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, 533 (7601), 110-4

EBI2 Augments Tfh Cell Fate by Promoting Interaction With IL-2-quenching Dendritic Cells

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EBI2 Augments Tfh Cell Fate by Promoting Interaction With IL-2-quenching Dendritic Cells

Jianhua Li et al. Nature.

Abstract

T follicular helper (Tfh) cells are a subset of T cells carrying the CD4 antigen; they are important in supporting plasma cell and germinal centre responses. The initial induction of Tfh cell properties occurs within the first few days after activation by antigen recognition on dendritic cells, although how dendritic cells promote this cell-fate decision is not fully understood. Moreover, although Tfh cells are uniquely defined by expression of the follicle-homing receptor CXCR5 (refs 1, 2), the guidance receptor promoting the earlier localization of activated T cells at the interface of the B-cell follicle and T zone has been unclear. Here we show that the G-protein-coupled receptor EBI2 (GPR183) and its ligand 7α,25-dihydroxycholesterol mediate positioning of activated CD4 T cells at the interface of the follicle and T zone. In this location they interact with activated dendritic cells and are exposed to Tfh-cell-promoting inducible co-stimulator (ICOS) ligand. Interleukin-2 (IL-2) is a cytokine that has multiple influences on T-cell fate, including negative regulation of Tfh cell differentiation. We demonstrate that activated dendritic cells in the outer T zone further augment Tfh cell differentiation by producing membrane and soluble forms of CD25, the IL-2 receptor α-chain, and quenching T-cell-derived IL-2. Mice lacking EBI2 in T cells or CD25 in dendritic cells have reduced Tfh cells and mount defective T-cell-dependent plasma cell and germinal centre responses. These findings demonstrate that distinct niches within the lymphoid organ T zone support distinct cell fate decisions, and they establish a function for dendritic-cell-derived CD25 in controlling IL-2 availability and T-cell differentiation.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. EBI2 and 7α,25-OHC promote positioning of newly activated CD4 T cells in the outer T zone
(a) Flow cytometric analysis of EBI2 expression on splenic OTII T cells and endogenous B cells in transfer recipients at 0 and 12 h after SRBC-OVA immunization. EBI2 KO cells were used as a staining control. Left histograms show example FACS data and right panel shows summary data across the indicated time points as geometric mean fluorescence intensity (geoMFI). (b) EBI2 expression on OTII and endogenous T cells in transfer recipients 2 days after saline or LPS-OVA immunization. Left histograms show example flow cytometric data and right panel shows summary geoMFI data for 4 mice. (c) Summary geoMFI time course data of EBI2 expression on LN OTII T cells in transfer recipients at the indicated times after alum-OVA immunization. (d) GFP expression in EBI2GFP/+ CD4 T cells that were unstimulated (naïve) or treated with anti-CD3 plus anti-CD28 for 2 days. Left histograms show example flow cytometric data and right panel shows summary geoMFI data for 3 mice. (e) Ebi2 mRNA abundance in cells of the type in d, determined by RT-qPCR and shown relative to the naïve cells. (f) Migration of OTII T cells and endogenous cells to the indicated amounts of 7α,25-OHC in transwell assays. Cells were from unimmunized (0 h) or immunized (day 1, 2) transfer recipient mice in one experiment (left panel) or from 12 hour immunized transfer recipients in a second experiment (right panel). Data are shown as % of input cells of each type that migrated. (g) Immunofluorescence analysis of spleen showing the distribution of co-transferred WT CD45.1+ (red) and EBI2 het or KO (GFP+, green) OTII T cells and endogenous B cells (IgD, blue) at 12 h and 1 day after immunization. (h, i) Immunohistochemical analysis of WT spleens (h) and inguinal LNs (i) showing the distribution of transferred control (WT) or EBI2 deficient (KO) OTII CD45.1+ T cells (blue) and endogenous B cells (IgD, brown) at day 2 after LPS-OVA immunization (h) or day 1 after alum-OVA immunization (i). (j) Immunohistochemical analysis of Cyp7b1, Ch25h or Hsd3b7 control (het, upper panels), or KO (lower panels) spleens showing the distribution of transferred WT OTII T cells (CD45.1, blue) and endogenous B cells (IgD, brown) at day 2 after SRBC-OVA immunization. (k) CCR7 expression on WT and EBI2 KO OTII T cells in transfer recipient spleens at the indicated days after SRBC-OVA immunization. **, p<0.01 by ANOVA (a, c) or student’s t-test (b, d, f). Data are representative of two (a–i, k) or three (j) independent experiments with at least three (a–c, k) or two (d–j) mice per group (error bars (e), s.e.m.).
