The TRIM37 variants in Mulibrey nanism patients paralyze follicular helper T cell differentiation

Figures

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  Fig. 1. Loss of Trim37 fails to mount protective humoral immunity against the influenza virus. a The pipeline of Burden Test for recurrent infections. b Volcano plot of the genetic burden test results for cases with recurrent infections. Points indicate a certain gene with the corresponding log2 (OR) and –log10 (P value) of the Fisher test results. The threshold of OR > 2 and the threshold of P < 0.05 are marked by two red dashed lines. Genes above the threshold are shown as green dots, while TRIM37 is shown as a red dot. c The schematic design of FINmajor mice. d Sanger sequencing of the WT and FINmajor mice. e Schematic presentation of WT Trim37 and FINmajor mutant (up) and immunoblot analysis of Trim37 levels in spleen samples obtained from the WT and FINmajor mice (down). f The weight loss of WT (n = 4) and FINmajor (n = 4) mice following PR8 influenza (0.5 LD50) infection. g Histopathological analysis was performed by H&E staining 13 days after PR8 influenza (0.5 LD50) infection. h WT (n = 3) and FINmajor (n = 3) mice were infected with PR8 influenza (0.5 LD50), and the sera were obtained 13 days post-infection. Viral-specific anti-HA IgG in the sera was measured by ELISAs. i–l WT…

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  Fig. 2. Trim37 is required for TFH cell differentiation. a–e WT (n = 3) and FINmajor (n = 3) mice were infected intranasally with PR8 H1N1 influenza virus (0.5 LD50). These mice were sacrificed on day 12 after infection. a Representative flow cytometry plots illustrate the frequency of CXCR5+PD1+ TFH cells as a percentage of CD4+Foxp3– T cells in the lung draining lymph nodes, and quantification of CXCR5+PD1+ TFH cells. b Representative flow cytometry plots illustrate the frequency of CXCR5+Bcl6+ TFH cells as a percentage of CD4+Foxp3– T cells in the lung-draining lymph nodes, and quantification of CXCR5+Bcl6+ TFH cells. c Representative flow cytometry plots illustrate the frequency of Fas+PNA+ GC B cells as a percentage of B220+ B cells in the lung-draining lymph nodes, and quantification of GC B cells. d Representative flow cytometry plots illustrate the frequency of B220loCD138hi plasma cells as a percentage of live cells in the lung-draining lymph nodes, and quantification of plasma cells. e Confocal microscopy of the spleen’s germinal center (B220+GL7+). f, g WT (n = 5) and FINmajor (n = 5) mice were vaccinated with 10 µg of PR8 vaccine adjuvanted with alum intraperitoneally.…

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  Fig. 3. Trim37 promotes TFH cell differentiation in a T-cell-intrinsic manner. a Sorting of CXCR5–PD1– non-TFH cells and CXCR5+PD1+ TFH cells from lung-draining lymph nodes and spleens in the C57BL/6 mice infected with PR8 influenza virus. b Cxcr5, Bcl6, and Trim37 mRNA expressions in non-TFH cells and TFH cells were measured by RT-PCR. c Schematic design of the Trim373×Flag mice. d CXCR5–PD1- non-TFH cells and CXCR5+PD1+ TFH cells were sorted from lung-draining lymph nodes and spleens in the Trim373×Flag mice infected with PR8 influenza virus. Flag, Bcl6, and β-actin protein expression was measured by Western blotting. e, f FINmajor bone marrow chimeric mice were infected intranasally with PR8 H1N1 influenza virus (0.5 LD50) and sacrificed on day 12 after infection. Representative flow cytometry plots showing the frequency of CD45.1+ (WT, n = 4) or CD45.2+ (FINmajor, n = 4) CXCR5+ PD1+ TFH cells (e) and CXCR5+ GL7+ GC-TFH cells (f) as a percentage of CD4+ T cells in the spleens. g–j Flow cytometry analysis of lymphocytes from WT (n = 3) and Trim37cko (n = 4) mice at day 10 after infection of PR8 H1N1 influenza virus (0.5 LD50). g, h The frequency and quantification of CXCR5+ PD1+ (g)…

