Research ArticleT CELL DIFFERENTIATION

Mutual inhibition between Prkd2 and Bcl6 controls T follicular helper cell differentiation

See allHide authors and affiliations

Science Immunology  24 Jan 2020:
Vol. 5, Issue 43, eaaz0085
DOI: 10.1126/sciimmunol.aaz0085
  • Fig. 1 Elevated serum IgE, IgG1, IgA, and IgM in Prkd2-deficient mice.

    (A and B) Phenotypic screening data. Serum antibodies in mice on day 14 after immunization with OVA/alum. (A) Total IgE and (B) OVA-specific IgE in wild-type C57BL/6J mice (B6) or the G3 descendants of a single ENU-mutagenized male mouse with REF (+/+), HET (Purnama/+), or VAR (Purnama/Purnama) genotypes for Prkd2 (left). Manhattan plot showing the P values of association between the total IgE (A) or OVA-IgE phenotype (B) and mutations identified in the affected pedigree calculated using a recessive model of inheritance (right). –Log10 P values are plotted versus the chromosomal positions of mutations. Horizontal red and purple lines represent thresholds of P = 0.05 with or without Bonferroni correction, respectively. The P values for linkage of the Prkd2 mutation are indicated. (C to P) Serum antibodies were measured before immunization (−) and on day 10 after immunization with OVA/alum (+). (C) Total IgE, (D) OVA-specific IgE, (E) total IgA, (F) total IgM, (G) total IgG1, (H) total IgG2b, (I) total IgG2a, (J) total IgG2c, and (K) total IgG3 concentration in serum from Prkd2+/+, Prkd2+/−, and Prkd2−/− mice. (L to P) Total IgE (L), OVA-specific IgE (M), total IgG1 (N), total IgA (O), and total IgM (P) concentration in serum from Rag2−/− mice engrafted with Prkd2+/+ or Prkd2−/− BM. Each symbol represents an individual mouse. Data are representative of three independent experiments with at least five mice per genotype (mean ± SD); P values were determined by one-way ANOVA with Tukey’s multiple comparisons test (A to K) or unpaired Student’s t test (L to P) (*P < 0.05, **P < 0.01, and ***P < 0.001).

  • Fig. 2 Increased numbers of TFH and GC B cells in Prkd2−/− mice.

    (A and B) IL-4 concentration in the culture medium of splenic CD4+ T cells left unstimulated (−) or stimulated (+) with PMA/ionomycin (A) or anti-CD3/CD28–coated beads (B) for 72 hours. n = 6 cultures from independent mice per genotype and condition. (C to E) The indicated cell populations were analyzed by flow cytometry in unimmunized mice (−) or in immunized mice on day 7 after immunization with OVA/alum (+). (C) Representative flow cytometric scatter plots (left) and quantification (right) of the frequency of splenic IL-4–GFPhighCD4+ T cells (CD3+CD4+GFPhigh) among CD4+ T cells. (D and E) Representative flow cytometric scatter plots (left) and quantification (right) of the frequency of splenic TFH cells (CD3+CD4+CXCR5highPD-1high) among CD4+ T cells (D) or GC B cells (CD3B220+GL-7highCD95high) among B220+ cells (E). (F) Confocal images of spleen cryosections from unimmunized Prkd2+/+ or Prkd2−/− mice (left). The sections were stained with anti-IgD (green) and PNA (red). Scale bar, 100 μm. Quantification of number of follicles containing PNA+ cells per spleen (right). (G) Representative flow cytometric scatter plots (left) and quantification (right) of the frequency of plasma cells (CD19+CD138high) in BM. Numbers adjacent to outlined areas indicate percent cells in each (C to E and G). Each symbol represents an individual mouse (C to G). Data are representative of three independent experiments with at least five mice per genotype (mean ± SD); P values were determined by unpaired Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).

  • Fig. 3 Excessive cell-autonomous Prkd2−/− TFH development.

    (A) Flow cytometric gating strategy. Splenic IL-4–GFP+CD4+ T cells (CD3+CD4+GFPhigh) were analyzed using PD-1 and CXCR5 markers to detect IL-4–GFP+ TFH (PD-1highCXCR5high) and IL-4–GFP+ non-TFH (TH2) cells (PD-1lowCXCR5low). (B) TFH and TH2 cells were analyzed by flow cytometry in unimmunized mice (−) or in immunized mice on day 7 after immunization with OVA/alum (+). Representative flow cytometric scatter plots (left) and quantification (right) of the frequency of IL-4–GFP+ TFH and IL-4–GFP+ TH2 cells among splenic IL-4–GFP+CD4+ T cells. Ratio between IL-4–GFP+ TFH and IL-4–GFP+ TH2 cells within GFPhighCD4+ T cells was also calculated. (C and D) Reconstitution of TFH in spleens of irradiated Rag2−/− recipient mice engrafted with a mixture containing equal numbers of Prkd2+/+ (CD45.1+) and Prkd2−/− (CD45.2+) BM cells (C) or naïve CD4+ T cells (D). Representative flow cytometric scatter plots (left) and quantification (right) of the frequency of TFH cells (CD3+CD4+CXCR5highPD-1high) derived from each donor among CD4+ T cells. Data are representative of three independent experiments with at least five mice per genotype (mean ± SD); P values were determined by unpaired Student’s t test (**P < 0.01 and ***P < 0.001).

