Immunology and Inflammation

A Germinal Center Checkpoint of AIRE in B Cells Limits Antibody Diversification

Department of Obstetrics and Gynecology, Wayne State University, Detroit, United States
Center for Molecular Medicine and Genetics, Wayne State University, Detroit, United States
The Seventh Affiliated Hospital of Sun Yat-Sen University, Shenzhen, China
School of Applied Science, Republic Polytechnic, Singapore, Singapore
Beijing Genomics Institute (BGI)-Shenzhen, Guangdong, China
Department of Virology, University of Helsinki, Helsinki, Finland
Department of Chemistry, Wayne State University, Detroit, United States
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
Barbara Ann Karmanos Cancer Institute, Department of Oncology, Wayne State University, Detroit, United States
Department of Pathology, Children’s Hospital of Michigan, Detroit, United States
Department of Dermatology and Allergic Diseases, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
Helsinki University Hospital Research Institute, Biomedicum, Helsinki, Finland
Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, United States
Complete Genomics Inc, Mountain View, United States
Department of Biochemistry, Microbiology and Immunology, Wayne State University, Detroit, United States
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, United States
Department of Molecular Pathology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia
Department of Biology, City College of New York, New York, United States
Mucosal Immunology Studies Team, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, United States

https://doi.org/10.7554/eLife.110530.1

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Figures and data

GC B Cells Express AIRE.
(A and B) Immunofluorescence analysis of the tonsillar tissue of a healthy donor for IgD, CD19, AIRE and DAPI-stained DNA. The dotted line outlines the follicles. Bars: 100 μm (A) or 25 μm (B). (C and D) Immunofluorescence analysis of tissues of a healthy donor for IgD, AIRE, CD19 and DNA. Arrow heads indicate follicular IgD⁺ plasmablasts. Dotted lines mark the boundary between follicular mantle zone and the follicle. Bars: 40 μm (C) and 15 μm (D). (E) Flow cytometric analysis of AIRE expression in tonsillar total CD19⁺ B cells, IgD⁺CD38⁻ naive B cells, IgD⁺CD38⁺ founder GC (FGC) B cells, IgD⁻CD38⁺ GC B cells and IgD⁻CD38⁻ memory B cells. The data represent 5 donors. (F and G) Flow cytometric and statistical analyses of Aire (GFP) expression in splenic and ILN viable CD19⁺B220⁺FAS⁺GL7⁺ GC B cells, CD19⁺B220⁺FAS⁻GL7⁻ non-GC B cells and CD19^loB220^loCD138⁺ PCs of B6 mice (shaded histograms, n = 5 for spleen and n = 4 for ILN) or B6.Aire^Adig mice (colored histograms, n = 5 for spleen and n = 4 for ILN) after 4 i.p. immunizations with NP₃₂-KLH with CFA and subsequently with IFA. Data are represented as mean ± SEM. **P < 0.01, ***P < 0.001, by 1-way ANOVA with Tukey’s post hoc test. (H) qPCR analysis of Aire transcript levels in CD19⁺B220⁺FAS⁻GL7⁻ non-GC B cells (n = 5) and CD19⁺B220⁺FAS⁺GL7⁺GFP⁺ Aire-expressing GC B cells (n = 4) of Aire^Adig mice after 1 i.p. immunization with SRBC and CFA. Data are represented as mean ± SEM. ***P < 0.001, by 2-tailed unpaired t-test. (I) Flow cytometric and statistical analyses of CD83⁺CXCR4^lo LZ and CD83⁻CXCR4^hi DZ B cells in splenic total GC and GFP⁺ GC B cells of immunized B6.Aire^Adig mice, by 2-tailed paired t-test. (J and K) Immunofluorescence analysis of the tonsillar tissue of a healthy donor for IgD, CD23, Pax5 and AIRE. The dotted line outlines the follicles and delineates the border between LZ and DZ. Bars: 200 μm (J) or 30 μm (K).

