Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis

Abstract

Regulatory T cells (Treg cells) have a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis. However, they also represent a major barrier to effective anti-tumour immunity and sterilizing immunity to chronic viral infections. The transcription factor Foxp3 has a major role in the development and programming of Treg cells. The relative stability of Treg cells at inflammatory disease sites has been a highly contentious subject. There is considerable interest in identifying pathways that control the stability of Treg cells as many immune-mediated diseases are characterized by either exacerbated or limited Treg-cell function. Here we show that the immune-cell-expressed ligand semaphorin-4a (Sema4a) and the Treg-cell-expressed receptor neuropilin-1 (Nrp1) interact both in vitro, to potentiate Treg-cell function and survival, and in vivo, at inflammatory sites. Using mice with a Treg-cell-restricted deletion of Nrp1, we show that Nrp1 is dispensable for suppression of autoimmunity and maintenance of immune homeostasis, but is required by Treg cells to limit anti-tumour immune responses and to cure established inflammatory colitis. Sema4a ligation of Nrp1 restrained Akt phosphorylation cellularly and at the immunologic synapse by phosphatase and tensin homologue (PTEN), which increased nuclear localization of the transcription factor Foxo3a. The Nrp1-induced transcriptome promoted Treg-cell stability by enhancing quiescence and survival factors while inhibiting programs that promote differentiation. Importantly, this Nrp1-dependent molecular program is evident in intra-tumoral Treg cells. Our data support a model in which Treg-cell stability can be subverted in certain inflammatory sites, but is maintained by a Sema4a-Nrp1 axis, highlighting this pathway as a potential therapeutic target that could limit Treg-cell-mediated tumour-induced tolerance without inducing autoimmunity.

Figure 1. Sema4a binds Nrp1 to potentiate T_reg function and survival in vitro

a, Transwell suppression assay in which T_reg cells were cocultured in the top chamber of a transwell plate with anti-CD3/anti-CD28 coated beads in the presence or absence of CD4⁺ or CD8⁺ T_conv cells that had been previously transfected with scrambled or siRNA to Sema4a. Proliferation of T_conv cells stimulated with anti-CD3/anti-CD28 coated beads in the bottom chambers was measured by [³H]-thymidine uptake. b, Transwell suppression assay with T_reg cells cocultured in top chamber with CD4⁺, CD8⁺, or CD11c⁺ cells including anti-Sema4a or its isotype control. c, Transwell suppression assay in which T_reg were cocultured in the absence of T_conv but in the presence of beads coated with Sema4a-Ig or its isotype control. d, ELISA based binding assay in which plates coated with Nrp1 were incubated with Sema4a-Ig or IgG1 in the presence of various blocking antibodies. Sema4a-Ig was detected using an isotype specific antibody. e, Transwell suppression assay where T_reg were purified by flow cytometry from Foxp3^Cre or Nrp1^f/fFoxp3^Cre mice. f, Annexin V-7-AAD staining of T_reg stimulated for 48 h in vitro in the presence of Sema4a-Ig or its isotype control. Results represent the mean of five independent experiments. *, p < 0.05, **, p < 0.01, ***, p < 0.001 by unpaired t-test. Error bars indicate s.e.m.