Extended Data Figure 2
Extended Data Figure 2. Defective differentiation of EBI2-deficient T cells to follicular helpers
(a) ICOS and OX40 expression on WT, EBI2-deficient (KO) OTII and endogenous CD4 T cells in transfer recipient spleens two days after SRBC-OVA immunization. (b, c) In vitro proliferation of WT and EBI2 KO T cells in response to anti-CD3 plus anti-CD28 in the presence of the indicated amounts of 7α,25-OHC, shown as violet tracer dye dilution profiles (b) and total CD4 T cell numbers (c) at day 3 of culture. Numbers in b indicate frequency of cells that have undergone 2 or more divisions. (d) Flow cytometric analysis of co-transferred WT and EBI2 KO OTII T cells for CXCR5 and intracellular Bcl6 expression at the indicated days following SRBC-OVA immunization. Numbers indicate frequency of cells in gated region. (e) Summary of data of the type in d. Upper plot shows frequency and lower plot number of CXCR5+Bcl6hi OTII T cells. (f) Il21 mRNA abundance in CXCR5+ PD-1hi control (Het) or EBI2 KO OTII T cells sorted from recipient spleens at day 3 after immunization with SRBC-OVA, determined by RT-QPCR and shown relative to the Het control. (f) Frequency and number of CXCR5+ PD-1hi (left panels) or CXCR5+ Bcl6hi (right panels) WT and EBI2 KO OTII T cells in mice that received the cells as separate transfers, at day 3 following SRBC-OVA immunization. **, p<0.01 by ANOVA (e) or student’s t-test (f, g). Data are representative of three (a, d, e, g) or two (b, c, f) independent experiments with at least three mice per group (error bars (f), s.e.m.).
Extended Data Figure 3
Extended Data Figure 3. EBI2-deficient T cells support reduced plasma cell and GC response
(a) Frequency and number of CXCR5+PD-1hi CD4+ control (WT) and EBI2 KO OTII T cells in spleens of day 3 Listeria-OVA immunized transfer recipients. (b) PD-1 and CXCR5 flow cytometric analysis of control (Het) and EBI2 KO polyclonal CD4 T cells co-transferred to OTII recipients, 8 days after immunization with unconjugated SRBCs. (c) Summary of data of the type in b shown as CXCR5+PD-1hi cell frequency and number. (d) Flow cytometric analysis for the GC markers FAS and GL7 on endogenous B cells in OTII TCR transgenic mice that received no cells, control (HET) CD4 T cells or EBI2 KO CD4 T cells, or in WT B6 control mice, 12 days after immunization with unconjugated SRBCs. (e) Summary of data from d shown as number of FAS+GL7+ GC B cells. (f) Flow cytometric analysis for CD138hi B220lo plasma cells (upper plots) and intracellular IgM and IgG1 staining of these cells (lower plots) in mice of the type in d. (g) Summary of data from f shown as number of cells. (h) Serum anti-SRBC antibody levels in mice of the type in d, determined by flow cytometric analysis of SRBCs stained with immune sera, plotted as geoMFI. (i, j) Number of Fas+GL7+ GC cells and CD138hiB220int plasma cells in Listeria-OVA immunized CD28 KO mice that had received control (het) or EBI2 KO OTII cells, analyzed at day 5. **, p<0.01 by ANOVA (g, h) or student’s t-test (a, e, c, i, j). Data are representative of two independent experiments with at least three mice per group.
