Research ArticleIMMUNOLOGY

MiR-23~27~24–mediated control of humoral immunity reveals a TOX-driven regulatory circuit in follicular helper T cell differentiation

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Science Advances  11 Dec 2019:
Vol. 5, no. 12, eaaw1715
DOI: 10.1126/sciadv.aaw1715
  • Fig. 1 Members of the miR-23~27~24 family collaboratively limit TFH cell responses.

    (A) Fluorescence-activated cell sorting (FACS) analyses, (B) frequencies and numbers of CXCR5+PD1+TFH cells and PNA+GL7+ GC B cells in the spleen from ~8-week-old T-DKO mice or their WT littermates 8 days after LCMV infection. (C) Immunohistological analyses of GC reactions in LCMV-infected spleen that were cryocut and stained with CD4 (red), GL7 (green), and IgD (blue). (D) Enzyme-linked immunosorbent assay analyses of total serum LCMV-specific IgG levels from LCMV-infected T-DKO mice or WT littermates. OD450 nm, optical density at 450 nm. Frequencies of (E) CXCR5+PD1+TFH cells and (F) PNA+GL7+ GC B cells in the spleen from ~8-week-old 23CTg mice or their WT littermates 8 days after LCMV infection. Frequencies of (G) CXCR5+PD1+TFH cells and (H) PNA+GL7+ GC B cells in the spleen from ~8-week-old 23Tg, 24Tg, or 27Tg mice and their corresponding WT littermates 8 days after LCMV infection. Data are representative of three to four independent experiments. Each symbol represents a mouse, and the bar represents the mean. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 2 Multiple TFH cell–associated genes are targeted by the miR-23~27~24 family.

    (A) HITS-CLIP analysis and (B) sequence alignment of putative miR-23 site in the 3′UTR of c-MAF. (C) Ratios of repressed luciferase activity of cells in the presence of c-MAF 3′UTR with or without mutations in the seed sequences in the presence of miR-23 compared with cells transfected with control miRNA. (D) Percentage of c-MAF geometric mean fluorescence intensity (gMFI) in CXCR5+PD1+TFH cells from T-DKO or 23Tg mice over WT littermates. (E) HITS-CLIP analysis and (F) sequence alignment of the putative miR-27 site in the 3′UTR of ICOS. (G) Ratios of repressed luciferase activity of cells in the presence of ICOS 3′UTR with or without mutations in the seed sequences in the presence of miR-27 compared with cells transfected with control miRNA. (H) Percentage of ICOS gMFI in CXCR5+PD1+TFH cells from T-DKO mice or 27Tg mice over WT littermates. (I) FACS analysis, (J) frequencies, and (K) percentage of IL-21 gMFI in in vitro–activated CD4+ T cells from T-DKO or 24Tg mice over WT littermates. (L) Sequence alignment of the putative miR-24 site in the 3′UTR of IL-21. (M) Ratios of repressed luciferase activity of cells in the presence of IL-21 3′UTR with or without mutations in the seed sequences in the presence of miR-24 compared with cells transfected with control miRNA. (N) FACS analysis and percentage of BCL6 gMFI in CXCR5+PD1+TFH cells from T-DKO or 24Tg mice over WT littermates. Data are representative of three independent experiments. Each symbol represents a mouse or cell sample, and the bar represents the mean. *P < 0.05, **P < 0.01, and ***P < 0.001. nt, nucleotide.

  • Fig. 3 miR-23 and miR-27 jointly repress TOX, a transcription factor that is highly up-regulated by BCL6 in TFH cells.

    (A) RNA-seq analysis of genes that were differentially expressed in Tn1, TH1, TFH, and GC-TFH cells isolated from WT B6 mice 8 days after LCMV infection. (B) Genes that were up-regulated in different T-DKO T cell subsets compared to their corresponding WT counterparts were shown. (C) Venn diagram analysis of genes enriched in TFH and/or GC-TFH cells, further up-regulated in T-DKO TFH and/or GC-TFH cells, containing putative targets of the miR-23~27~24 family by HITS-CLIP and bound by BCL6 in TFH cells. FACS analysis and gMFI of TOX protein amounts in (D) different T cell subsets from WT B6 mice or (E) PSGLloCXCR5+ GC-TFH cells from T-DKO mice or WT littermates. (F) HITS-CLIP analysis and (G) sequence alignment of putative miR-23 and miR-27 sites in the 3′UTR of TOX. Ratios of repressed luciferase activity of cells in the presence of TOX 3′UTR with or without corresponding mutations in the seed sequences in the presence of (H) miR-23 or (I) miR-27 compared with cells transfected with control miRNA. (J) gMFI of TOX protein amounts in PSGLloCXCR5+ GC-TFH cells from 23CTg, 23Tg, 24Tg, or 27Tg mice and their corresponding WT littermates. (K) FACS analysis of TOX protein expression in BCL6-deficient T cells transduced with BCL6-expressing or control retroviral vectors. Cells that expressed different BCL6 amounts (lo, int., or hi) were individually gated. (L) FACS analysis and percentage of TOX gMFI in BCL6 lo, int., and hi populations from BCL6-reexpressing T cells over T cells transduced with control vector were shown. Data are representative of three independent experiments. Each symbol represents a mouse or cell sample, and the bar represents the mean. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 4 BCL6 targets TOX in human TFH cells.

