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.
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We thank members of I.T.’s laboratory for technical advice and discussions, Y. Iizuka for microinjection of RNA for genome editing in mice, H. Asahara and T. Chiba for the advice on genome editing in human cell lines, and P. Burrows for critical reading of the manuscript. We apologize to colleagues whose work we could not cite because of space restrictions. This work was supported by RIKEN IMS PID project (to I.T.), MEXT KAKENHI (no. JP18H02778 to T.M.) and Health Labour Sciences Research grant (no. 20FC1053 to T.M.), JSPS Grant-in-Aid for Young Scientists (no. JP20K16884 to M.Y.), JSPS Fund for the Promotion of Joint International Research (no. JP18KK0228 to S.O.), AMED The Practical Research Project for Rare/Intractable Diseases (to S.O.), JSPS KAKENHI (no. JP18H02395 to K.Y.J.Z.), JSPS research fellowship (to A.K.P.), and the National Institute of Allergy and Infectious Diseases (P01AI061093 to B.B. and J.-L.C.).
The authors declare no competing interests.
Peer review information Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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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 TH1* 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 TCRαβ, 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.
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.
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 knock-out 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 AIOLOSWT and AIOLOSG159R in the IKZF3-KO NALM-6 cell line reconstituted with FLAG-tagged AIOLOSWT or AIOLOSG159R. 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 AIOLOSWT and AIOLOSG159R 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 AIOLOSG159R peaks (GGAGC, GGAGG, and GCAGG) are delineated by the blue square. GGGAA and TGGAA motifs were consistently associated with AIOLOSWT, whereas GGAGC, GGAGG, GCAGG, and CCCAGA motifs were repeatedly shown association with AIOLOSG159R. Peaks with non-differential binding between AIOLOSWT and AIOLOSG159R were enriched with relatively low accumulation of AIOLOS canonical binding motifs. e, EMSA showing binding of AIOLOSWT and AIOLOSG159R binding to AIOLOS consensus sequence (indicated in red font, IK-BS4 probe) or AIOLOSG159R specific motif (GGAGC, indicated in blue font) containing probe designed from genome regions with high AIOLOSG159R peaks. Direct binding of AIOLOSG159R 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 Ikzf3G158R/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 Ikzf3G158R/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.
a, Flow cytometric analysis of thymocyte and lymph node T cells in Ikzf3+/+, Ikzf3+/G158R, and Ikzf3G158R/G158R mice. Expression of indicated surface markers in total thymocytes, lymphocyte gated lymph node cells and CD3ε+ lymph node cells are shown. Numbers indicate the percentage of cells in each gate or each quadrant. Mature T cells in lymph node of Ikzf3G158R/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 Ikzf3G158R/G158R mice showed skewing to CD44+ memory phenotype, which also recapitulated the patient’s phenotype. b, TCRβ, CD3ε, CD4, and CD8α expression levels in thymocyte and lymph node T cell subsets of Ikzf3+/+, Ikzf3+/G158R, and Ikzf3G158R/G158R mice. Numbers represent relative MFI against Ikzf3+/+ mice. Similar to the human patients, Ikzf3+/G158R and Ikzf3G158R/G158R mice showed decreased expression of TCRβ and CD3ε expressions in thymocytes and lymph node T cells, respectively. c, Emergence of CD4loCD8+ cells in thymus of Ikzf3G158R/G158R mice. CD4 expression in CD8α+ thymocytes (delineated by red line) is shown in the histogram. Numbers represent relative MFI against Ikzf3+/+ mice.
FLAG-ChIP-seq was performed in IKZF3-KO NALM-6 cell line reconstituted with FLAG-tagged AIOLOSWT or AIOLOSG159R. 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, AIOLOSWT predominantly bound to TGGAA-rich regions.
Extended Data Fig. 7 C-terminal sequence of Ikzf3G158R:Δc-ZF (Ikzf3G158R:D461fs) allele and T cell phenotypes of Ikzf3+/G158R:Δc-ZF mouse.
a, Direct sequencing of Ikzf3 mRNA by Ikzf3G158R allele-specific PCR amplification of cDNA amplified using an Ikzf3G158R 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 Ikzf3G158R 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.
Unprocessed immunoblot images for Fig. 2d,e.
Unprocessed immunoblot images for Fig. 3b.
Statistical source data for Fig. 4c–f.
Graph source data for Fig. 5d.
Unprocessed immunoblot images for Fig. 6a.
Statistical source data for Fig. 6g.
Unprocessed immunoblot images for Fig. 7b.
Statistical source data for Fig. 7d,e.
Graph source data for Extended Data Fig 2d.
Unprocessed immunoblot images for Extended Data Fig 3b,e.
Unprocessed immunoblot images for Extended Data Fig 4a,c.
Statistical source data for Extended Data Fig. 4b,d. Graph source data for Extended Data Fig. 4e.
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Yamashita, M., Kuehn, H.S., Okuyama, K. et al. A variant in human AIOLOS impairs adaptive immunity by interfering with IKAROS. Nat Immunol 22, 893–903 (2021). https://doi.org/10.1038/s41590-021-00951-z