Extended Data Figure 4
Extended Data Figure 4. T cell EBI2 is required for CD4+ DC-mediated augmentation of Tfh cell induction
(a) Frequency and number of CXCR5+PD-1hi OTII T cells in μMT recipients determined by flow cytometric analysis. (b) Flow cytometric analysis for CD11c and MHC class II on splenocytes from Zbtb46-DTR mice treated with saline or DT for one day. Graph shows summary data for DC number in 4 mice of each type. (c) Frequency and number CXCR5+PD-1hi WT and EBI2 KO OTII T cells in spleens from Zbtb46-DTR BM chimeras treated with saline or diphtheria toxin (DT), at day 3 after immunization with SRBC-OVA. (d) Immunohistochemical analysis of spleen sections from WT mice without immunization (saline) or SRBC immunized for the indicated times, stained to detect IgD+ B cells (blue) and DCIR2+ DCs (brown). (e) Immunohistochemical analysis of spleen sections from recipients of WT or EBI2 KO OTII T cells at 12 h and 1 day after immunization SRBC-OVA immunization, stained for OTII CD45.1+ T cells (blue) and DCIR2+ DCs (brown). (f) Immunohistochemical analysis of spleen sections from WT:Zbtb46-DTR or CCR7 KO:Zbtb46-DTR mixed BM chimeras treated with DT, at day 2 after immunization. (g) Frequency and number of CXCR5+PD-1hi WT and EBI2 KO OTII T cells in spleens from WT:Zbtb46-DTR (control) or CCR7 KO:Zbtb46-DTR mixed BM chimeras treated with DT, at day 3 after immunization. (h) Frequency and number of CXCR5+PD-1hi control (het) and EBI2 KO co-transferred OTII T cells in spleens of CD47 KO recipients at day 3 after SRBC-OVA immunization. (i) Number of total and CD4+ DC in spleens from Irf4f/f CD11c-Cre or + mice. (j) As for g but in Irf4f/f CD11c-Creor + recipient mice. (k) As for g but in Batf3 KO recipient mice. (l) ICOSL surface levels for DCs from mice immunized 12 h earlier with saline or SRBCs and also treated with control or ICOS blocking antibody. (m) ICOSL surface levels for CD4+ or CD8+ DCs from CD28 KO mice immunized 12 h earlier with saline or SRBCs. (n) Il6 and Tgfβ mRNA abundance in sorted CD4+ and CD8+ splenic DCs from mice treated with saline or SRBC six hours earlier, determined by RT-QPCR, shown relative to the control CD8+ DC. **, p<0.01 by ANOVA (g, k) or student’s t-test (a–c, i, j, m). Data are representative of three (a–e) or two (f–n) independent experiments with at least three (a–c, g–n) or two (d–f) mice per group (error bars (n), s.e.m.).
Extended Data Figure 5
Extended Data Figure 5. DCs produce membrane and soluble CD25 and inhibit IL2R signaling
(a) Heat map of RNAseq data from sorted CD4+ splenic DC showing the top 15 most induced genes at 1 h following SRBC versus saline immunization. (b) CD25 surface levels in CD11b+ migratory and resident DCs from LNs of mice immunized with saline or alum-OVA 2 days earlier. Graph on right shows summary data for total number of migratory CD25+ DCs. (c) Immunohistochemical analysis of spleen sections from TCRβδ KO mice immunized one day earlier with saline or SRBC, stained to detect IgD (blue) and CD25 (brown). (d, e) CD122 (Il2rb) mRNA determined by RT-QPCR (d) and surface staining (e) on the indicated cell types isolated from spleens of WT OTII T cell recipients at day 0, 1 and 2 after SRBC-OVA immunization. Transcript data are plotted relative to the signal in CD4+ DCs at day 0. (f) Intracellular flow cytometric analysis of pSTAT5 in CD4+ DCs or, as a positive control CD4+ T cells, that were untreated or incubated with IL2 (200 pg/ml) or, as a further positive control, GM-CSF (100 pg/ml). (g) Il2 mRNA in control (Het) and EBI2 KO OTII T cells isolated from recipient mice at the indicated times after SRBC-OVA immunization. (h) Intracellular flow cytometry for IL2 in cells of the type in f at 0, 12 and 24 h. Percentages show mean (±SEM) for 3 mice at each time point. (i) Flow cytometric analysis of CD25 expression on co-transferred WT and EBI2 KO OTII T cells in WT recipients at the indicated days after SRBC-OVA immunization. (j) Prdm1 (encoding Blimp1) transcript levels in sorted CXCR5+PD-1hi control (het) and EBI2 KO OTII T cells from SRBC-OVA immunized mice at day 3, plotted relative to the mean level in the Het group. (k) Summary of pSTAT5 staining data for OTII T cells from mice immunized one day earlier with SRBC-OVA, incubated with the indicated amounts of 7α,25-OHC plus IL2 (200 pg/ml) for one hour. (l) Flow cytometry of pSTAT5 in CD25+ (regulatory) T cells exposed to the indicated amounts of IL2 that had been pre-mixed with supernatants (s/n) from 8 h cultures of splenic CD4+ DCs from WT or CD25 KO mice immunized with saline or SRBCs 1 day before. Graph on right shows summary data from one experiment. **, p<0.01 by ANOVA (b, l) or student’s t-test (j). Data are representative of one (a) or two (b–l) independent experiments with at least two (a) or three (b–l) mice per group (error bars (g, j), s.e.m.).
