R. The distance data were obtained from the molecular dynamics simulation trajectories of WT AIOLOS and AIOLOS^G159R mutant. (d) EMSA showing in vitro DNA binding of WT AIOLOS and the AIOLOS^G159R mutant. Nuclear lysates of HEK293T cells transfected with an expression vector encoding each protein were incubated with a radiolabeled IK-BS4 probe. Anti-AIOLOS antibody (Ab) generated a supershift of the DNA–protein complexes (red arrow). Cell lysates were tested for AIOLOS expression with an anti-AIOLOS Ab (lower panel). Data are representative of three independent experiments. (e) Competitive EMSA using an IK-BS1 probe with nuclear lysates of HEK293T cells transfected with WT AIOLOS and/or the AIOLOS^G159R mutant at the indicated ratios. Cell lysates were tested for AIOLOS expression with an anti-AIOLOS Ab (lower panel). Data are representative of three independent experiments.

Figure 3.. Altered DNA binding specificity of the AIOLOS^G159R variant.

(a) A schematic of AIOLOS ChIP-seq of human NALM-6 pre-B cells. IKZF3 knocked-out in NALM-6 pre-B cells by targeting exon 2 using CRISPR-Cas9. Dox-inducible, FLAG-tagged AIOLOS^WT or AIOLOS^G159R was retrovirally transduced into the IKZF3 knock-out NALM-6 cells. AIOLOS was induced by doxycycline treatment and FLAG ChIP-seq was performed. (b) A immunoblot of transduced AIOLOS^WT and AIOLOS^G159R before and after induction by doxycycline. (c) ChIP-seq tracks showing representative loci with unique and common binding by AIOLOS^WT and AIOLOS^G159R. Numbers represent the signal values of binding enrichment of the detected peaks. Structures of the corresponding genes are shown below the ChIP-seq tracks. Locations of binding motifs (GGGAA and GGAGC) within the ChIP-seq track regions are indicated at the bottom in red and blue. The upper and lower rows indicate motifs of the plus and minus strands, respectively. (d) A correlation heatmap and clustering of the quadruplicate ChIP-seq samples of AIOLOS^WT and AIOLOS^G159R induced in IKZF3 knock-out NALM-6 cells. Hierarchical clustering is shown above the heatmap. (e) A binding affinity heatmap and clustering of the quadruplicate ChIP-seq samples. Rows represent the binding loci and the color intensity reflects binding strength. Hierarchical clustering of the differentially bound sites is indicated to the left of the heatmap. (f) A Venn diagram representing differential and non-differential bindings of AIOLOS^WT and AIOLOS^G159R, calculated from quadruplicate ChIP-seq samples. (g, h) The top significant DNA binding motifs with p-values for AIOLOS^WT and AIOLOS^G159R. Top 1000 highly enriched peaks bound by representative ChIP-seq samples of AIOLOS^WT or AIOLOS^G159R (g), and top 1000 statistically different bindings between AIOLOS^WT and AIOLOS^G159R quadruplicate ChIP-seq samples (h). The AIOLOS consensus binding sequence (GGGAA) is delineated by the red square, whereas binding motifs specific to the AIOLOS^G159R peaks (GGAGC, GGAGG, and GCAGG) are delineated by the blue square. The TGGAA sequence (delineated by black square) is AIOLOS^WT enriched peaks. Abbreviations: MCS, multiple cloning site; MMLV, Moloney murine leukemia virus

Figure 4.. Ikzf3^G158R knock-in mice recapitulate impaired B cell differentiation in the patient.