Follicular B Cell AIRE Expression Requires CD40 Signaling.
(A and B) Immunofluorescence analysis of tonsillar tissues of a HIGM3 patient for IgD, AIRE, CD19 and DNA. The area outlined in A is shown with a higher magnification in B. The dotted line outlines the follicles. Bars: 100 μm (A) or 25 μm (B). (C) Flow cytometric analysis of Aire (GFP) expression in splenic B cells of a B6 or B6.Aire^Adig mouse treated for 3 d with medium or CD40L with or without IL-4 in the absence (vehicle) or presence of CAPE. The data represent the results from 3 B6 and 3 B6.Aire^Adig mice. (D and E) qRT-PCR and Western Blot analyses of AIRE transcript and protein levels, the protein levels of total and Ser536-phosphorylated NF-κB p65, as well as total and Thr202/Tyr204-phosphorylated Erk1/2 in human peripheral blood IgD⁺ B cells treated with medium or CD40L, or CD40L and IL-4, in the presence of vehicle or CAPE for 3 d. Data are represented as mean ± SEM. ***P < 0.001, by 2-tailed unpaired t-test. (F and G) qRT-PCR and Western Blot analyses of Aire transcript and Aire protein levels in mouse CH12 cells treated with anti-CD40, TGF-β and 100 ng/ml IL-4 for 3 d. Data are represented as mean ± SEM. P < 0.001, by 2-tailed unpaired t-test.

AIRE Inhibits CSR and SHM in B cells.
(A) The generation and immunization of Aire^+/+ and Aire^−/– BM and B cell chimeric mice. (B) The percentage of CD45.1⁺ Aire^+/+ and CD45.2⁺ Aire^−/– B cells in the splenic GC B cells of the secondary μMT recipient mice (n = 6) after the immunizations. ***P < 0.001, by 2-tailed paired t-test. (C and D) Flow cytometric and statistical analyses of the ratio of NP₈-to NP₃₆-binding (C) and NP₈-to total NP-binding (D) GC B cells after immunizations of the secondary μMT recipient mice (n = 6). **P < 0.05, **P < 0.01, by 2-tailed paired t-test. (E) The generation and immunization of Aire^+/+ and Aire^−/– B cell chimeric mice. (F) Flow cytometric analysis of surface IgD and IgM on NP₃₆-binding B cells in μMT recipients of Aire^+/+ or Aire^−/– B cells immunized with NP₃₂-KLH. The result represents 3 age- and sex-matched μMT recipients each of B cells from 3–5 age- and sex-matched littermate donor Aire^+/+ or Aire^−/– mice. (G) The ratios of the titers of circulating NP₄-binding to NP₂₉-binding IgM, IgG1, IgG2b and IgG3 in immunized μMT recipient mice of Aire^+/+ or Aire^−/– B cells. The results represent 4 experiments, each consisting of B cells from 3–5 age- and sex-matched littermate donor mice and 6–8 age- and sex-matched littermate μMT recipient mice. *P < 0.05, **P < 0.01, by 2-tailed unpaired t-test. (H) The SHM landscape across IgHV, including FR2, CDR2, FR3, CDR3 and FR4, of NP₃₆-binding IgM^‒IgD^‒ or IgM⁺IgD⁺ Aire^+/+ and Aire^‒/‒ donor B cells in μMT recipients after immunizations with NP₃₂-KLH. The result represents 3 μMT recipients of Aire^+/+ donor B cells and 3 μMT recipients of Aire^‒/‒ donor B cells. (I) Frequencies of C-to-T transitions in SHMs in IgHV of NP-specific IgG⁺, IgA⁺ or IgE⁺ splenic B cells from μMT recipient mice of Aire^+/+ or Aire^‒/‒ B cells after immunizations with NP₃₂-KLH. Data are represented as median ± upper/lower quartile. *P < 0.05, **P < 0.01, by 1-tailed unpaired t-test. (J) qRT-PCR analysis of the fold induction of Iμ-Cγ1 and Iμ-Cγ3 post-switch transcript levels in peripheral blood IgD⁺CD27^‒ naïve B cells from healthy subjects (n = 5) or APS-1 patients (n = 5) stimulated for 3 d with CD40L and IL-4 or IFN-γ over the respective unstimulated control B cells. *P < 0.05, by 1-tailed unpaired t-test (upper panel) or 1-tailed Mann-Whitney U test (lower panel).