  • Fig. 4 Prkd2-dependent phosphorylation of Bcl6 limits Bcl6 access to the nucleus in CD4+ T cells.

    (A) MFI of Bcl6 in non-TFH (CD3+CD4+PD-1lowCXCR5low) and TFH (CD3+CD4+PD-1highCXCR5high) in the spleen. Prkd2+/+ splenocytes were used to stain with isotype control IgG. Representative flow cytometric histogram plots (left) and quantification (right). (B) Frequency of Bcl6 expressing CD4+ T cells (CD3+CD4+Bcl6high) in the spleen. (C) MFI of Bcl6 in splenic CD4+ T (CD3+CD4+) cells. Prkd2+/+ splenocytes were used to stain with isotype control IgG. Representative flow cytometric histogram plots (above) and quantification (below). (D) Immunoblot analysis of Bcl6 in whole-cell lysates (WCL), cytosolic extracts (Cyt), or nuclear (Nuc) extracts of pooled CD4+ T cells from Prkd2−/− or Prkd2+/+ littermates. (E) Immunoblot analysis of HA-tagged Bcl6 in whole-cell lysates, cytosolic extracts, or nuclear extracts of HEK293T cells transfected with or without FLAG-tagged Prkd2. Mouse (left) and human proteins (right) are shown. (F) FLAG-tagged human Prkd2 was transfected into HEK293T cells with or without HA-tagged human Bcl6, and whole-cell lysates were subjected to immunoprecipitation with anti-FLAG M2 magnetic beads, followed by immunoblotting with anti-FLAG or anti-HA. (G) FLAG-tagged human Bcl6 was transfected into HEK293T cells with or without HA-tagged human Prkd2, and whole-cell lysates were subjected to immunoprecipitation with anti-FLAG M2 magnetic beads, followed by immunoblotting with anti-FLAG or anti-HA. (H) Pull-down assay. FLAG-tagged human Bcl6 was transfected into HEK293T cells, and whole-cell lysates were subjected to immunoprecipitation with anti-FLAG M2 magnetic beads. Immunoprecipitates were incubated with recombinant GST-tagged human Prkd2, followed by immunoblotting with anti-FLAG or anti-Prkd2. (I) FLAG-tagged human Bcl6 was transfected into HEK293T cells with or without HA-tagged human full-length or kinase domain–deleted (ΔK) Prkd2. Cell lysates were subjected to Phos-tag or normal immunoblot analysis using anti-FLAG or anti-HA. (J) Immunoblot analysis of HA-tagged Bcl6 in whole-cell lysates, cytosolic extracts, or nuclear extracts of HEK293T cells transfected with or without FLAG-tagged human full-length or kinase domain–deleted (ΔK) Prkd2. α-Tubulin and histone H3 were used as cytoplasmic and nuclear markers, respectively, and as loading controls (D to G, I, and J). Data are representative of three independent experiments. At least five mice per genotype were used per experiment [mean ± SD in (A) to (C)]. P values were determined by unpaired Student’s t test (**P < 0.01).

  • Fig. 5 Bcl6 down-regulates Prkd2 in CD4+ T cells.