    (A) ChIP-seq analysis of BCL6 (two replicates) or histone H3 acetylated at Lys27 (H3K27ac), monomethylated at Lys4 (H3K4me1), or trimethylated at Lys4 (H3K4me3) with input controls at TOX in human GC-TFH cells presented as reads per million per nucleotide (RPM/bp). Previously identified BCL6-positive bindings were marked. Fold changes of TOX expression in (B) T cells transduced with BCL6-expressing lentiviral vector compared to non-TFH cells or T cells transduced with control vector and in (C) human non-TFH, TFH, or GC-TFH cells, respectively. FACS analysis of (D) CD4+CD45RO T cells and CD4+CD45RO+ T cells from human lymph nodes (LNs). CXCR5hiPD1hi GC-TFH, CXCR5intPD1intTFH, and CXCR5PD1 non-TFH cells (in CD4+CD45RO+ T cells) were gated for (E) FACS analysis, and percentages of TOX gMFI over CD4+CD45RO T cells are shown. (F) CXCR5hiPD1hi GC-TFH cells that expressed different BCL6 amounts (lo, int., or hi) were individually gated. (G) FACS analysis and percentage of TOX gMFI in BCL6 lo, int., and hi populations from CD4+CD45RO+CXCR5hiPD1hi GC-TFH cells over CD4+CD45RO T cells are shown. Data are representative of three independent experiments. Each symbol represents a human donor, and the bar represents the mean. *P < 0.05 and ***P < 0.01.

  • Fig. 5 Modulation of TOX levels in T cells affects TFH cell responses.

    (A) FACS analysis and percentage of TOX gMFI in CD4+ SMARTA T cells electroporated with a Tox guide RNA (gRNA)/Cas9–expressing plasmid over cells transduced with a control plasmid are shown. (B) FACS analysis and frequencies of SLAMloCXCR5+TFH or BCL6+CXCR5+ GC-TFH cells in the B6 recipients transferred with congenically marked GFP+Tox-gRNA+CD4+ SMARTA T cells or control cells 7 days after LCMV infection. (C) FACS analysis and percentage of TOX gMFI in CD4+ SMARTA T cells retrovirally transduced with TOX-expressing vector over cells transduced with control vector are shown. (D) FACS analysis and frequencies of SLAMloCXCR5+TFH cells or BCL6+CXCR5+ GC-TFH cells in the B6 recipients transferred with congenically marked GFP+pTOX+CD4+ SMARTA T cells or control cells 7 days after LCMV infection. Data are representative of two independent experiments. Each symbol represents a mouse, and the bar represents the mean. ***P < 0.001.

  • Fig. 6 TOX promotes the expression of multiple genes crucial for TFH cell differentiation and function.

    (A) Venn diagram analysis of genes that were differentially expressed between TOX-deficient and TOX-sufficient cells in CD8+ T cells and common ILC progenitors. RNA-seq data for CD8+ T cells and common ILC progenitors were derived from GenBank under accession GSE93804 and GSE65850, respectively. KO, knockout. (B) TFH-associated genes that were down-regulated in both TOX-deficient CD8+ T cells and common ILC progenitors (compared to their respective control groups) are shown. (C) qPCR analysis of expression of Tcf7, Lef1, and Pdcd1 in T cells with TOX overexpression. FACS analysis of (D) TCF1, (E) LEF1, and (F) PD1 protein expression in TOX-overexpressing T cells compared to corresponding controls. Cells that expressed different TOX amounts (lo, int., or hi) were individually gated. FACS analysis and percentage of (G) TCF1, (H) LEF1, and (I) PD1 gMFI in TOX lo, int., and hi populations from TOX-overexpressing T cells over control cell populations are shown. Data are representative of three independent experiments. Each symbol represents a mouse, and the bar represents the mean. *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/12/eaaw1715/DC1

    Fig. S1. Mice with T cell–specific ablation of the miR-23~27~24 family exhibited enhanced TFH and GC B cell responses during airway allergic reaction.

