TGF-β Signaling Initiated in Dendritic Cells Instructs Suppressive Effects on Th17 Differentiation at the Site of Neuroinflammation

While the role of Transforming Growth Factor β (TGF-β) as an intrinsic pathway has been well established in driving de novo differentiation of Th17 cells, no study has directly assessed the capacity of TGF-β signaling initiated within dendritic cells (DCs) to regulate Th17 differentiation. The central finding of this study is the demonstration that Th17 cell fate during autoimmune inflammation is shaped by TGF-β extrinsic pathway via DCs. First, we provide evidence that TGF-β limits at the site of inflammation the differentiation of highly mature DCs as a means of restricting Th17 cell differentiation and controlling autoimmunity. Second, we demonstrate that TGF-β controls DC differentiation in the inflammatory site but not in the priming site. Third, we show that TGF-β controls DC numbers at a precursor level but not at a mature stage. While it is undisputable that TGF-β intrinsic pathway drives Th17 differentiation, our data provide the first evidence that TGF-β can restrict Th17 differentiation via DC suppression but such a control occurs in the site of inflammation, not at the site of priming. Such a demarcation of the role of TGF-β in DC lineage is unprecedented and holds serious implications vis-à-vis future DC-based therapeutic targets

A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells

Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1\ua0(IDO1) are immunoregulatory enzymes catalyzing the degradation of L-arginine and L-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1-dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid-derived suppressor cells, conditioned DCs toward an IDO1-dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1-phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings

Involvement of the Fas (CD95) system in peripheral cell death and lymphoid organ development

International audienceFas‐mediated apoptosis is a form of cell death that operates through a Fas‐Fas ligand (FasL) interaction. In this study we investigated the role of the Fas system during development of normal and Fas‐mutated lymphocytes. Irradiated RAG2–/– recipients were reconstituted with bone marrow cells from B6 and lpr mice (Fas defective) or from B6 and gld mice (FasL defective), and analyzed for long‐term development. The results showed a primary role of the Fas system in peripheral cell death and thymic colonization. In the periphery, the interaction in vivo between Fas+ and Fas– T cell populations indicated that cellular homeostasis was defective. Indeed, we observed a FasL‐mediated cytotoxic effect on normal‐derived T cells, explaining the dominance of lpr T cells in the mixed chimeras. The Fas mutation affected neither cell activation nor cell proliferation, as the effector (Fas–) and target (Fas+) cells behaved similarly with regard to activation marker expression and cell cycle status. However, Fas– T cells failed to seed the periphery and the thymus in the long term. We suggest that this could be due to the fact that FasL is involved in the structural organization of the lymphoid compartment

Transfer of Maternal Immune Cells by Breastfeeding: Maternal Cytotoxic T Lymphocytes Present in Breast Milk Localize in the Peyer’s Patches of the Nursed Infant

<div><p>Despite our knowledge of the protective role of antibodies passed to infants through breast milk, our understanding of immunity transfer via maternal leukocytes is still limited. To emulate the immunological interface between the mother and her infant while breast-feeding, we used murine pups fostered after birth onto MHC-matched and MHC-mismatched dams. Overall, data revealed that: <b>1)</b> Survival of breast milk leukocytes in suckling infants is possible, but not significant after the foster-nursing ceases; <b>2)</b> Most breast milk lymphocytes establish themselves in specific areas of the intestine termed Peyer’s patches (PPs); <b>3)</b> While most leukocytes in the milk bolus were myeloid cells, the majority of breast milk leukocytes localized to PPs were T lymphocytes, and cytotoxic T cells (CTLs) in particular; <b>4)</b> These CTLs exhibit high levels of the gut-homing molecules α4β7 and CCR9, but a reduced expression of the systemic homing marker CD62L; <b>5)</b> Under the same activation conditions, transferred CD8 T cells through breast milk have a superior capacity to produce potent cytolytic and inflammatory mediators when compared to those generated by the breastfed infant. It is therefore possible that maternal CTLs found in breast milk are directed to the PPs to compensate for the immature adaptive immune system of the infant in order to protect it against constant oral infectious risks during the postnatal phase.</p></div

Transfer of breast milk leukocytes to suckling pups.

