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A p85α-osteopontin axis couples the receptor ICOS to sustained Bcl-6 expression by follicular helper and regulatory T cells

Nature Immunology volume 16, pages 96–106 (2015)Cite this article

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Abstract

Follicular helper T cells (TFH cells) and follicular regulatory T cells (TFR cells) regulate the quantity and quality of humoral immunity. Although both cell types express the costimulatory receptor ICOS and require the transcription factor Bcl-6 for their differentiation, the ICOS-dependent pathways that coordinate their responses are not well understood. Here we report that activation of ICOS in CD4+ T cells promoted interaction of the p85α regulatory subunit of the signaling kinase PI(3)K and intracellular osteopontin (OPN-i), followed by translocation of OPN-i to the nucleus, its interaction with Bcl-6 and protection of Bcl-6 from ubiquitin-dependent proteasome degradation. Post-translational protection of Bcl-6 by OPN-i was essential for sustained responses of TFH cells and TFR cells and regulation of the germinal center B cell response to antigen. Thus, the p85α–OPN-i axis represents a molecular bridge that couples activation of ICOS to Bcl-6-dependent functional differentiation of TFH cells and TFR cells; this suggests new therapeutic avenues to manipulate the responses of these cells.

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Figure 1: OPN regulates the differentiation of TFH cells and TFR cells.
Figure 2: The OPN-i-deficient phenotype of TFH and TFR cells is cell intrinsic.
Figure 3: OPN-i deficiency impairs Bcl-6 protein expression.
Figure 4: Costimulation via ICOS promotes interaction between OPN-i and p85α.
Figure 5: p85α is required for the differentiation of TFH and TFR cells.
Figure 6: p85α chaperones the translocation of OPN-i to the nucleus.
Figure 7: Intranuclear OPN-i interacts with and stabilizes Bcl-6.
Figure 8: The p85α–OPN-i interaction regulates the responses of TFH and TFR cells in vivo.

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Acknowledgements

We thank H. von Boehmer (Dana-Farber Cancer Institute) for B6.Foxp3GFP mice; L. Cantley (Weill Cornell Medical College) for p85α and Flag-p85α plasmids; T. Dawson (Johns Hopkins University) for the HA-Ub plasmid; S. Yamanaka (Kyoto University) for the HA-p110δ plasmid; J. Zhao (Dana-Farber Cancer Institute) for the HA-p110α plasmid; K. Wucherpfennig for critical reading; L. Cameron for help with image preparation and analysis; Y. Shao for microarray analysis; and A. Angel for preparation of the manuscript and figures. Supported by the US National Institutes of Health (AI48125 to H.C.; and T32 CA070083 to J.W.L.), The LeRoy Schecter Research Foundation (H.C.), the Benacerraf Society (J.W.L.) and the Belgian-American Educational Foundation (B.V.).

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Authors and Affiliations

  1. Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Jianmei W Leavenworth, Bert Verbinnen, Jie Yin, Huicong Huang & Harvey Cantor

  2. Department of Microbiology & Immunobiology, Division of Immunology, Harvard Medical School, Boston, Massachusetts, USA

    Jianmei W Leavenworth, Bert Verbinnen & Harvey Cantor

  3. Department of Cell Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China

    Jie Yin

  4. Department of Parasitology, Wenzhou Medical College, Wenzhou, China

    Huicong Huang

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Contributions

J.W.L. and H.C. conceived of and planned experiments, analyzed data and wrote the paper; B.V. made the OPN-i-KI genomic construct; and J.W.L., J.Y. and H.H. performed experiments.

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Correspondence to Harvey Cantor.

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Integrated supplementary information

Supplementary Figure 1 Sorting and gating strategy.