Aire^−/– CH12 Cells Undergo Elevated CSR.
(A and B) Flow cytometric and statistical analyses of IgA CSR in WT and Aire^−/– CH12 cells treated with medium (Control) or anti-CD40, TGF-β and IL-4 for 3 d. Data in F was determined as the difference between the percentages of IgA⁺IgM⁻ cells in stimulated samples to unstimulated samples. The results (mean ± SEM) represent or compare 16 experiments involving WT CH12 cells, 8 experiments involving clones 43, 6 experiments involving clone 53, and 13 experiments involving clone 69. **P < 0.01, ***P < 0.001, by 2-tailed unpaired t-test. (C) qRT-PCR analysis of the Iα-Cμ circle transcript levels (mean ± SEM) in WT and Aire^−/– CH12 cells treated with medium (Control) or anti-CD40, TGF-β and IL-4 for 3 d. The results compare 3 experiments. *P < 0.05, **P < 0.01, by 1-tailed unpaired t-test. (D) qRT-PCR analysis of the Iγ1-Cμ circle transcript level in Aire^+/+ CH12 cells and Aire^−/– CH12 cell clones 43, 53 and 69 that were either unstimulated or stimulated with anti-CD40, TGF-β and IL-4 for 3 d. The result was normalised using the respective Actb transcript level and expressed as fold of induction (mean ± SEM) relative to unstimulated Aire^+/+ CH12 cells. (E and F) Western Blot analysis of AID in WT and Aire^‒/‒ CH12 cells that were either unstimulated or stimulated with anti-CD40, TGF-β and IL-4 for 3 d. Lamin B1 and GAPDH were used as the control for nuclear and cytoplasmic proteins, respectively. The data are presented as mean ± SEM and represent 3 experiments. (G) qRT-PCR analysis of Aicda and the Iμ-Cμ and Iα-Cα germline transcript levels (mean ± SEM) in Aire^+/+ CH12 cells and Aire^−/– CH12 cell clones 43, 53 and 69 that were either unstimulated or stimulated with anti-CD40, TGF-β and IL-4 for 3 d. (H) Flow cytometric analysis of IgA CSR in Aire^−/– CH12 cells (clone 69) transfected with a plasmid expressing WT (AIRE^WT-GFP) AIRE-GFP and treated with medium (Control) or anti-CD40, TGF-β and IL-4 for 3 d. The results represent 3 experiments.

AIRE Interacts with AID in GC B Cells.
(A and B) Imaging flow cytometric analysis of AIRE and AID in tonsillar IgD⁻CD38⁺ GC B cells of a healthy donor. Bars: 7 μm (A). The results represent 3 donors. (C and D) Co-IP of AIRE and AID in tonsillar CD19⁺ total, IgD⁺ naive and FGC and CD19⁺IgD^‒ GC and memory B cells of a healthy donor, and in splenic CD19⁺ B cells of a B6 mouse after 3 doses of immunization with SRBCs. The results are representative of tonsils of 4 donors and spleens of 3 mice. (E) The domain structures of recombinant WT and mutant human AIRE and AID molecules. Dotted lines indicated the deleted regions in the mutant proteins. (F) Co-IP of WT AID and WT or mutant AIRE in HKB-11 cells 24 h after transfection of plasmid(s) encoding WT AID and WT or mutant AIRE proteins. (G) The domain structures of recombinant WT and mutant human AID proteins. (H) Co-IP of WT AIRE and WT or mutant AID in HKB-11 cells 24 h after transfection of plasmid(s) encoding WT AIRE and WT or mutant AID proteins. The results in E and G are representative of 3 experiments. (I) Flow cytometric analysis of IgA CSR in Aire^−/– CH12 cells (clone 69) transfected with a plasmid expressing either WT (Aire^WT-GFP) or CARD-deficient (Aire^ΔCARD-GFP) AIRE-GFP and treated with medium (Control) or anti-CD40, TGF-β and IL-4 for 3 d. The results represent 3 experiments.