<p><b>(A)</b> Breeding of wild type (WT) C57BL/6 and C57BL/6-GFP^tg (GFP^tg) mice was coordinately mated. At day 0–2, WT neonates were transferred to be continually nursed by GFP^tg dams until their weaning (21 days). PPs and other organs from suckling infants were surgically excised, purified, and put into cell suspensions for flow cytometry analysis. FACS plots show the level of GFP expression by CD45.2+ splenocytes of GFP^tg (<i>left</i>, positive control) and WT (<i>right</i>, negative control) mice. <b>(B)</b> Leukocytes were identified using the following orientating gates: The first orientating gate was selected using a SSC vs. CD45 plot where the cutoff for CD45+ cells was set using FMO control. Subsequently, annexin V vs. propidium iodide (PI) plot of CD45+ cells separated events into a major gate of annexin V^- PI^- events which represents viable CD45+ cells. Subsequent specific gate for other populations that were preselected as CD45+ annexin V^- PI^- are shown in CD45 vs. GFP plots. Dot Plots show the presence of breast milk leukocytes (GFP+CD45.2+) in the peyer’s patches (PPs), spleen (SPL), thymus (THY), mesenteric lymphnodes (MLN), and intestinal mucosa (IM). Percent of transferred leukocytes was assessed 18 days post-fostering. Panels show WT pups breastfed by WT mothers (<i>upper</i>, <i>negative control</i>) and WT pups breastfed by MHC-matched GFP^tg dams (<i>lower</i>). <b>(C)</b> Bar graphs summarize the presence of GFP+ cells in organs as a % of total CD45.2+ cells at day 6, 14, and 18 post-fostering, and one week post-weaning (day 28). Data were obtained from three experiments using a total of 24 pups with <i>n</i> = 6–10 pups per experiment (DAY 6); three experiments using a total of 16 pups with <i>n =</i> 4–6 pups per experiment (DAY 14); five experiments using a total of 26 pups with <i>n =</i> 4–6 pups per experiment (DAY 18); and three experiments using a total of 9 mice with <i>n = 3</i> animals per experiment (DAY 28). <b>(D)</b> BALB/c neonates were transferred to be foster-nursed by GFP^tg dams until their weaning. Bar graph summarizes the presence of GFP+ cells as a % of total CD45.2+ cells in organs at day 18 post-fostering and one week post-weaning (day 30). Data were generated from three experiments using a total of 12 pups (DAY 18) and 6 mice (Day 30) with <i>n</i> = 2–5 animals per experiment. <b>(C and D)</b> Data are shown as means of % GFP+ CD45.2+ cells ± s.e.m. Error bars represent s.e.m. A 2-tailed Student’s <i>t</i> test distribution with paired groups was evaluated for statistical significance. P > 0.05 was considered not significant (NS), *<i>P</i> < 0.05 was considered significant, and **<i>P</i> < 0.005.</p

Characterization of breast milk leukocytes.

<p><b>(A)</b> Different GFP+ cell subtypes were identified in milk bolus and PPs of C57BL/6 pups nursed by GFP^tg dams based on the co-expression of GFP and CD45.2 in combination with CD3, CD8, CD4, CD11b, CD19, and Gr1. Bar graphs show the average contribution of cell subset(s) to the total of GFP+CD45.2+ cells in milk bolus (<i>left</i>) and PPs (<i>right</i>). <b>(B)</b> Bar graphs show the % of each cell subset (gated on total CD45.2+ GFP+ cells) in milk bolus versus PPs: CD11b+ (CD3-CD19-CD11b+, <i>upper left</i>), GR1+ (CD3-CD19-Gr-1+, <i>upper middle</i>), CD19+ (CD3-CD19+, <i>upper right</i>), CD3+ (CD19-CD11b-CD3+, <i>lower left</i>), CD8+ (CD3+CD8+, <i>lower middle</i>), and CD4+ (CD3+CD4+, <i>lower right</i>) cells. Data are shown as means of % cell subset gated on GFP+CD45.2+ cells ± SD. Error bars represent SD. <b>(A and B)</b> Data were obtained from four experiments using a total of 16 (milk bolus) and 25 (PPs) animals with a combined material of 3 to 5 (milk) and 5 to 8 (PPs) mice per experiment. (<b>C</b>) Contour Plots show expression of α4β7, CCR9, and CD62L by breast milk CD8+GFP+ T cells that localize in PPs of suckling pups vs. blood CTLs of the birth mother. (<b>D</b>) Bar graphs show the % of CD8+CCR9+, CD8+α4β7+, and CD8+CD62L+ in PPs (gated on milk CD3+CD8+GFP+ cells) versus adult blood (gated on total CD3+CD8+ cells). Data are shown as means of % cell subset gated on CD8+GFP+ (PPs) or CD8+ (blood) T lymphocytes ± SD. Error bars represent SD. Bar graphs summarize the data from four experiments using a total of 25 (PPs) and 5 (blood) animals with a combined material of 5 to 8 (PPs) and 1 to 2 (blood) mice per experiment. A 2-tailed Student’s <i>t</i> test distribution with paired groups was evaluated for statistical significance. *<i>P</i> < 0.05 was considered significant, **<i>P</i> < 0.005, ***<i>P</i> < 0.0005, and P > 0.05 was considered not significant (NS).</p

Increased extracellular pressure enhances cancer cell integrin-binding affinity through phosphorylation of β1-integrin at threonine 788/789

Increased extracellular pressure stimulates β1-integrin-dependent cancer cell adhesion. We asked whether pressure-induced adhesion is mediated by changes in β1-integrin binding affinity or avidity and whether these changes are phosphorylation dependent. We evaluated integrin affinity and clustering in human SW620 colon cancer cells by measuring differences in binding between soluble Arg-Gly-Asp (RGD)-Fc ligands and RGD-Fc-F(ab′)2 multimeric complexes under ambient and 15-mmHg increased pressures. Phosphorylation of β1-integrin S785 and T788/9 residues in SW620 and primary malignant colonocytes was assessed in parallel. We further used GD25-β1-integrin-null murine fibroblasts stably transfected with either wild-type β1A-integrin, S785A, TT788/9AA, or T788D mutants to investigate the role of β1-integrin site-specific phosphorylation. SW620 binding of RGD-Fc-F(ab′)2 multimeric complexes, but not soluble RGD-Fc ligands, was sensitive to integrin clustering. RGD-Fc ligand binding was significantly increased under elevated pressure, suggesting that pressure modulates β1-integrin affinity. Pressure stimulated both β1-integrin S785 and T788/9 phosphorylation. GD25-β1A-integrin wild-type and S785A cells displayed an increase in adhesion to fibronectin under elevated pressure, an effect absent in β1-integrin-null and TT788/9AA cells. T788D substitution significantly elevated basal cell adhesion but displayed no further increase under pressure. These results suggest pressure-induced cell adhesion is mediated by β1-integrin T788/9 phosphorylation-dependent changes in integrin binding affinity