T-cell-expressed proprotein convertase furin is essential for maintenance of peripheral immune tolerance

Abstract

Furin is one of seven proprotein convertase family members that promote proteolytic maturation of proproteins. It is induced in activated T cells and is reported to process a variety of substrates including the anti-inflammatory cytokine transforming growth factor (TGF)-beta1 (refs 2-4), but the non-redundant functions of furin versus other proprotein convertases in T cells are unclear. Here we show that conditional deletion of furin in T cells allowed for normal T-cell development but impaired the function of regulatory and effector T cells, which produced less TGF-beta1. Furin-deficient T regulatory (Treg) cells were less protective in a T-cell transfer colitis model and failed to induce Foxp3 in normal T cells. Additionally, furin-deficient effector cells were inherently over-active and were resistant to suppressive activity of wild-type Treg cells. Thus, our results indicate that furin is indispensable in maintaining peripheral tolerance, which is due, at least in part, to its non-redundant, essential function in regulating TGF-beta1 production. Targeting furin has emerged as a strategy in malignant and infectious disease. Our results suggest that inhibiting furin might activate immune responses, but may result in a breakdown in peripheral tolerance.

Figure 1. Normal thymic T cell development, but activated/memory phenotype of peripheral T cells in CD4cre-fur^f/f mice

a, b, Proportions of CD4⁺, CD8⁺, TCR-β⁺ and Foxp3⁺ thymocytes in 8 week old mice. Panels on the right show CD4⁺/CD8⁺ proportions in TCR-β rearranged cells. Three mice per group were analyzed. Representative flow cytometry blots and plotted mean values are shown. c, Activated/memory splenic T cells in 7–9 week old mice. Representative flow cytometry blots and plotted mean values are shown (n=3 per group). d, Cytokine production. Mesenteric lymph node cells were stimulated with plate-bound CD3 and soluble CD28 antibodies for 48 hours (n=4, nine weeks old CD4cre-fur^f/f and fur^f/f mice).

Figure 2. Development of age-related autoimmunity in CD4cre-fur^f/f mice

a, Lymphoid organs, colon and stomach/duodenum of CD4cre-fur^f/f and age-matched wild-type animals (6 months). b, Haematoxylin- and eosin-stained sections of colon, stomach and liver (6 months old, CD4cre-fur^f/f and fur^f/f mice). c, Anti-dsDNA (DA) and nuclear antibody (NA) titers in CD4cre-fur^f/f animals compared with age-matched wild-type and fur^f/f animals (n=8, 5–7 months). d, Serum cytokines in CD4cre-fur^f/f animals compared with age-matched wild-type and fur^f/f animals (n=6–8, 5–7 months). e, ELISA for serum immunoglobulins of CD4cre-fur^f/f animals compared with age matched wild-type and fur^f/f animals (n=6–13, 5–7 months).

Figure 3. Deletion of furin in T cells results in T cell expansion/activation, and impairs TGF-β1 production, and CD103 expression

a,b, Absolute CD4⁺Foxp3, CD4⁺Foxp3⁺ and CD8⁺ cell numbers and the proportion of CD4⁺Foxp3CD69⁺ cells in the mesenteric lymph nodes of CD4cre-fur^f/f animals were compared with age-matched wild-type and fur^f/f animals (n=3, 5–6 months). c, TGFβ-1 production. Purified CD4cre-fur^f/f and fur^f/f CD4⁺CD25 and CD4⁺CD25⁺ cells were activated with plate-bound CD3 and soluble CD28 antibodies; TGFβ-1 was measured by ELISA in duplicate and a representative of three experiments is shown. d, Expression of Foxp3 and CD103 in lamina propria and intraepithelial CD4⁺ cells; shown is a representative flow cytometry plot of three mice per group analyzed (5–6 months).

Figure 4. Furin deficiency results in increased T effector cells aggressiveness and impaired T regulatory cell protection in suppressing colitis

Purified wild-type (Wt) CD4cre-fur^f/f (Ko) CD4⁺CD25CD45Rb^hi naïve T cells were transferred alone or in combination with wild-type or furin-deficient CD4⁺CD25⁺ T regulatory cells into TCRα^−/− recipients. Mice were analyzed on week 10 (n=5 per group). a, Representative images of colons. b, Representative colon histology. c, Body weight change during the experiment .d, Absolute CD4⁺ cell numbers in mesenteric lymph nodes. e, Spontaneous conversion of adoptively transferred naïve CD4⁺CD25CD45Rb^hi cells into CD4⁺Foxp3⁺ cells. Representative flow cytometry blots and plotted mean values are shown. In e data are pooled from two identical experiments.