Kidins220 regulates the development of B cells bearing the λ light chain
- Faculty of Biology, Albert-Ludwigs-University of Freiburg, Germany
- Signalling Research Centers BIOSS and CIBSS, University of Freiburg, Germany
- Center of Chronic Immunodeficiency CCI, University Clinics and Medical Faculty, Germany
- Department of Dermatology and Allergy Biederstein, School of Medicine, Technical University of Munich, Germany
- Institute of Medical Engineering and Medical Informatics, School of Life Sciences, FHNW 15 University of Applied Sciences and Arts Northwestern Switzerland, Switzerland
- Max Planck Institute of Immunobiology and Epigenetics, Germany
- CIBSS – Centre for Integrative Biological Signalling Studies, University of Freiburg, Germany
- Division of Clinical and Experimental Immunology, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Austria
- Department of Rheumatology and Clinical Immunology, University Medical Center Freiburg, Faculty of Medicine, University of Freiburg, Germany
- aiNET GmbH, Switzerland
- SIB Swiss Institute of Bioinformatics, Switzerland
Figures
Figure 1 with 1 supplement
Kidins220 immature B-KO cells show a skewed primary BCR repertoire.
(A) Schematics of the genotypes of the Kidins220 locus and cre-recombinase expression for control (CTRL) and B cell specific knockout (B–KO) mice (left). Representative flow cytometric BM analysis …
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Figure 1—source data 1
Kidins220 immature B-KO cells show a skewed primary BCR repertoire (Figure 1 and Figure 1—figure supplement 1).
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Immature B-KO B cells show a skewed primary BCR repertoire.
(A, C, D) Immature B cells (B220+IgM+IgD-) of three individual mice per genotype were pooled and subjected to single cell sequencing analyzing full-length Ig-gene V(D)J recombination status and BCR …
Figure 2 with 1 supplement
Kidins220 is required for the opening of the Igl locus in pro-/pre-B cells.
(A) Experimental setup to generate primary BM derived pro-/pre-B cell cultures. Total BM was isolated from CTRL and B-KO mice and cultured for 7 days in medium supplemented with IL-7. Next, IL-7 was …
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Figure 2—source data 1
Kidins220 is required for the opening of the Igl locus in pro-/pre-B cells.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
The transcriptional network controlling LC recombination as well as RAG protein activity are independent of Kidins220.
(A) Schematic representation of the primer position (black arrows) for the detection of the Igk0.8 and Igl1 germline transcripts. (B) RNA isolated from pro-/pre-B cell cultures after IL-7 withdrawal …
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Figure 2—figure supplement 1—source data 1
Kidins220 is required for the opening of the Igl locus in pro-/pre-B cells.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 1 supplement
Kidins220 is required for the generation of λLC-bearing B cells even in κ-KO mice.
(A, I) Representative flow cytometric analysis of the BM (A) and spleen (I) of CTLR and B-KO mice in κ-sufficient (κ-CTRL) and -deficient (κ-KO) backgrounds. (B-H; J–Q) Total cell numbers for each B …
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Figure 3—source data 1
Kidins220 is required for the generation of λLC-bearing B cells even in κ-KO mice.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Kidins220 is required for the generation of λLC bearing B cells even in κ-KO mice.
(A) Genotypes of the Kidins220 locus, the mb1 Cre-recombinase expression, and the Igκ locus for CTRL and B-KO mice carrying a neomycin resistance gene (T) heterozygously (κ-CTRL) or homozygously …
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Figure 3—figure supplement 1—source data 1
Kidins220 is required for the generation of λLC bearing B cells even in κ-KO mice.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
BCL2-mediated survival partially rescues λLC deficiency in vitro.
(A) Experimental setup to generate primary BM-derived pro-/pre-B cell cultures overexpressing BCL2. Total BM was isolated from CTRL and B-KO mice and cultured for 7 days in medium supplemented with …
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Figure 4—source data 1
BCL2-mediated survival partially rescues λLC deficiency in vitro.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Kidins220 influences B cell survival and IL-7R signaling.