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  Fig. 4. Trim37 binds directly to Bcl6. a, b CXCR5–PD1– non-TFH cells CXCR5+PD1+ TFH cells were sorted from lung-draining lymph nodes and spleens in the WT and Trim37ko mice infected with PR8 influenza virus. a Bcl6 protein expression was measured by Western blot analysis. b Bcl6 mRNA expression was measured by RT-PCR. c The WT and Trim37ko CD4+ T cells were cultured under TFH-like conditions and stimulated with anti-CD3 and anti-ICOS for 4 h. Then, CHX was added to the medium for the indicated hours. Immunoblot analysis of Bcl6 in lysates of CD4+ T cells was performed. Residual Bcl6 protein was normalized to actin and presented relative to that before the addition of CHX (right). d Immunoblot analysis of lysates from the HEK293T cells transfected with HA-tagged Trim37 and Flag-tagged Bcl6 plasmids, followed by immunoprecipitation with anti-Flag affinity gel and immunoblot analysis with anti-HA or anti-Flag antibodies (Abs). e Confocal microscopy of the HEK293T cells transfected with HA-tagged Trim37 and Flag-tagged Bcl6 plasmids. Immunofluorescence was performed with anti-HA (green) and anti-Flag (red) Abs. Nuclei were stained with DAPI (blue). f The schematic design of Bcl6TST mice…

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  Fig. 5. Trim37 targets Bcl6 for K27/29-linked ubiquitination. a Reconstituting the E. coli ubiquitination system by transforming the pACYC vector that expresses HA-tagged Ub, E1, and E2, with or without Trim37WT or Trim37C18R, along with the pET22b-His-Bcl6 vector into BL21 cells. This process was followed by affinity enrichment (Ni-NTA pulldown) of ubiquitinated Bcl6 and immunoblot analysis of Bcl6 expression with anti-His, anti-HA, and anti-Trim37 Abs. b Immunoblot analysis of lysates obtained from the HEK293T cells transfected with Flag-tagged Bcl6, His-tagged ubiquitin, and HA-tagged empty vector, Trim37WT, or Trim37C18R. This process was followed by immunoprecipitation with an anti-Flag affinity gel and analysis with anti-HA, anti-Flag, and anti-His Abs. c Immunoblot analysis of lysates obtained from the HEK293T cells transfected with various combinations of plasmids such as HA-tagged Trim37 and Flag-tagged Bcl6 with His-tagged WT-Ub, K6-Ub, K11-Ub, K27-Ub, K29-Ub, K33-Ub, K48-Ub, K63-Ub, and all K-R mutated Ub. Then, assays were performed as in b. d Immunoblot analysis of Bcl6 in lysates of the HEK293T cells transfected with HA-tagged Bcl6 and increasing doses of His-tagged…

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  Fig. 6. Trim37 ubiquitinates Bcl6 at the K227, K302, K327, K535, and K689 residues. a Reconstituting the E. coli ubiquitination system by transforming the pACYC vector that expresses HA-tagged Ub, E1, and E2, with or without Trim37WT, along with the pET22b-His-Bcl6 vector into BL21 cells. This step was followed by two-step affinity enrichment. The supernatant containing ubiquitinated Bcl6 was analyzed by mass spectrometry. Site mapping for ubiquitination of Bcl6 is shown (right). b Immunoblot analysis of lysates obtained from the HEK293T cells transfected with Flag-tagged Bcl6WT or Bcl65KR and plasmids as indicated. This step was followed by immunoprecipitation with an anti-Flag affinity gel and analysis with anti-HA, anti-Flag, and anti-His Abs. c HEK293T cells were transfected with HA-tagged Bcl6WT or Bcl65KR and Trim37WT. Then, the cells were treated with CHX for the indicated hours. Immunoblot analysis of Bcl6 in lysates was performed. Residual Bcl6 protein was normalized to actin and presented relative to that before the addition of CHX (right). d OT-II Bcl6-deficient CD4+ T cells transduced with RV, Bcl6WT, or Bcl65KR retroviruses, and then transfer to WT mice (RV, n = 4, Bcl6WT, n…

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  Fig. 7. Trim37 promotes the stability of Bcl6 and differentiation of TFH cell dependent on the MATH domain. a Schematic presentation of full-length Trim37 and its mutants. RING, ring-finger domain; Bbox, B-box domain; CC, coiled-coil domain; MATH, meprin and TRAF–C homology domain. b Immunoblot analysis of lysates obtained from the HEK293T cells transfected with Flag-tagged Bcl6 and HA-tagged full-length Trim37 or Trim37 mutants as indicated, followed by immunoprecipitation with anti-Flag affinity gel and immunoblot analysis with anti-HA or anti-Flag Abs. c Immunoblot analysis of lysates obtained from the HEK293T cells transfected with Flag-tagged Bcl6, His-tagged ubiquitin, and the HA-tagged vectors Trim37WT, Trim37△MATH, Trim37G322V. This process was followed by immunoprecipitation with an anti-Flag affinity gel and analysis with anti-HA, anti-Flag, and anti-His Abs. d Immunoblot analysis of Bcl6 in lysates of the HEK293T cells transfected with HA-tagged Bcl6 and empty vector, Trim37WT, Trim37△MATH, and Trim37G322V. Then, the cells were treated with CHX for the indicated hours. Residual Bcl6 protein was normalized to actin and presented relative to that before the addition of CHX…