(a) Genomic sequences of the Ikzf3^G158R and Ikzf3 knock-out alleles. Nucleotide and amino acid sequences are indicated. Orange font indicates altered amino acids. Purple font indicates the amino acid coded by the synonymous mutation. (b) Flow cytometric analysis of BM B cell progenitors and splenic B cell subsets with the indicated genotypes of mice at 9–16 weeks of age. (c, d) Frequencies of B cell progenitor subsets in BM (c) and splenic B cell subsets (d) of mice with the indicated genotypes (n = 5 for Ikzf3^+/+ and Ikzf3^+/G158R, n = 4 for Ikzf3 ^G158R/G158R (c); n = 6 for Ikzf3^+/+ and Ikzf3^+/G158R, n = 4 for Ikzf3 ^G158R/G158R (d)). Bar graphs show mean with SD. (e) Frequencies of B cell progenitor subsets and splenic B cells of Ikzf3^+/+ and Ikzf3^+/G158R mice at the indicated (BM: n = 3 for Ikzf3^+/+ and Ikzf3^+/G158R (6–8 weeks of age), n = 5 for Ikzf3^+/G158R (9–16 weeks of age), spleen: n = 6 for Ikzf3^+/+(6–8 weeks of age), n = 3 for Ikzf3^+/G158R (6–8 weeks of age), n = 6 for Ikzf3^+/G158R (9–16 weeks of age)). Bar graphs show mean with SD. * p <0.05, ** p <0.005, determined by one-way ANOVA (c–e). (f) BM chimera experiment using BM of Ikzf3^+/+ and Ikzf3^+/G158R mice. CD45.2⁺ Ikzf3^+/+ or Ikzf3^+/G158R BM was mixed with CD45.1⁺ Ikzf3^+/+ BM at a 1:1 ratio and injected to sublethally irradiated CD45.1⁺ Ikzf3^+/+ mice. Percentages of CD45.2⁺ cells within BM B-lineage cells, splenic B cells (B220⁺), T cells (TCRβ⁺), NK cells (TCRβ^-CD56⁺), and myeloid cells (CD11b⁺) at 8 weeks after transplantation are shown. Moreover, the percentages of CD45.2⁺ cells among B cell progenitors in the BM are indicated (n = 5 for Ikzf3^+/+ and n = 3 Ikzf3^+/G158R). Bar graphs and line graphs show mean with SD. P values were determined by t-test.

Figure 5.. Genes involved in B cell development are dysregulated in Ikzf3^+/G158R pre-B cells.

(a) Principal component analysis of the pre-B cell transcriptomes of Ikzf3^+/+ and Ikzf3^+/G158R mice (n = 3/genotype). (b) MA plot (M, log ratio; A, mean average) of gene expression of Ikzf3^+/+ vs. Ikzf3^+/G158R pre-B cells assessed by RNA-Seq. Each symbol represents one gene and red symbols indicate differentially expressed genes (DEGs) with an FDR of <0.05. The numbers of down- and up-regulated genes are indicated in the upper- and lower-right corners, respectively. The vertical lines represent log₂0.5 of fold change in expression and the horizontal line represents an average logCPM of 3. These thresholds identified 608 and 778 DEGs up- and down-regulated in Ikzf3^+/G158R pre-B cells, respectively. (c) A volcano plot of gene expression in Ikzf3^+/+ vs. Ikzf3^+/G158R pre-B cells. Red symbols indicate DEGs with an FDR of <0.05. (d) Gene ontology (GO) analysis of DEGs between Ikzf3^+/+ and Ikzf3^+/G158R pre-B cells. DEGs with log₂ fold change of >0.5 with an average logCPM of >3 were included for analysis. Each row represents a GO term with p-values of up- or down-regulated genes in Ikzf3^+/G158R pre-B cells. (e) A Venn diagram showing overlaps of 3401 DEGs between Ikzf3^+/+ and Ikzf3^+/G158R pre-B cells with known Ikaros binding sites in the WT pre-B cells (42655 total peaks and 7504 peaks in the TSS, GSE86897). Red and blue semicircles represent up- and down-regulated DEGs in Ikzf3^+/G158R pre-B cells, respectively. Abbreviations: FDR, false discovery ratio; TSS, transcription start site

Figure 6.. AIOLOS^G159R and Aiolos^G158R interfere with Ikaros function.