(A) BM cells were freshly isolated from CTRL and B-KO mice and analyzed for apoptosis and cell viability. The frequency of apoptotic (AnnexinV+PI-) and dead (AnnexinV+PI+) cells was analyzed by flow …
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Figure 4—figure supplement 1—source data 1
Kidins220 influences B cell survival and IL-7R signaling.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 1 supplement
Ectopic BCL2-expression partially rescues λLC expression in Kidins220 B-KO mice.
(A, H) Representative flow cytometry plots of CTRL and B-KO mice in the absence or presence of the BCL2 transgene of BM (A) and spleen (H). Total cell numbers for each B cell compartment from BM (B–G…
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Figure 5—source data 1
Ectopic BCL2-expression partially rescues λLC expression in Kidins220 B-KO mice.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Ectopic BCL2 expression partially rescues λLC expression in B-KO mice.
(A) The genotypes of the Kidins220 locus, the mb1 Cre-recombinase expression, and vav-BCL2 transgene (vav-BCL2Tg) expression for CTRL and B-KO mice are depicted. (B-F; H–N) Quantification of the …
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Figure 5—figure supplement 1—source data 1
Ectopic BCL2 expression partially rescues λLC expression in B-KO mice.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 1 supplement
Kidins220 is dispensable for the elimination of autoreactive BCRs but necessary for the expression of innocuous λLC during tolerance induction.
(A) Experimental setup for BM transfer into CD45.1 mice. HSC from CTRL and B-KO mice were isolated by negative magnetic purification. A total of 5x105 cells were injected intravenously into …
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Figure 6—source data 1
Kidins220 is dispensable for the elimination of autoreactive BCRs but necessary for the expression of innocuous λLC during tolerance induction.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Kidins220 is dispensable for the elimination of autoreactive BCRs but is necessary for the expression of innocuous λLC during tolerance induction.
Quantification of the relative cell numbers of B cell subpopulations in the BM (A–D) and spleen (E–H) of WT and κ-macroself transgenic mice reconstituted with BM of CTRL or B-KO mice. The …
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Figure 6—figure supplement 1—source data 1
Kidins220 is dispensable for the elimination of autoreactive BCRs but is necessary for the expression of innocuous λLC during tolerance induction.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig6-figsupp1-data1-v2.xlsx
Figure 7 with 1 supplement
BCL2 overexpression fails to rescue λLC expression during tolerance induction.
(A) Experimental setup for BM transfer of virally transduced HSC into CD45.1 WT or CD45.1 κ-macroself transgenic mice. HSC from CTRL and B-KO mice were isolated by negative magnetic purification and …
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Figure 7—source data 1
BCL2 overexpression fails to rescue λLC expression during tolerance induction.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
BCL2 overexpression partially rescues λLC expression during tolerance induction.
Quantification of the relative cell numbers of B cell subpopulations in the BM (A–D) and spleen (E–H) of WT and κ-macroself transgenic mice reconstituted with BM of BCL2 overexpressing HSCs of …
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Figure 7—figure supplement 1—source data 1
BCL2 overexpression fails to rescue λLC expression during tolerance induction.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig7-figsupp1-data1-v2.xlsx
Figure 8 with 1 supplement
Rewired pre-BCR signaling in the absence of Kidins220.
(A) Representative flow cytometry plots of the BM of CTRL and B-KO mice showing the surface expression of CD25, MHCII and CD43 (left). For CD25, quantification of nine independent experiments with …
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Figure 8—source data 1
Rewired pre-BCR signaling in the absence of Kidins220.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
Kidins220 does not overall alter the mitochondrial function of B cells.
B cell subpopulations in the BM and spleens of CTRL and B-KO mice were analyzed by flow cytometry using specific antibodies against B220, CD117 (c-kit), CD25, κLC, and λLC. Cells were additionally …
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Figure 8—figure supplement 1—source data 1
Kidins220 does not overall alter the mitochondrial function of B cells.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig8-figsupp1-data1-v2.xlsx
Figure 9
Overexpressing BTK partially rescues λLC development.
Pro-/pre-B cell cultures were retrovirally transduced with an overexpression plasmid coding for BTK at day 7 of IL-7 culture. Two days later, IL-7 withdrawal was performed. Cells transduced with the …
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Figure 9—source data 1
Overexpressing BTK partially rescues λLC development.
- https://cdn.elifesciences.org/articles/83943/elife-83943-fig9-data1-v2.xlsx