AIRE Inhibits AID Function by Interfering with AID Targeting to Its IgH DNA Substrate.
(A and B) The principle, chemistry and calibration of the dot blot assay for the quantitation of genomic uracil content. (C) The genomic uracil levels in WT, Aire^‒/‒, Aicda^‒/‒, or Ung^‒/‒ CH12 cells after 72 h of treatment without or with anti-CD40, TGF-β and IL-4. The results are presented as mean ± SEM and represent 3 experiments. ***P < 0.001, by 2-tailed unpaired t-test. Bisulfite-treated E. coli DNA was included as a positive control. (D) The genomic uracil content in WT and Aire^‒/‒ CH12 cells after 48 or 72 h of treatment without or with anti-CD40, TGF-β and IL-4. The results are presented as mean ± SEM and represent 3 experiments. **P < 0.01, ***P < 0.001, by 2-tailed unpaired t-test. (E) ChIP-qPCR analysis for the interaction of AID with Sμ, Iμ and Sγ1 regions in WT and Aire^‒/‒ CH12 cells after 72 h of treatment without or with anti-CD40, TGF-β and IL-4. The results are presented as mean ± SEM and represent 3 experiments. *P < 0.05, by 2-tailed unpaired t-test. (F) Co-IP of AID with pSer5-Pol II, total Pol II, Spt5 and Aire in WT and Aire^‒/‒ CH12 cells after 72 h of treatment without or with anti-CD40, TGF-β and IL-4. The results represent 3 experiments.

Aire Deficiency in B Cells Promotes Humoral Autoimmunity and Compromises Cutaneous Anti-Candida Defense.
(A) The cutaneous candidiasis infection model. (B and C) GMS stain of cutaneous C. albicans and skin fungal burden (CFU per mg of tissue) (mean ± SEM) in μMT recipient mice of Aire^+/+ or Aire^‒/‒ donor B cells 4 d after infection. Bars: 1 mm (B, upper panels) or 100 μm (B, lower panels). **P < 0.01, by 1-tailed unpaired t-test. (D) ELISA of the levels (mean ± SEM) of autoantibodies binding to IL-17A, IL-17F and IL-22 in the sera of μMT recipient mice of Aire^+/+ or Aire^‒/‒ donor B cells 4 d after infection. *P < 0.05, **P < 0.01, by 1-tailed unpaired t-test. (E) ELISA of the levels (mean ± SEM) of blocking activity of IL-17A, IL-17F and IL-22 to their receptors in the sera of μMT recipient mice of Aire^+/+ or Aire^‒/‒ donor B cells 4 d after infection. *P < 0.05, **P < 0.01, ***P < 0.001, by 1-way ANOVA with Tukey’s post hoc test. (F and G) Flow cytometric and statistical analyses of IL-17A and IL-22 expression in cutaneous T cells of μMT recipient mice of Aire^+/+ or Aire^‒/‒ donor B cells (n = 4 in each group) 4 d after infection and after ex vivo re-stimulation. The data are represented as mean ± SEM. *P < 0.05, by 1-tailed unpaired t-test. (H) Immunofluorescence analysis of Ly-6G (red) and DNA (blue) in cutaneous tissues surrounding the C. albicans infection site in μMT recipient mice of Aire^+/+ or Aire^‒/‒ donor B cells 4 d after infection. The results in B–H represent 2 experiments, with 4 mice per group in each experiment. Bars: 160 μm (upper panels) or 40 μm (lower panels). (I) A simplified schematic of AIRE-mediated GC checkpoint of antibody diversification in B cells. At the T–B cell border of secondary lymphoid organs, B cells present antigens to and receive co-stimulation from DC-activated T cells, which also induce AIRE expression in B cells via CD40. The activated B cells enter the GC DZ and undergo SHM, proliferation and subsequent affinity selection by interacting with antigens on the surface of follicular dendritic cells (FDCs) in LZ. Low-affinity B cells will undergo apoptosis, whereas high-affinity B cells receive help from T follicular helper (T_FH) cells to undergo CSR, and subsequently either re-enter the SHM‒proliferation cycle in the DZ or exit the GC as plasma cells or memory B cells. AIRE in B cells limits autoantibody generation by restraining excessive AID activity in the GC.

