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. 2013 Jul 25;39(1):160-70.
doi: 10.1016/j.immuni.2013.06.010. Epub 2013 Jul 11.

T Cell-Derived Protein S Engages TAM Receptor Signaling in Dendritic Cells to Control the Magnitude of the Immune Response

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Free PMC article

T Cell-Derived Protein S Engages TAM Receptor Signaling in Dendritic Cells to Control the Magnitude of the Immune Response

Eugenio A Carrera Silva et al. Immunity. .
Free PMC article

Abstract

Dendritic cell (DC) activation is essential for the induction of immune defense against pathogens, yet needs to be tightly controlled to avoid chronic inflammation and exaggerated immune responses. Here, we identify a mechanism of immune homeostasis by which adaptive immunity, once triggered, tempers DC activation and prevents overreactive immune responses. T cells, once activated, produced Protein S (Pros1) that signaled through TAM receptor tyrosine kinases in DCs to limit the magnitude of DC activation. Genetic ablation of Pros1 in mouse T cells led to increased expression of costimulatory molecules and cytokines in DCs and enhanced immune responses to T cell-dependent antigens, as well as increased colitis. Additionally, PROS1 was expressed in activated human T cells, and its ability to regulate DC activation was conserved. Our results identify a heretofore unrecognized, homeostatic negative feedback mechanism at the interface of adaptive and innate immunity that maintains the physiological magnitude of the immune response.

