An essential role for the Zn2+ transporter ZIP7 in B cell development

Abstract

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

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Fig. 1: A novel autosomal recessive agammaglobulinemia caused by mutations in ZIP7.
Fig. 2: Multiple loss-of-function mutations in ZIP7.
Fig. 3: Generation of an allelic series of ZIP7 mutant mice.
Fig. 4: ZIP7 deficiency leads to a B cell-intrinsic failure in development.
Fig. 5: ZIP7 deficiency leads to developmental arrest at the late pre-B to immature B cell transition.
Fig. 6: Reduced cytoplasmic zinc in the presence of ZIP7 mutation.
Fig. 7: Impaired ZIP7 function results in reduced BCR signaling.
Fig. 8: ZIP7-dependent inhibition of B cell phosphatase activity

Data availability

RNA-sequencing data generated for this study (Fig. 5 and Supplementary Figs. 4 and 5) have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE108178. Other data that support the findings of this study (including raw data supporting Fig. 1 and Supplementary Fig. 1) are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank colleagues in the Newcastle University Flow Cytometry and Bioimaging Facilities for assistance. We acknowledge N. Ashley, A. Mead, P. Sopp and C. Waugh for assistance with single cell experiments and flow cytometry, D. Biggs and C. Preece for generation of the mouse models and staff at the Oxford Functional Genomics Facility for animal care. We also thank the National Diagnostic Epidermolysis Bullosa Laboratory (St Thomas’ Hospital, London) and the NIHR Newcastle Biomedical Research Centre. We thank K. Taylor for helpful discussions. This work was supported by the Medical Research Council (MR/J0003042/1, MR/N00275X/1 and MR/L020149/1: DIVA) (C.A., R.J.C., E.F., J.C.C. and G.A.R.); the Sir Jules Thorn Trust (12/JTA) (S.H., D.S., T.S.D. and K.E.); the St Giles Foundation, the Rockefeller University, INSERM, Paris Descartes University, Howard Hughes Medical Institute, National Institutes of Health (5P01AI061093 and 5R01AI104857) and the French National Research Agency (ANR 14-CE15-0009-01) (B.B., S.J.d.J., J.-L.C. and M.E.C.); the Wellcome Trust (WT098424AIA; 090532/Z/09/Z and 207556/Z/17/Z) (P.C., G.A.R., J.R.C., S.P.-P., B.D. and S.H.); Cancer Research UK (C52690/A19270) (C.O. and J.R.C.); Diabetes UK (BDA11/0004210 and BDA/15/0005275) (P.C., G.A.R.); the Northern Counties Kidney Research Fund (14.06) (A.F. and A.W.); the National Health and Medical Research Council of Australia (C.S.M., S.G.T.) and the Ludwig Institute for Cancer Research (E.J.F. and J.C.C.). S.H. is a Wellcome Investigator and R.J.C. is a Principal Investigator of the MRC Human Immunology Unit.

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C.A. designed and performed experiments, analyzed data and wrote the paper. R.J.C., M.E.C. and S.H. designed experiments, analyzed data and wrote the paper. D.J.S., B.B., T.S.D., M.D.-L., C.O., B.C.L., L.A., R.C., R.B.-P., P.C., M.v.d.B., B.D., A.W. and S.P.-P. designed and performed experiments and analyzed data. K.R.E., J.R.C., J.C.C., G.A.R., S.G.T., J.A.M., S.P. and J.-L.C. designed experiments and analyzed data. B.B., B.v.d.B., K.R.B., A.P.C., D.S., X.X., Y.X. and M.S.K. performed bioinformatic analysis. E.C., T.L.C., A.F., E.J.F., S.J.d.J. and C.S.M. performed experiments. A.J.C., G.K., T.R.L., M.T.d.l.M., J.M.P., R.S.S., M.E.C. and S.H. cared for patients and provided clinical data. All authors reviewed the manuscript

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Correspondence to Richard J. Cornall or Mary Ellen Conley or Sophie Hambleton.

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Supplementary Figure 1 Allele frequency and predicted severity and evolutionary conservation of mutated residues in SLC39A7.

(a) Population frequency of putative pathogenic alleles of SLC39A7 (ZIP7) from the GNOMAD database13. (b) Combined Annotation Dependent Depletion (CADD)14 scores vs maximum mean allele frequencies of the SLC39A7 variants recorded in this study (red circles) and/or GNOMAD Database (blue circles for predicted null mutations (frameshift/stop/essential splicing), green circle (in-frame insertions and deletions) or gray circle (missense mutations)). None of the predicted null mutations or Inframe INDELS were found in the homozygous state. (c) Alignments of human ZIP7, mouse ZIP7 and Bordetella pertussis ZIP, generated as two pairwise alignments with human ZIP7 in BLAST-P, highlighting residues mutated in patients with agammaglobulinemia (missense, yellow; nonsense, pink) and predicted transmembrane segments (gray)15.