Aire^−/– CH12 Cell Clones, Related to Figure 4 and Figure 5.

Aire^−/– CH12 Cell Clones, Related to Figure 4 and Figure 5.

Human AIRE and AID Constructs, Related to Figure 4 and Figure 5.

Cloning Primers Used to Generate Aire^−/– CH12 Clones and AIRE and AID Mutant Molecules, Related to Figure 4 and Figure 5.

Cloning Primers Used to Generate Aire^−/– CH12 Clones and AIRE and AID Mutant Molecules, Related to Figure 4 and Figure 5.

AIRE Is Expressed Specifically in GC B Cells of Human and Mouse Secondary Lymphoid Organs, Related to Figure 1.
(A) Immunofluorescence analysis of the thymus of a healthy donor for EpCAM, AIRE and DNA. Bars: 20 μm. (B and C) Immunofluorescence analysis of tonsillar tissues of healthy donors for IgD, AIRE, CD19, Pax5, Bcl-6 and DNA. The dotted lines mark the boundary between tonsil follicular mantle zone and the follicle. Arrow heads point to follicular IgD⁺ plasmablasts (B) and Pax5⁺Bcl-6⁺AIRE⁺ GC B cells (C). Bars: 15 μm (B) and 30 μm (C). (D) Flow cytometric analysis of AIRE expression in human peripheral blood naive (IgD⁺CD27⁻), MZ (IgD⁺CD27⁺), switched memory (IgD⁻CD27+), double-negative (IgD⁻CD27⁻) B cells, and transitional (CD24^hiCD38^hi), mature (CD24^intCD38^int), memory (CD24^hiCD38⁻) B cells and plasma cells (CD24⁻CD38^hi). (E) Immunofluorescence analysis of the thymic tissue of a B6 mouse for UEA-1, AIRE and DNA, and the splenic tissue of a B6 mouse immunized with 3 doses of sheep red blood cells (SRBCs) for IgD, Aire, CD19 and DNA, and Bar: 20 μm. (G) Flow cytometric gating strategy for identifying mouse splenic non-GC (CD19⁺B220⁺GL7⁻ FAS⁻), GC (CD19⁺B220⁺GL7⁺FAS⁺) B cells and plasma cells (CD19^loB220^loCD138⁺). (H) Genotypes and Aire expression in ILN, splenic, peripheral blood and peritoneal B cells of a litter of Aire^Adigmice after 1 dose of i.p. SRBC immunization with or without CFA. (I) Percentage of GFP⁺ B cells (mean ± SEM) in splenic GC B cells of Aire^Adig transgene-positive mice (n = 5) after 1 dose of i.p. SRBC immunization with CFA. The dotted line indicates of mean value of GFP⁺ B cells in splenic non-GC B cells of these mice. (J) AIRE expression in mouse peripheral blood, splenic, MLN, PP, peritoneum and thymic B cells of B6.Aire^Adigmice after 1 dose of i.p. SRBC immunization without CFA. The data are representative of 6 B6.Aire^Adig and 6 B6 mice that were age- and sex-matched and housed in the same SPF room.