(a) Co-IP of FLAG-AIOLOS (WT or G159R) and HA-IKAROS. Immunoprecipitation with anti-FLAG Ab was performed following transfection of HEK293T cells with FLAG-AIOLOS (WT or G159R) and HA-IKAROS. A immunoblot with anti-HA Ab is shown. Representative of three independent experiments. (b) NIH3T3 cells were transfected with FLAG-tagged AIOLOS (WT or G159R) with or without HA-tagged IKAROS. The cells were stained with anti-HA and/or anti-Aiolos Abs and then incubated with secondary Abs conjugated with Alexa Fluore 488 for AIOLOS or Alexa Fluore 568 for IKAROS (left). COS-7 cells were transiently transfected with RFP-IKAROS and either GFP-tagged AIOLOS or GFP-AIOLOS^G159R (right). The speckled foci represent heterochromatin formation. Representatives of three independent experiments for each condition. Scale bar, 10 μm. (c-f) ChIP-seq analysis of Aiolos and Ikaros binding in thymocytes of Ikzf3^+/+ and Ikzf3^G158R/G158R mice. Summary of two independent experiments. Representatives binding loci are shown along with the corresponding chromosomal positions and gene structures. Numbers represent signal values of binding enrichment of the identified peaks. Locations of binding motifs within the ChIP-seq track regions are indicated at the bottom (c). A Venn diagram showing overlapped binding by Aiolos and Ikaros in thymocytes of Ikzf3^+/+ and Ikzf3^G158R/G158R mice. Figures represent distinct peak numbers identified in each sample (d). A correlation heatmap and clustering of the genome-wide binding by Aiolos and Ikaros in Ikzf3^+/+ and Ikzf3^G158R/G158R thymocytes in duplicate (e). The top significant DNA binding motifs for Aiolos and Ikaros in Ikzf3^+/+ and Ikzf3^G158R/G158R thymocytes are shown with p-values. Peaks with >20-fold enrichment were subjected to analysis. Binding motifs of the top 1000 enriched peaks of Ikaros not co-bound by Aiolos in Ikzf3^G158R/G158R thymocytes are also shown. The consensus binding sequence of Ikaros and Aiolos is delineated by the red squares, whereas the binding motifs specific to Aiolos^G158R and Ikaros in Ikzf3^G158R/G158R thymocytes are delineated by the blue squares (f). (g) The luciferase reporter gene assay results of HEK293T cells transfected with various combinations of vectors are indicated below the plot. The results were normalized to those of cells transfected with empty vectors. Data are presented as the mean with SEM of three independent experiments. Representative of three independent experiments. Abbreviation: TF, transcription factors

Figure 7.. Restored B cell development and T cell abnormalities by removal of the dimerization domain of the Aiolos^G158R mutant.

(a) A schematic of the C-terminal ZF deleting experiments in Ikzf3^+/G158R mice. Guide RNA targeting exon 8 of Ikzf3 and Cas9 mRNA were co-injected to the fertilized eggs. Founder offspring harboring a C-terminal deletion on the Ikzf3^G158R allele (Ikzf3^{+/G158R:Δc-ZF} mice) were selected by mouse crossing. (b) Co-immunoprecipitation of FLAG-tagged WT or Δc-ZF mutant AIOLOS and HA-IKAROS using total cell lysates of HEK293T cells transfected with FLAG-AIOLOS (WT or Δc-ZF mutant) and HA-IKAROS. Immunoblots with anti-HA Ab and anti-AIOLOS Ab are shown. Representative of three independent experiments. (c) Flow cytometric analysis of BM B cell progenitors, splenic B cell subsets, thymocytes, and lymph node T cells of 6-week-old Ikzf3^+/+, Ikzf3^+/G158R, and Ikzf3^{+/G158R:Δc-ZF} mice. Numbers adjacent to the outlined areas indicate the percent of cells in each gate. (d, e) Frequency of BM B-lineage cells (d), splenic B cells, and FO B cell subsets (e) in Ikzf3^+/+, Ikzf3^+/G158R, and Ikzf3^{+/G158R:Δc-ZF} mice (n = 4 for Ikzf3^+/+, n = 3 for Ikzf3 ^+/G158R, and n = 6 for Ikzf3^{+/G158R:Δc-ZF}). Bar graphs show mean with SD. * p <0.05, ** p <0.005, determined by one-way ANOVA (d, e). (f) Expression of TCRβ, CD3ε, and CD44 in thymocytes and lymph node T cell subsets of Ikzf3^+/+, Ikzf3^+/G158R, and Ikzf3^{+/G158R:Δc-ZF} mice. Numbers represent relative mean fluorescence intensity against Ikzf3^+/+ mice.