Extended Data Figure 6
Extended Data Figure 6. DC CD25 expression reduces IL2 signaling in activated CD4 T cells, favoring their differentiation to follicular helpers
(a) Diagram of CD25 KO:Zbtb46-DTR BM chimera generation and time line of experiment. DTx, DT treatment. (b–d) Numbers (b), surface marker expression (c) and outer T zone positioning (d) of CD4+ DCIR2+ DCs in WT:Zbtb46-DTR and CD25 KO:Zbtb46-DTR mixed BM chimeras pretreated with DT, at day 1 after saline or SRBC immunization. (e) Number of Foxp3+ CD25+ regulatory T cells in mice of the type in k except that the mice were immunized for three days. (f) Immunohistochemical analysis of spleen sections from mice of the type in a, stained to detect IgD (blue) and CD25 (brown). (g, h) Frequency and number of CXCR5+PD-1hi control (EBI2 Het) and EBI2 KO OTII T cells in spleens (g) or LNs (h) from WT:Zbtb46-DTR or CD25 KO:Zbtb46-DTR mixed BM chimeras pretreated with saline or DT, at day 3 after immunization with Listeria-OVA (g) or alum-OVA (h). (i, j) Flow cytometric analysis for HEL-binding CD138+ plasma cells (i) and HEL-binding GL7+ Fas+ GC B cells (j) in spleens from WT:Zbtb46-DTR (control) or CD25 KO:Zbtb46-DTR mixed BM chimeras that had received Hy10 B cells and been treated with DT, at day 5 after immunization with HEL-SRBC. (k) Soluble CD25 detected by ELISA in spleen extracts taken from 12 h SRBC immunized mice, at day 1 after saline or recombinant CD25 treatment. **, p<0.01 by student’s t-test (h, k). Data are representative of two independent experiments with at least three (b, c, g–k) or two (d, f) mice per group (error bars (k), s.e.m.).
Extended Data Figure 7
Extended Data Figure 7. Model of how EBI2-dependent positioning of activated T cells in association with CD25+ DCs in the outer T zone favors Tfh cell differentiation
Initially, cognate T cells throughout the T zone are activated by antigen recognition and promptly start upregulating EBI2 and making IL2. EBI2 guides cells to the 7α,25-OHC high outer T zone and in this location they interact with activated DCs producing membrane and shed CD25 that binds and quenches IL2. This limits IL2R signaling on the T cell via pSTAT5 and allows induction of Bcl6 by other inputs such as ICOSL. T cells that lack EBI2 or remain in the inner T zone for other reasons are exposed to autocrine IL2 and this induces Blimp1, a repressor of Bcl6, disfavoring the Tfh cell fate.
Figure 1
Figure 1. EBI2 promotes positioning of newly activated CD4 T cells in the outer T zone
(a) Immunohistochemical analysis of spleens for transferred WT or EBI2 KO OTII CD45.1+ T cells (blue) and endogenous B cells (IgD, brown) at 12 h, 1 day and 2 days after SRBC-OVA immunization. (b) Fraction of WT and EBI2 het or KO OTII T cells in the outer ¼ of the splenic T zone at 12 h and 1 day after SRBC-OVA. Sections were stained as in Extended Data Fig. 1g. See Methods for details. (c, d) As for a except mice were immunized with Listeria-OVA (c) or alum-OVA and inguinal LNs were analyzed (d). **, p<0.01 by student’s t-test. Data are representative of three (a, b) or two (c–e) experiments with at least three (a) or two (b–e) mice per group.
Figure 2
Figure 2. Defective differentiation of EBI2-deficient T cells to follicular helpers
(a) CD69 expression on WT OTII, EBI2 KO OTII and endogenous CD4 T cells in transfer recipient spleens 2 days after SRBC-OVA immunization. Histograms show representative FACS and graphs show summary geoMFI data for 3 mice of each type. (b) Proliferation of co-transferred WT and EBI2 KO OTII T cells monitored by violet tracer dye dilution at days 2 and 3 after immunization. Numbers indicate mean % (±SD) of cells in the indicated gate (n=6). (c) Summary of data from b shown as a ratio of KO/WT OTII T cell number at the indicated days. (d) Flow cytometric analysis of co-transferred WT and EBI2 KO OTII T cells for PD-1 and CXCR5 at days 2 and 3 following immunization. Numbers indicate frequency of cells in gated region. (e) Summary of data of the type in d. Upper plot shows frequency and lower plot number of CXCR5+PD-1hi CD4+ OTII T cells. (f) Frequency and number of CXCR5+PD-1hi CD4+ OTII T cells in peripheral LNs of mice of the type in d, immunized with alum-OVA. *, p<0.05 and **, p<0.01 by ANOVA (d, f) or student’s t-test (g). Data are representative of three (a–f) or two (g) experiments with at least three mice per group.