Upper left, FACS plots show isolation of different CD4+ TH populations from B6 or OPN-i-KI mice after immunization with KLH in CFA. TN: CD4+CD44loCXCR5loPD-1loGITR− naïve cells; TFH: CD4+CD44hiCXCR5+PD-1+GITR− cells; TFR: CD4+CD44hiCXCR5+PD-1+GITR+ cells; Non-TFH: CD4+CD44hiCXCR5loPD-1loGITR− cells; Treg: CD4+CD44medCXCR5−PD-1−GITR+ cells. Bottom left, FACS plots show isolation of CD4+ TFH or TFR cells from OPN-i-KI or OPN-KO mice 5 d after immunization with KLH in CFA, shown in Fig. 2e. Gating control stains that lack (–) either anti-PD-1 or biotin-anti-CXCR5 using OPN-i-KI cells are shown. Upper right, gating strategy for TFH (CD4+CD44hiCXCR5+PD-1+Foxp3−), TFR (CD4+CD44hiCXCR5+PD-1+Foxp3+) and non-TFH (CD4+CD44hiCXCR5loPD-1lo) cells in Fig. 3a. Bottom right, Gating controls for defining the PD-1+CXCR5+ surface phenotype of CD4+ TFH and TFR cells in Fig. 3c. Negative control: CD4+CD44lo cells; Positive control (for CXCR5 stains): Fas+B220+ B cells; FMO PD-1 control: all antibodies except (–) anti-PD-1; CXCR5 control: all antibodies except (–) biotin-anti-CXCR5 (streptavidin-APC alone).

Supplementary Figure 2 Generation and confirmation of OPN-i-KI mice.

a, Spp1 genomic locus and targeting strategy. Boxes represent exons; exon 2 (gray) indicates the mutation site with deletion of the 45 nucleotides after the translational start site (ATG) that encode an N-terminal signal sequence while sparing other endogenous elements. A transcriptional STOP element flanked by loxP sites (black triangles) was inserted upstream of this mutation site to prevent OPN-i expression. Germline transmitted Spp1flstop/+ mice were backcrossed to B6 mice for at least 5 generations before crossing with mice carrying the Cre recombinase to allow OPN-i expression. neor, neomycin-resistance gene. b, PCR of genomic DNA showing wild-type, OPN-i-KI and OPN-KO mice after crossing with EIIa-Cre mice using genotyping primers indicated as gray triangles in a. OPN-KO mice gained the STOP element (194 bp) compared to wild-type allele. wild-type: 324 bp, OPN-i-KI (after Cre recombination): 453 bp, OPN-KO: 518 bp. c, Secreted OPN protein measured by ELISA from supernatants of purified DC, NK, T cells and peritoneal macrophages from each mouse strain after stimulation with the indicated reagents for 24 h or 2 d for macrophages. d, Immunoblot analysis of splenocyte lysates from the indicated mouse strains, probed with anti-OPN and anti-actin. Right, quantification of ratio of OPN to actin (n = 5 mice per group). e, Secreted IFN-α protein in pDC after stimulation by CpG-B (ODN-1668) (n = 3 mice per group) (***P < 0.001; error bars, mean ± s.e.m).

Supplementary Figure 3 OPN-i deficiency does not affect B cell activity or other helper T cell differentiation.

a, Quantification of CD44 expression (MFI) by CD4+ T cells, percent CD4+ T cells and Foxp3+CD44+CD4+ Treg cells from OT-II, OT-II OPN-KO and OT-II OPN-i-KI mice (as in Fig. 1b) 7 d post-challenge. Data represent at least three independent experiments with 6 mice per group (error bars, mean and ± s.e.m). b, Titer of total (NP23) and high-affinity (NP4) NP-specific IgG in the serum of Rag2−/− Prf1−/− hosts transferred with OT-II CD4+ T cells from OPN wild-type or OPN-KO mice and OPN wild-type or OPN-KO B cells followed by immunization with NP13-OVA in CFA and analysis 10 d later. Data represent two independent experiments with 4 mice per group. c, Frequency and numbers of donor CD45.2+Vβ5+CD4+ T cells and surface expression of CD44 by these cells from spleens of CD45.1 congenic recipients 7 d post-immunization with OVA in CFA. d, Flow cytometry of donor Vβ5+CD4+ T cells in c. Numbers adjacent to outlined areas indicate percent Bcl-6+CXCR5+ TFH cells and Vβ5+CD4+ T cells expressing intracellular cytokines. Below, frequency of TFH cells and cytokine-producing cells (n = 6 mice per group). *P < 0.05 (unpaired two-tailed Student’s t-test); NS, not significant. Error bars indicate mean ± s.e.m. e, Cytokine production by naïve CD44loCD25−CD4+ T cells purified from the indicated OT-II mice and differentiated for 5 d under TH1, TH2, TH17 and TFH conditions. *P < 0.05 (error bars, mean ± s.e.m of triplicate wells).