    Fig. S2. Elevation in the frequency of TFH cells in the LCMV-specific T cell population upon LCMV infection.

    Fig. S3. Enhanced LCMV-specific Ab responses in mice with T cell–specific ablation of the miR-23~27~24 family.

    Fig. S4. TFH cell–intrinsic role of the miR-23~27~24 family in regulating TFH cell responses.

    Fig. S5. Elevated expressions of the miR-23~27~24 family in GC-TFH cells.

    Fig. S6. Treg cell–specific ablation of the miR-23~27~24 family led to any alteration in TFH and GC B cell responses upon LCMV infection.

    Fig. S7. Exaggerated regulation by the miR-23~27~24 family in T cells led to reduced TFH cell responses.

    Fig. S8. Presence of distinct T cell subsets in mice during LCMV infection.

    Fig. S9. TOX was repressed by miR-23 and miR-27 but not miR-24.

    Fig. S10. TOX knockdown led to impaired TFH cell responses.

    Fig. S11. Modulations of TOX amounts in T cells did not affect T cell homeostasis.

    Fig. S12. Id2, Icos, and Maf are not regulated by TOX in TFH cells.

    Table S1. Gene list III: Genes are significantly up-regulated in both TFH and GC-TFH cells.

    Table S2. Gene list IV: Genes are significantly up-regulated in TFH cells.

    Table S3. Gene list V: Genes are significantly up-regulated in GC-TFH cells.

    Table S4. Gene list I: Genes are significantly up-regulated in T-DKO GC-TFH cells.

    Table S5. Gene list II: Genes are significantly up-regulated in T-DKO TFH cells.

    Table S6. miR-23 targets by HITS-CLIP.

    Table S7. miR-24 targets by HITS-CLIP

    Table S8. miR-27 targets by HITS-CLIP.

    Table S9. Gene list: Common elements in “GSE93804” and “GSE65850.”

    Table S10. Primer list.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. Mice with T cell–specific ablation of the miR-23~27~24 family exhibited enhanced TFH and GC B cell responses during airway allergic reaction.
    • Fig. S2. Elevation in the frequency of TFH cells in the LCMV-specific T cell population upon LCMV infection.
    • Fig. S3. Enhanced LCMV-specific Ab responses in mice with T cell–specific ablation of the miR-23~27~24 family.
    • Fig. S4. TFH cell–intrinsic role of the miR-23~27~24 family in regulating TFH cell responses.
    • Fig. S5. Elevated expressions of the miR-23~27~24 family in GC-TFH cells.
    • Fig. S6. Treg cell–specific ablation of the miR-23~27~24 family led to any alteration in TFH and GC B cell responses upon LCMV infection.
    • Fig. S7. Exaggerated regulation by the miR-23~27~24 family in T cells led to reduced TFH cell responses.
    • Fig. S8. Presence of distinct T cell subsets in mice during LCMV infection.
    • Fig. S9. TOX was repressed by miR-23 and miR-27 but not miR-24.
    • Fig. S10. TOX knockdown led to impaired TFH cell responses.
    • Fig. S11. Modulations of TOX amounts in T cells did not affect T cell homeostasis.
    • Fig. S12. Id2, Icos, and Maf are not regulated by TOX in TFH cells.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Gene list III: Genes are significantly up-regulated in both TFH and GC-TFH cells.
    • Table S2 (Microsoft Excel format). Gene list IV: Genes are significantly up-regulated in TFH cells.
    • Table S3 (Microsoft Excel format). Gene list V: Genes are significantly up-regulated in GC-TFH cells.
    • Table S4 (Microsoft Excel format). Gene list I: Genes are significantly up-regulated in T-DKO GC-TFH cells.
    • Table S5 (Microsoft Excel format). Gene list II: Genes are significantly up-regulated in T-DKO TFH cells.
    • Table S6 (Microsoft Excel format). miR-23 targets by HITS-CLIP.
    • Table S7 (Microsoft Excel format). miR-24 targets by HITS-CLIP.
    • Table S8 (Microsoft Excel format). miR-27 targets by HITS-CLIP.
    • Table S9 (Microsoft Excel format). Gene list: Common elements in “GSE93804” and “GSE65850.”.
    • Table S10 (Microsoft Excel format). Primer list.

    Files in this Data Supplement:

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