Supplementary Figure 2 Mutant forms of ZIP7 are expressed but display reduced Zn transporter activity.

(a) Fluorescence micrographs revealing expression of endogenous ZIP7 in primary dermal fibroblasts from healthy control or P1, stained with antibody against ZIP7 (red) and DAPI (blue). Scale bar, 20µm. (b) Impaired Zn2+ conductance of mutant forms of ZIP7 expressed in Xenopus oocytes, visualized by zinquin fluorescence. Left, fluorescence micrographs showing Zn2+-related zinquin signal and right, pairwise image analysis in ImageJ, as described in Methods. Images are representative of 4 independent experiments as exemplified in Fig 2h. (c) Western blot of detergent extracts of Xenopus oocytes injected in parallel with those visualized in (b), revealing expression of the recombinant ZIP7 proteins. Images are representative of 3 independent experiments. (d-e) Cytoplasmic Zn2+ concentration in HEK-293T cells stably expressing the genetically encoded cytoplasmic Zn2+ sensor eCALWY-4 and transfected with empty vector (EV), or vectors encoding WT, E363K (d) or P190A (e) ZIP7 proteins. Cytoplasmic Zn2+ concentration was calculated by comparing the steady state live cell fluorescence intensity with maximum and minimum signals obtained in the presence of TPEN and zinc pyrithione respectively, as described in Methods. Total number of cells analyzed was 80 (EV), 55 (WT), 26 (E363K) and 42 (P190A) across 2–4 experiments. Columns show means and bars the standard error. Comparisons were by one-way ANOVA with Bonferroni’s correction; * indicates p=0.0258 and ** p=0.0051.

Supplementary Figure 3 Normal T cell development in ZIP7 P198A/P198A mice.

(a-b) Representative flow cytometry of T cell subsets in the thymus (a) and spleen (b) of WT and ZIP7P198A/P198A (P198A-Hom) mice. Representative of 3 independent experiments (c-d) The percentage of B220+ B cells, naïve (CD62L+CD44-), activated (CD62L-CD44+) and memory (CD62L+CD44+) CD4+ and CD8+ T cells, NK cells (NK1.1+CD3-), neutrophils (B220-CD3-Ly6g+CD11b+Ly6c+) and monocytes (B220-CD3-Ly6c++F4/80-) within the leukocyte populations in wild-type (closed circles) and P198A-Hom (open circles) blood (c) and spleen (d). Representative of three separate experiments; circles represent individual mice, 5 WT and 5 P198A-Hom; bars means and 95% CI, and comparisons by two-way ANOVA with Bonferroni’s correction for multiple comparison; *p<0.0001. (e) The relative proportion of thymic or splenic CD4+ and CD8+ cells, in lethally irradiated mice reconstituted for 8 weeks with 70:30 mixtures of WT or P198A-Hom CD45.2+ and WT CD45.1+ BM. Filled columns show mean percentage CD45.2+ cells and data are representative of 3 experiments.

Supplementary Figure 4 Transcriptomic analysis of WT and ZIP7-deficient B cells from Hardy fraction B.

B220+CD43+CD24+BP1- pro-B cells were sorted flow-cytometrically and whole transcriptome analysis performed by RNAseq on 100 cell samples from WT and P198A-Hom mice. We identified genes whose transcription varied by at least two-fold between WT and P198A-Hom mice with Padj < 0.05 (see Methods). (a-c) Heatmaps show the relative expression level of the subset of these genes that are also differentially expressed during normal B cell development: (a) during the transition from Fractions A to B (reduced, purple and raised, blue), (b) during the transition from Fractions B to C (reduced, red and raised, green) and (c) between Fractions B and D (reduced, yellow and raised, orange), based on the ImmGen database (www.immGen.org). In each heatmap, the far left column indicates the direction of change expected from ImmGen; each of the remaining columns represents one mouse and rows individual transcripts.

Supplementary Figure 5 Transcriptomic analysis of WT and P198A-Hom B cells from Hardy fractions D and E.

B220+CD43-IgM-IgD- late pre-B (FrD) and B220+CD43-IgM+IgD- immature B cells (FrE) were flow-sorted and whole transcriptome analysis performed by RNA-seq on 100 cell samples from WT and P198A-Hom mice. We identified genes whose transcription varied by at least two-fold between WT and P198A-Hom mice with Padj < 0.05 (see Methods) and filtered these against sets of genes that are also differentially expressed at the indicated stages of normal B cell development (www.immGen.org). (a-b) Heatmaps showing the relative expression of the subset of genes that show differential expression between wild-type and P198A-Hom B cells in Hardy Fraction D and also (a) between Fractions C and D (reduced, yellow and raised, red) and (b) between Fractions D and E (reduced, green and raised, blue) of normal mice. (c) Heatmaps showing the relative expression level of the subset of genes that show differential expression between wild-type and P198A-Hom B cells in Hardy Fraction E and also between Fractions D and E (reduced, green and raised, blue) of normal mice. In each heatmap, the far left column indicates the direction of change expected from ImmGen; each of the remaining columns represents one mouse and rows individual transcripts.