Supplementary Figure 4 Effects of OPN-i deficiency on Bcl-6 expression and the differentiation of inducible Treg cells and TFR cells.

a, Flow cytometry of CD25+Foxp3+ iTreg differentiated from sorted naïve CD25−CD4+ T cells, stimulated with plate-bound anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) in the presence of TGF-β1 (5 ng/ml) and hIL-2 (100 U/ml) for 5 d. b, iTregs from (a) were co-cultured with CFSE-labeled naïve CD25−CD4+ T cells (responder) activated with anti-CD3 and irradiated APC at different ratios. Histograms of CFSE dilutions, analyzed by flow cytometry, as readout of responder proliferation. Serum titers of total (NP23) and high-affinity (NP4) IgG (c) and anti-KLH (d) IgG from recipients in Fig. 2c (n = 5 mice per group). *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired two-tailed Student’s t-test; error bars, mean ± s.e.m). e, Immunoblot analysis of enriched CD44+CD4+ T cells from the indicated mice at days 1-15 after immunization with KLH in CFA, probed with the indicated Abs. Below, ratio of Bcl-6 to actin. f, RT-PCR analysis of Bcl6 and Prdm1 mRNA in CD44+CD4+ T cells purified from OPN-i-KI or OPN-KO mice from e. Bcl6 or Prdm1 expression was normalized to the Rps18 control and results are presented relative to that of OPN-i-KI mice at d1, set as 1. Data are representative of two independent experiments (e) or one experiment with 3 mice per time point (f; error bars, mean ± s.e.m).

Supplementary Figure 5 Microarray analysis of genes upregulated in CD4+ T cells by costimulation with ICOS.

a, Multiplot of genes upregulated in CD4+ T cells after restimulation with anti-CD3 and anti-ICOS (duplicates) compared to anti-CD3 alone (quadruplicates) as described in Fig. 4a. 210 (red) genes upregulated and 9 (blue) genes downregulated after co-ligation of CD3 and ICOS (cut-off 1.5 fold and P < 0.01). b, Functional analysis performed by Ingenuity pathway analysis (IPA) of 210 genes upregulated by ICOS co-stimulation in a. Functional annotations that are related to T-cell activation, differentiation, antibody production and antibody-mediated autoimmune disease with P values and numbers of genes are listed. c, Heatmap analysis displays 31 genes upregulated in ICOS-activated CD4+ T cells that correlate with systemic autoimmune syndrome revealed by IPA in b (P = 2.65 × 10-11).

Supplementary Figure 6 OPN-i does not interact with p110, and p85α deficiency does not impair other helper T cell differentiation in vivo.

Immunoassay of lysates of 293T cells transfected with plasmids expressing HA-p110α (a) or HA-p110δ (b) and increasing concentrations of OPN-i, assessed by immunoprecipitation with anti-HA and immunoblot analysis with the indicated Abs. c, Purified CD44+CD4+ T cells from OPN-i-KI or OPN-KO mice 3 d after immunization with KLH and CFA were treated as in Fig. 4a. Quantification of ratios of phospho-Akt (pAkt) to total Akt by ELISA from cells after 30 min of crosslinking. d, Flow cytometry of splenocytes of OT-II OPN-i-KI or OT-II OPN-KO mice 3 d post-immunization with NP13-OVA in CFA, stimulated with (+) or without (–) IL-6 (20 ng/ml) for 15 min. Overlay of histograms of intracellular phospho-STAT1 and phospho-STAT3 among CD4+CD44+ T cells. e, Flow cytometry of splenocytes from p85α wild-type and p85α KO mice 3 d after injection with KLH and CFA. Numbers indicate percent Foxp3−Bcl-6+CXCR5+ TFH cells and Foxp3+Bcl-6+CXCR5+ TFR cells. Right, Bcl-6 MFI (n = 4 mice per group). **P < 0.01 (unpaired two-tailed Student’s t-test; error bars, mean ± s.e.m). Data represent two independent experiments. f, Quantification of numbers and surface CD44 expression of CD45.1−CD4+ donor cells from Fig. 5a. g, Gating controls for defining Bcl-6+CXCR5+ CD4+ TFH or TFR cells in Fig. 5a,b,d. CXCR5 control: all antibodies except (–) biotin-anti-CXCR5 (streptavidin-APC alone); Bcl-6 control: all antibodies except anti-Bcl-6; in this case, an IgG isotype-matched control for anti-Bcl-6 was used; Negative control: splenic CD44loCD4+ T cells from B6 mice at day 8 post-injection with KLH in CFA; Positive control: splenic CD44hiCD25medCD4+ T cells from B6 mice at d8 post-immunization with KLH in CFA; or Bcl-6+CXCR5+ cells in CD19+ B cells from Tcrα−/– recipients of p85α KO Treg in Fig. 5b. h, Flow cytometry of donor CD45.1−CD4+ T cells from Fig. 5a, stimulated with PMA and Ionomycin for 5 h. Numbers indicate percent CD4+ T cells expressing intracellular cytokines. Right, frequency of cytokine-producing CD4+ T cells. Data represent two independent experiments with 3-4 mice per group (error bars, mean ± s.e.m). i, Immunoassay of lysates of 293T cells transfected with vectors expressing Flag-p85α and OPN-i, treated with calf intestinal phosphatase (CIP), and assessed by immunoprecipitation with anti-Flag followed by immunoblot analysis. j, Diagram of a short sequence motif of OPN with a tyrosine at position 166 that may interact with the p85α SH2 domain. k, Expression of surface ICOS receptor and intracellular Bcl-6 in CD44+CD4+ T cells from OPN-i-KI mice 3 d after immunization with KLH in CFA.