    (A) Frequency of Bcl6 expressing CD4+ T cells (CD3+CD4+Bcl6high) in the spleen from unimmunized Prkd2+/+ mice (−) or from Prkd2+/+ mice on day 6 after OVA/alum immunization (+). Each symbol represents an individual mouse. (B) MFI of Bcl6 in non-TFH (CD3+CD4+PD-1lowCXCR5low) and TFH (CD3+CD4+PD-1highCXCR5high) in the spleen from unimmunized Prkd2+/+ mice (None) or from Prkd2+/+ mice on day 6 after OVA/alum immunization. Splenocytes from unimmunized mice were used to stain with isotype control IgG. Representative flow cytometric histogram plots (left) and quantification (right). At least five mice were used per condition. (C) Immunoblot analysis of Prkd2 and Bcl6 (left) and qRT-PCR analysis of Prkd2 mRNA (right) in splenic CD4+ T cells from unimmunized Prkd2+/+ mice (−) or from Prkd2+/+ mice on day 6 after OVA/alum immunization (+). Prkd2 mRNA was normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA level in CD4+ T cells from unimmunized mice. (D) Immunoblot analysis of Prkd2 and Bcl6 (left) and qRT-PCR analysis of Prkd2 mRNA (right) in non-TFH (PD-1lowCXCR5low) or TFH (PD-1highCXCR5high) sorted from the same pool of Prkd2+/+ CD4+ T cells. Prkd2 mRNA was normalized to GAPDH mRNA level in non-TFH. Data for qRT-PCR are average of three independent experiments. (E) Immunoblot analysis of Prkd2 and Bcl6 (left) and qRT-PCR analysis of Prkd2 mRNA (right) in splenic CD4+ T cells from unimmunized mice (−) or from mice on day 6 after OVA/alum immunization (+). Prkd2 mRNA was normalized to GAPDH mRNA level in CD4+ T cells from unimmunized Prkd2+/+ mice. Data are representative of three independent experiments [mean ± SD in (A) to (E)]. P values were determined by unpaired Student’s t test (**P < 0.01 and ***P < 0.001).

  • Fig. 6 dsDNA antibodies in Prkd2−/− mice.

    (A to D) dsDNA-specific IgG (A), dsDNA-specific IgM (B), dsDNA-specific IgA (C), and dsDNA-specific IgE (D) concentrations in serum from unimmunized 4- to 24-week-old Prkd2+/+ and Prkd2−/− mice. NZB/NZW F1 hybrid mice (24 weeks old) known to develop autoimmune disease are shown as a positive control. Each symbol represents an individual mouse. O.D., optical density. Data are representative of three independent experiments with at least five mice per genotype (mean ± SD); P values were determined by unpaired Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/43/eaaz0085/DC1

    Fig. S1. Effect of the Purnama mutation on IgE responses.

    Fig. S2. Accelerated IgE responses in Prkd2-deficient mice after immunization with papain.

    Fig. S3. Immune cells in Prkd2-deficient mice.

    Fig. S4. Increased number of TFH and GC B cells in Prkd2−/− spleens.

    Fig. S5. Increased number of TFH in Prkd2−/− lymph nodes.

    Fig. S6. Prkd2−/− TH2 cells.

    Fig. S7. TFH, GC B cells, and antibody responses in Prkd2−/− Bcl6fl/flCD4-Cre+ mice.

    Fig. S8. IL-2 production and IL-2–regulated immune cells in Prkd2−/− mice.

    Fig. S9. Bcl6 expression in Prkd2−/− B cells.

    Fig. S10. Effect of B cell–intrinsic Prkd2 deficiency on GC B cell development.

    Fig. S11. Flow cytometric gating strategy to sort PD-1highCXCR5high and PD-1lowCXCR5low CD4+ T cells.

    Fig. S12. Frequencies of TFH or GC B cells and IgE response to immunization in Il12a−/− mice.

    Fig. S13. Enlarged lymph nodes and spontaneous BALT formation in Prkd2−/− mice.

    Fig. S14. Regulation of TFH-dependent antibody responses by mutual inhibition between Prkd2 and Bcl6.

    Table S1. Raw data file (Excel spreadsheet).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Effect of the Purnama mutation on IgE responses.
    • Fig. S2. Accelerated IgE responses in Prkd2-deficient mice after immunization with papain.
    • Fig. S3. Immune cells in Prkd2-deficient mice.
    • Fig. S4. Increased number of TFH and GC B cells in Prkd2−/− spleens.
    • Fig. S5. Increased number of TFH in Prkd2−/− lymph nodes.
    • Fig. S6. Prkd2−/− TH2 cells.
    • Fig. S7. TFH, GC B cells, and antibody responses in Prkd2−/− Bcl6fl/flCD4-Cre+ mice.
    • Fig. S8. IL-2 production and IL-2–regulated immune cells in Prkd2−/− mice.
    • Fig. S9. Bcl6 expression in Prkd2−/− B cells.
    • Fig. S10. Effect of B cell–intrinsic Prkd2 deficiency on GC B cell development.
    • Fig. S11. Flow cytometric gating strategy to sort PD-1highCXCR5high and PD-1lowCXCR5low CD4+ T cells.
    • Fig. S12. Frequencies of TFH or GC B cells and IgE response to immunization in Il12a−/− mice.
    • Fig. S13. Enlarged lymph nodes and spontaneous BALT formation in Prkd2−/− mice.
    • Fig. S14. Regulation of TFH-dependent antibody responses by mutual inhibition between Prkd2 and Bcl6.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Raw data file (Excel spreadsheet).

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article