Supplementary Figure 6 The B cell developmental arrest caused by ZIP7 deficiency is not associated with endoplasmic reticulum (ER) stress and is not corrected by expression of the pro-survival factor BCL2.

(a) RT-PCR assay for the ER-stress-associated shortened Xbp1-splice variant (Xbp1s). Results show RT-PCR of cDNA from WT (lanes 1–3) or P198A-Hom (lanes 4–6) flow-sorted B cells from FrD and FrE. The controls were tunicamycin treated (Tm) or untreated (Un) mouse embryonic fibroblasts (MEFs). Three mice were studied per genotype in the single experiment shown. (b-c) Representative flow cytometry analysis of B cell development and maturation in BM (b) and spleen (c) of WT and P198A-Hom mice, with and without co-expression of a Bcl2 transgene (upper and lower panels). Gates corresponding to Fr A-F in the BM and total (B220+CD19+), follicular (Fo, CD23+CD21+) and Marginal Zone (MZ, CD23-CD21hi) B cells in the spleen are highlighted. (d-e) Absolute numbers of B cells, gated as in (b-c), in Hardy Fractions in the BM (d) and in splenic subsets (e) from WT, P198A-Hom, WT BCL2 transgenic (BCL2) and P198A-Hom BCL2 transgenic (P198A-Hom BCL2) mice, here n=3 per group and representative of 4 independent experiments. Bars show means and 95% CI; statistical comparison was by two-way ANOVA with Bonferroni correction for multiple comparison, * =p<0.0001.

Supplementary Figure 7 Normal TCR signaling and threshold for activation ZIP7-deficient thymocytes.

(a-c) Mean phospho-specific antibody binding to indicated intracellular signaling molecules downstream of the TCR, 5 min after stimulation of WT (closed circles) and P198A-Hom (open circles) thymocytes with anti-CD3, gated on CD4+CD8+ (a), CD4+ (b) and CD8+ cells (c). (d-f) Mean phospho-specific antibody binding to indicated intracellular signaling molecules 30 min after treatment of WT (closed circles) and P198A-Hom (open circles) thymocytes with the PTEN specific inhibitor BpV(phen), in the absence of TCR stimulation, gated CD4+CD8+ (d), CD4+ (e) and CD8+ cells (f). In this figure, circles represent values from individual mice (n=3 mice per genotype), bars are means of groups and 95% CI. Data are representative of 3 independent experiments.

Supplementary Figure 8 PTEN contributes to phosphatase activity, but PTEN haploinsufficiency does not rescue B cell development in ZIP7 deficiency; normal PLCγ2 kinase expression in P198A-Hom cells.

(a) Reduced phosphatase activity in B cells from PTEN-deficient PTENffMb1Cre (n=2) mice compared with wild-type Mb1Cre controls (n=4). Plots show gating on the pre-B (Fraction D) population in these animals (left panel) and reduced phosphatase activity thereof in the absence of PTEN (middle panel, red fill: PTENffMb1Cre; blue fill: PTENwtMb1Cre); quantification in right panel. Bars show means and 95% CI; n=4 PTENwtMb1Cre and 2 PTENf/fMb1Cre mice; experiment done once. (b) Mean anti-PLCγ2 antibody staining in wild-type/P198A-heterozygote (closed circles) and P198A-Hom (open circles) SWHEL immature (B220+CD43-HEL+IgM+IgD-) B cells and B220-CD43- controls; bars show means and 95% CI (n=3 mice per group, experiment done once). (c-d) B cell numbers in irradiated CD45.1+ B6 mice reconstituted with WT (open triangles), PTENf/+Mb1Cre heterozygote (open circles), P198A-Hom (filled triangles) or P198A-Hom PTENf/+Mb1Cre compound mutant BM (filled circles), gated on Hardy Fractions A-F in the BM (c) and total B220+CD19+, follicular B220+CD19+CD23+CD21+ and marginal B220+CD19+CD23-CD21++ B cells in the spleen (d). Each symbol represents an individual mouse (n= 5 chimeric mice generated per genotype). Bars show means and 95% CI.

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Anzilotti, C., Swan, D.J., Boisson, B. et al. An essential role for the Zn2+ transporter ZIP7 in B cell development. Nat Immunol 20, 350–361 (2019). https://doi.org/10.1038/s41590-018-0295-8

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