Supplementary Figure 7 Wild-type OPN-i interacts with and stabilizes Bcl-6, but the Y166F OPN-i mutant does not.

a, Confocal microscopy of 293T cells transfected with plasmids encoding p85α, Flag–Bcl-6 and OPN-i–GFP wild-type or OPN-i–GFP Y166F mutant, assessed by pre-extraction of soluble nuclear proteins with 0.5% Triton X-100 after 24 h of transfection followed by immunostaining as indicated. Yellow in the merged image shows colocalization of Bcl-6 and OPN-i wild-type. b, Immunoassay of nuclear and cytosolic fractions of 293T cells transfected with plasmids encoding Flag–Bcl-6, OPN-i wild-type or OPN-i Y166F mutant, assessed by immunoprecipitation (IP) with anti-Flag and then immunoblot analysis. c, Top, Illustration of Bcl-6 protein deletion mutants. Immunoassay of lysates of 293T cells transfected with plasmids encoding OPN-i and Flag–Bcl-6 wild-type or deletion mutants, assessed by IP and immunoblot analysis as in b. Right, immunoassay of lysates of 293T cells transfected with plasmids encoding OPN-i–Flag and Bcl-6 ZF deletion mutant (no Flag tag) followed by IP with anti-Flag and immunoblot with anti-Bcl-6 and anti-Flag. Arrowhead: IgG heavy chain. d, Immunoblot analysis of lysates of 293T cells transfected with plasmids encoding OPN-i (100 ng) and graded concentrations of Flag–Bcl-6 wild-type or deletion mutants (lane 1,4,7,10: 450 ng; 2,5,8,11: 300 ng; 3,6,9,12: 150 ng). Cell lysates from lanes 1,4,7,10 were used for immunoassay in Fig. 7b. e, Immunoblot analysis of lysates of 293T cells transfected with plasmids encoding Flag–Bcl-6 and/or OPN-i, treated with (+) or without (–) DUBi for 8 h, probed with anti-Flag and anti-actin. Below lanes, ratio of Flag (Bcl-6) to actin.

Supplementary Figure 8 Schematic of sustenance of Bcl-6-dependent follicular T cell differentiation mediated by the p85α–OPN-i axis.

Engagement of ICOS and TCR on CD4+ T cells by APC (e.g., DC) promotes p85α–OPN-i complex formation that requires the tyrosine site 166 of OPN-i. p85α chaperones OPN-i entry into the nucleus, where intranuclear OPN-i interacts with Bcl-6 via the sequences within the RD2 and protects Bcl-6 from ubiquitination-mediated degradation. This p85α–OPN-i axis connects ICOS signals to stable Bcl-6 expression (highlighted in blue) and ensures functional follicular T cell differentiation program.

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Leavenworth, J., Verbinnen, B., Yin, J. et al. A p85α-osteopontin axis couples the receptor ICOS to sustained Bcl-6 expression by follicular helper and regulatory T cells. Nat Immunol 16, 96–106 (2015). https://doi.org/10.1038/ni.3050

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