Figure 2. Nrp1-deficient T_reg fail to suppress antitumor immune responses

a, Tumor growth curve (top) and survival plot (bottom) of Foxp3^Cre, Nrp1^f/fFoxp3^Cre, or Foxp3^DTR.gfp mice receiving 1.25 × 10⁵ MC38 melanoma cells s.c. and (for Foxp3^DTR.gfp) 100 μg diphtheria toxin (DT) i.p. twice weekly. b, As in a, but mice received 1.25 × 10⁵ EL4 thymoma i.d. c, As in a, but mice received 1.25 × 10⁵ B16 melanoma i.d. d, Tumor growth curve of C57/BL6 mice receiving 1.25 × 10⁵ B16 melanoma i.d. concomitant with injections of isotype control, anti-Sema4a, or anti-Nrp1 (100 μg) twice weekly. e, Tumor growth curve as in d except mice received Sema4a-Ig twice weekly. f, Tumor growth curve of C57/BL6 mice receiving 1.25 × 10⁵ B16 melanoma i.d. When tumors were palpable (day 5, indicated by arrow), mice began receiving injections of anti-Nrp1 or its isotype control (400 μg initially, 200 μg every 3 d). g, Lung metastasis counts from Foxp3^Cre or Nrp1^f/fFoxp3^Cre mice injected with 2.5-10 × 10⁵ B16 cells i.v. 17-20 d earlier. h, Tabulation of flow cytometric analysis of tumor-infiltrating lymphocytes from Foxp3^Cre or Nrp1^f/fFoxp3^Cre mice injected i.d. with B16 18 d earlier. i, Sema4a expression of various immune cells in ndLN, dLN, or TIL. Results represent the mean of five (a-c, n=10-25 mice), three (d-h n=8-20 mice), or three (i) experiments. *, p < 0.05, **, p < 0.01, ***, p < 0.001, by (a-f) one-way ANOVA or (g-i) unpaired t-test. Error bars indicate s.e.m.

Figure 3. Ligation of Nrp1 by Sema4a promotes T_reg stability through modulation of Akt-mTOR signaling

a, TIRF microscopic analysis of IS Akt phosphorylation in T_regs stimulated 20 min on a lipid bilayer coated with anti-TCR antibodies in the presence or absence of Sema4a-Ig. b, Transwell suppression assay using T_regs retrovirally expressing WT or DN-Akt. Transductants were selected and expanded using puromycin and IL-2. c, Immunoprecipitation (IP) analysis of Nrp1 using in vitro expanded T_regs serum starved for 3h, then stimulated as indicated for 6 h prior to IP. d, Transwell suppression assay using Foxp3^Cre or Pten^f/fFoxp3^Cre T_regs. e, TIRF microscopic analysis of IS Akt phosphorylation as in a of Nrp1^f/fFoxp3^Cre T_reg cells retrovirally reconstituted with WT or ΔSEA Nrp1. f, Foxo3a cytoplasmic (top) and nuclear (bottom) localization signals, as defined by masking using actin and DNA staining. Arbitrary Units (AU) represent fluorescence intensity calculated volumetrically through 20-30 slices of T_reg cells. n=70-93 g, Heat map (right) of genes regulated by Nrp1. Foxp3^Cre and Nrp1^f/fFoxp3^Cre CD45RB^lo Foxp3 (YFP)⁺ CD4⁺ T cells were stimulated for 48h with anti-CD3, anti-CD28, 100 U/mL rhIL-2, and immobilized IgG1 or Sema4a-Ig. RNA was subjected to Affymetrix gene profiling. T_reg signature genes are in bold. All genes shown met FDR < 0.10 and compared using two-way ANOVA. Results represent at least three independent experiments (a, c, e, f) or represent means of three (b, d) or seven (g) experiments. *, p < 0.05, ** p < 0.01 by unpaired t-test. Error bars indicate s.e.m.

Figure 4. Tumor-infiltrating T_reg bear a signature similar to Sema4a:Nrp1 ligation

a, Akt phosphorylation in T_reg. Tumor-bearing Foxp3^Cre or Nrp1^f/fFoxp3^Cre mice were sacrificed and ndLN and TIL were harvested. Cells were immediately fixed and stained for phospho-Akt. Shaded histogram indicates isotype control. Results are tabulated normalized to isotype control staining. IRF4/RORγt (b), Ki67/BrdU (c), cleaved caspase-3 (d), Bcl2 (e), ICOS (f), IL-10 (g) and CD73 (h) staining from ndLN, dLN, or TIL from tumor-bearing Foxp3^Cre or Nrp1^f/fFoxp3^Cre mice. Ki67/BrdU analysis included injection with BrdU 14 h prior to harvest. IL-10 staining included restimulation with PMA and ionomycin for 16h in the presence of brefeldin A. Results represent the mean of 3-5 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by paired t-test (a, n=7) or unpaired t-test (b-h n=9-25). Error bars indicate s.e.m.