Figure 3
Figure 3. T cell EBI2 is required for CD4+ DC-mediated augmentation of Tfh cell induction
(a) Frequency and number of CXCR5+PD-1hi WT and EBI2 KO OTII T cells in control or MD4 recipient spleens at day 3 after immunization with SRBC-OVA. (b) Immunohistochemical analysis of consecutive sections from WT transfer recipient spleens at day 2 after immunization, stained for : left panels, WT or EBI2 KO OTII CD45.1+ T cells (blue) and B cells (IgD, brown); center panels, DCIR2+ DCs (brown) and B cells (IgD, blue); right panels, OTII CD45.1+ T cells (blue) and DCIR2+ DCs (brown) (c) Frequency of WT or EBI2 KO OTII T cells contacting DCIR2+ DCs determined in sections of the type in b and Extended Data Fig. 4e, at the indicated times after immunization. (d) Icosl mRNA abundance in splenic CD4+ and CD8+ CD11c+ DCs from mice immunized 12 h earlier with saline or SRBCs, shown relative to the saline control. (e) Summary data of ICOSL surface levels for DCs of the type in d from mice also treated with control or ICOS blocking antibody. (f) ICOSL surface levels on CD4+ splenic DCs from mice that had received WT or EBI2 KO OTII T cells, 12 h after immunization with SRBC-OVA. Recipient mice were CD28 KO. See Supplementary Information for details. (g) Frequency and number CXCR5+PD-1hi WT and EBI2 KO OTII T cells in spleens from mice treated with ICOS blocking antibody, analyzed at day 3 after immunization. *, p<0.05 and **, p<0.01 by ANOVA (a–d, e) or student’s t-test (f, g). Data are representative of three (a–c) or two (d–g) experiments with at least three (a, c–g) or two (b) mice per group (error bars (d), s.e.m.).
Figure 4
Figure 4. DC CD25 expression reduces IL2 signaling in activated CD4 T cells, favoring their differentiation to follicular helpers
(a, b) CD25 transcript (a) and surface (b) levels on CD4+ and CD8+ splenic DCs from mice immunized with saline or with SRBCs 12 h, 1 or 2 days earlier. Transcript levels are plotted relative to the day 0 mean for each DC type. (c) Immunohistochemical analysis of spleens from WT mice immunized with saline or SRBCs, stained to detect IgD (blue) and CD25 (brown). Inset shows 12 h SRBC immunized CD25 KO. (d) Flow cytometry of pSTAT5 in WT, EBI2 Het or EBI2 KO OTII T cells in mice that received mixtures of CD45.2 and CD45.1/2 marked cells, stained ex vivo 1 day after SRBC-OVA immunization. Left panels show example histogram plots of gated OTII T cells and right graph shows summary geoMFI data for 3 mice of each type in one experiment, including mice injected with IL2 as a positive control. Orange histogram indicates endogenous CD4+ T cells. (e) Summary of pSTAT5 levels in control (Het) and EBI2 KO OTII T cells in LNs at day 3 after alum-OVA immunization. (f) Soluble CD25 detected by ELISA in culture supernatants of splenic CD4+ DCs from WT, TCR KO or CD25 KO mice immunized 1 day earlier with saline or SRBCs, or medium alone. (g, h) Soluble CD25 detected by ELISA in spleen extracts taken from WT, CD25 KO, TCR KO, or Zbtb46-DTR treated with DT, immunized as indicated (g) or 12 h (h) prior to analysis. (i) Frequency and number of CXCR5+PD-1hi control (EBI2 Het) and EBI2 KO OTII T cells in spleens from WT:Zbtb46-DTR or CD25 KO:Zbtb46-DTR mixed BM chimeras pretreated with saline or DT, at day 3 after SRBC-OVA. (j) HEL-binding plasma cell and GC B cell numbers in spleens from WT:Zbtb46-DTR (control) or CD25 KO:Zbtb46-DTR chimeras treated with saline or DT and transferred with Hy10 B cells, at day 5 after immunization with HEL-SRBC. (k) Serum IgG1 and IgG2b anti-HEL antibody in mice of the type in j analyzed by FACS of HEL-conjugated mouse RBCs. (l, m) Summary geoMFI of pSTAT5 levels (l) and CXCR5+PD-1hi cell frequencies and numbers of control (Het) and EBI2 KO OTII T cells in WT mice at day 1 (l) and day 3 (m) after SRBC-OVA immunization and treatment with saline or recombinant CD25 (rCD25). TKO, TCRβδ KO. *, p<0.05 and **, p<0.01 by ANOVA (a, b, f–h, k) or student’s t-test (d, e, i, j, l). Data are representative of three (a, b, d) or two (c, e–m) experiments with at least three (a, b, d–f, h–m) or two (c, g) mice per group (error bars (a, f–h), s.e.m.).

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