Aire^+/+ and Aire^−/– Donor BM and B Cells Had a Similar Phenotype Before Transfer, Related to Figure 3.
(A) Flow cytometric analysis of CD45.1⁺ Aire^+/+ and CD45.2⁺ Aire^−/– donor BM before and after B220 cell depletion. (B) Flow cytometry analysis of splenic naive resting B cells that were purified from the spleens of primary μMT chimeras of CD45.1⁺ Aire^+/+ and CD45.2⁺ Aire^−/– BM and used as donor B cells for the secondary μMT chimeric hosts. The ratio of CD45.2⁺ Aire^+/+ and CD45.2⁺ Aire^−/– splenic B cells were adjusted to be 1:1 prior to the secondary transfer. (C) Representative purity of CD45.2⁺ Aire^+/+ and CD45.2⁺ Aire^−/– littermate donor B cells before adoptive transfer into μMT hosts. (D) Cell surface expression of CD21, CD23, CD38, CD40, CD62L, CD80, CD86, CD93, I-A^b, BAFF-R and IgM and IgD on purified CD45.2⁺ Aire^+/+ and CD45.2⁺ Aire^−/– littermate donor B cells before adoptive transfer, as determined by flow cytometry. (E) NP₈-to-NP₃₆ binding ratios (mean ± SEM) of pre-immune splenic naive resting donor B cells of CD45.2⁺ Aire^−/–, CD45.2⁺ Aire^+/+ and CD45.1⁺ Aire^+/+ mice, by 1-way ANOVA with Tukey’s post hoc test. (F) Percentage of GL7⁺FAS⁺ GC B cells in the spleens of μMT recipients of either Aire^+/+ or Aire^−/– B cells that were immunized i.p. with NP₃₂-KLH. Flow cytometry was performed 4 d after the last immunization. (G) Cell surface expression of the co-stimulatory or co-inhibitory molecules CD80, CD86, PD-L1, PD-L2 and ICOSL on GL7⁺FAS⁺ GC B cells in the spleens of μMT recipients after immunizations. Shaded histograms indicate the staining using isotype-matched control antibodies. (H and I) Percentage of splenic PD-1⁺CXCR5⁺ T_FH cells and PD-1⁺CXCR5⁺Foxp3⁺CD25⁺ T_FR cells in the spleens of immunized μMT recipients. The results shown represent 4 experiments, each consisting of B cells from 3–5 age- and sex-matched littermate donor mice and 6–8 age- and sex-matched littermate μMT recipient mice. (J and K) Flow cytometric and statistical analyses of the percentages of total and intravascular B cells in thymic cells of μMT mice that received donor B cells after all the immunizations with NP₃₂-KLH. Age and sex-matched unimmunized μMT mice were included as controls. The data are represented as mean ± SEM. (L) The sorting and sequencing strategies for Aire^+/+ and Aire^‒/‒ donor B cells in μMT recipients after immunizations with NP₃₂-KLH. NP-specific B cells were sorted based on NP₃₆ binding.

Aire^+/+ and Aire^−/–B Cells Showed Similar Proliferation and Apoptosis in vitro, Related to Figure 3.
(A and B) CFSE dilution in purified splenic B cells from age- and sex-matched littermate donor Aire^+/+ and Aire^−/– mice treated with medium (Control) or 5 μg/ml anti-CD40 and 100 ng/ml IL-4 for 4 or 6 d. Non-viable cells were excluded from the analysis. (C) Statistical comparison of the percentage (mean ± SEM) of CFSE^lo Aire^+/+ vs. Aire^−/– splenic B cells (n = 3) after 4 or 6 days of stimulation with 5 μg/ml anti-CD40 and 100 ng/ml IL-4, by 2-tailed unpaired t-test. The results represent 3 independent experiments. (D) Apoptosis of Aire^+/+ or Aire^−/– B cells treated with medium (Control) or 500 ng/ml CD40L and 100 ng/ml IL-4 for 3 or 7 d, as determined by Annexin V and 7-AAD staining by flow cytometry. All results shown are representative of 3 experiments, each consisting of cells from 2–3 age- and sex-matched littermate Aire^+/+ and Aire^−/– mice.