Figures

Figure 1
Figure 1. Activated T cells express Pros1
(A) Splenic CD4+ T cells from OT-II mice were co-cultured with BM-DC alone or in the presence of 200 μg/mL OVA for 24 h. Pros1 induction was analyzed after antigen-specific activation of CD4+ T cell (CD4+CD69+). Representative FACS plots (left) and MFI for Pros1 expression on CD4+ T cells (right) are shown. (B) CD45.2 T cells from indicated genotypes were transferred into CD45.1 recipients and 1 day after transfer recipient mice were injected into the rear footpad with 50 μg of OVA/IFA/LPS. Popliteal and inguinal LNs were collected 48 h after immunization. Representative plots show CD45.2 transferred T cells and Pros1 expression in Ctrl OT-II T cells compared with Pros1 KO OT-II T cells. (C) Splenic CD4+ cells were isolated and activated in vitro with anti-CD3/CD28. Pros1 mRNA expression was determined by qPCR and normalized to unstimulated cells. (D) Representative FACS histograms of Pros1 expression on resting and activated CD4+ T cells with anti-CD3/CD28 for 15 h. Gray histogram represents activated CD4+ cells from Pros1flox/flox Cd4-Cre+ (Pros1 KO) mice. Data are presented as representative individual samples or as mean ± SEM of at least 4 to 6 independent samples per group. * p < 0.05, ***p < 0.001.
Figure 2
Figure 2. Deficiency of Pros1 in transferred T cells leads to accelerated colitis in Rag1−/− recipient mice
Sorted CD4+CD25CD45RBhigh T cells from Pros1flox/flox Cd4-Cre (Ctrl) or Cre+ (Pros1 KO) were transferred i.p. into Rag1−/− mice and colitis development was monitored. (A) Colonoscopy score of disease severity and (B) representative pictures of colonoscopy at week 6 are shown. Note loss of translucency, stool inconsistency (*), and increased mucosal granularity (#) in the Rag1−/− recipient of Pros1 KO T cells. Intracellular IFN-γ and IL-17A production on CD4+ cells from mesenteric LN (C) and lamina propria leukocytes of large intestine (D). Data are presented as individual samples or as mean ± SEM and are representative of 2 independent experiments with at least 5 samples per group. * p < 0.05, **p < 0.01.
Figure 3
Figure 3. T cell-derived Pros1 limits DC activation
(A) CD45.2 T cells from indicated genotypes were transferred into CD45.1 recipients and 1 day after transfer recipient mice were injected with 50 μg of OVA/IFA/LPS into the rear footpad. Popliteal and inguinal LNs were collected after 72 h and CD86 and CD40 expression were measured on CD11c+ cells. Representative histograms for non-immunized and OVA immunized mice and independent data are shown. (B) Splenic CD4+CD25 T cells were isolated from Pros1flox/flox Cd4-Cre OT-II (Ctrl OT-II Tc) and Pros1flox/flox Cd4-Cre+ OT-II (Pros1 KO OT-II Tc) mice and co-cultured with BM-DCs in the presence of the indicated concentrations of OVA. Representative FACS histograms and percentage of CD86+CD11c+ and CD40+ CD11c+ cells are shown. (C) TNF-α and IL-6 production in the co-culture supernatants after 72 h, as measured by ELISA. (D) Representative plots showing intracellular staining for TNF-α and IL-6 in CD11c+ (left) and individual samples (right) are shown. Data are presented as representative individual samples or as mean ± SEM of 3–5 independent samples per group. * p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4. Deletion of Pros1 in T cell results in increased immune responses upon immunization with T-dependent antigens
(A) Serum TNP specific IgG1 in TNP-KLH-immunized Pros1flox/flox Cd4-Cre or Pros1flox/flox Cd4-Cre+ mice. (B) TNP-specific IgG3 in TNP-Ficoll-immunized mice. Data are presented as mean ± SEM and are representative of 2 independent experiments with at least 4 samples per group. **p < 0.01, ***p < 0.01.
Figure 5
Figure 5. Negative feedback of T cell-derived Pros1 depends of cell proximity and Axl and Mer signaling on DCs
(A) Splenic CD4+ CD25 T cells from Pros1flox/flox Cd4-Cre (Ctrl Tc) or Pros1flox/flox Cd4-Cre+ (Pros1 KO) mice were activated in vitro with anti-CD3/CD28 for 4 h. Activated T cells were then co-cultured with BM-DCs in presence of 30 μg/mL of Poly I:C for 15 h. To prevent close contact between activated CD4+ T cells and BM-DCs, BM-DCs were cultured on the upper chamber of a transwell system while activated T cells were cultured on the lower chamber. MFI of CD86 and CD40 expression on CD11c+ cells in the indicated conditions are shown. (B) Where indicated, 10 μg/mL of Annexin V (AnxV) was added to the T cell culture 1 h prior to co-culture with BM-DCs. Representative FACS histograms of CD86 and CD40 expression on CD11c+ cells are shown. (C) CD4+CD25 OT-II Ctrl T cells were co-cultured with WT or AM−/− BM-DC in the presence of 200 μg/mL of OVA for 48 h. Representative histograms for CD86 and CD40 expression on CD11c+ cells are shown. (D) Percentage of CD11c+SOCS3+ in activated BM-DCs upon co-culture with Ctrl or Pros1 KO T cells. (E) IkBα (top), Tubulin (bottom) in the co-culture were analyzed by western blot at the indicated time points. (F) Phospho-NF-κB p65 (Ser536) subunit was measured on activated CD11c+ cell co-cultured with Ctrl or Pros1 KO T cells by FACS. Data are presented as percentage of phospho-NF-κB p65 on CD11c+ cells, by histograms. Data are presented as individual samples or as mean ± SEM and are representative of at least 2 independent experiments with 3–4 independent samples per group. * p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6
Figure 6. PROS1 is expressed in activated human CD4 T cells and PROS1 blockade leads to enhanced activation of DCs in MLR
Human naïve CD4 T cells were isolated from PBMCs by negative selection and stimulated with anti-CD3/CD28 for 6 days. (A) Expression of PROS1 mRNA, normalized to unstimulated naïve CD4+ T cells, is shown. (B) Surface PROS1 expression on CD4+CD69+ cells after 6 days of stimulation with α-CD3/CD28. Representative histogram from at least 5–7 independent donors is shown. (C) Sections from non-reactive and reactive human lymph nodes (LNs) were stained with anti-CD3 and anti-PROS1. Representative PROS1 staining in the inter-follicular CD3+ T cell areas on non-reactive (n=6) and reactive (n=6) LNs are shown. (D) Schematic representation of MLR and PROS1 neutralization. (E and F) CD86 and CD40 expression were measured on CD11c+ cells at day 5. Representative histograms (left) and independent data with at least 3 independent samples per group (right) are shown. Each experiment was repeated at least twice employing 3 to 5 different donors. (G) Percentage of total PROS1 in healthy controls and IBD patients. Total PROS1 was determined using automated latex immunoassay methodology and normalized to an average value of PROS1 from a pool of healthy donors. * p < 0.05, **p < 0.01, ***p < 0.01.

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