Validation of Aire^−/– CH12 Cell Clones, Related to Figure 4.
(A) Verification of Aire mutations in CH12 clones by PCR using primers that only anneal to the WT sequence, giving no amplification in clones 43, 47 and 53. Clone 47 has a 3-bp deletion in both Aire alleles causing a single amino acid deletion, and hence was not used in experiments. (B) Verification of Aire mutations in both alleles of CH12 clone 69 by PCR showing no amplification using primer pair #2 which anneals to the WT but not the mutated sequence. Primer pair #1 amplifies a sequence immediately downstream of the mutation site, and primer pair #3 is specific for the single-stranded repair template used in CRISPR. (C) Western Blot analysis of AIRE protein expression in WT and Aire^−/– CH12 cells. (D) Flow cytometric analysis of apoptosis by Annexin V and 7-AAD staining of WT and Aire^−/– CH12 cells treated with medium (Control) or anti-CD40, TGF-β1 and IL-4 for 3 d. (E) Percentages of late (Annexin V⁺7-AAD⁺) and early (Annexin V⁺7-AAD⁻) apoptotic cells (mean ± SEM) in WT and Aire^−/– CH12 cells treated with medium (Control) or anti-CD40, TGF-β1 and IL-4 for 3 d. *P < 0.05, by 2-tailed t-test. The data in D and E represent 4 experiments.

AIRE and AID Co-localize in the Nuclei of GC B Cells, Related to Figure 5.
(A–E) Imaging flow cytometry of AIRE and AID in tonsillar IgD⁻CD38⁺ GC, IgD⁺CD38⁻ naive, IgD⁻CD38⁻ switched memory B cells, IgD⁻CD38^hi switched PCs and IgD⁺CD38⁺ founder GC (FGC) B cells of a healthy donor. DNA was counter stained with DAPI. Samples stained with isotype-matched control antibodies were used to define the fluorescence baseline for AIRE and AID. Four representative cells in each population stained with AIRE and AID or with isotype control antibodies were shown. Bars: 7 μm.

AID Interacts with AIRE in B Cells, Related to Figure 5 and Figure 6.
(A) Co-IP of AIRE and AID in tonsillar CD19⁺ total, IgD⁺ naive, and FGC and CD19⁺IgD^‒ GC and memory B cells of a healthy donor after treatment of the cell lysates with DNAse I. PCR amplification of β-Actin gDNA in DNAse I-treated or untreated cells was also performed. (B) Co-IP of AIRE and AID in splenic B cells of immunized WT or Aicda^−/– mice. The data represent 2 experiments. (C) Co-IP of AID with pSer5-Pol II, total Pol II, Spt5 and Aire in WT and Aire^‒/‒ CH12 cells after 72 h of treatment without or with anti-CD40, TGF-β and IL-4. The results represent 3 experiments.

AIRE Deficiency in B Dells Impairs Skin T_H17 Immunity against C. albicans, related to Figure 7.
(A) Levels of autoantibodies (mean ± SEM) binding to IL-17A, IL-17F and IL-22 in the sera of μMT recipient mice of Aire^+/+ or Aire^‒/‒ donor B cells 4 d after infection. *P < 0.05, **P < 0.01, by 1-tailed unpaired t-test. (B) Flow cytometric gating strategy for identifying mouse skin viable T cells after ex vivo re-stimulation. T cells downregulation CD3 or TCR after ex vivo stimulation with PMA and ionomycin; thus CD3⁺ or TCRβ⁺ events were gated for analysis. This gate also included TCRγδ⁺ T cells, which were CD3⁺.

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