Tolerogenic dendritic cells generated in vitro using a novel protocol mimicking mucosal tolerance mechanisms represent a potential therapeutic cell platform for induction of immune tolerance.

Dendritic cells (DCs) are mediators between innate and adaptive immunity and vital in initiating and modulating antigen-specific immune responses. The most important site for induction of tolerance is the gut mucosa, where TGF-ß, retinoic acid, and aryl hydrocarbon receptors collaborate in DCs to induce a tolerogenic phenotype. To mimic this, a novel combination of compounds - the synthetic aryl hydrocarbon receptor (AhR) agonist IGN-512 together with TGF-ß and retinoic acid - was developed to create a platform technology for induction of tolerogenic DCs intended for treatment of several conditions caused by unwanted immune activation. These in vitro-generated cells, designated ItolDCs, are phenotypically characterized by their low expression of co-stimulatory and activating molecules along with high expression of tolerance-associated markers such as ILT3, CD103, and LAP, and a weak pro-inflammatory cytokine profile. When co-cultured with T cells and/or B cells, ItolDC-cultures contain higher frequencies of CD25+Foxp3+ regulatory T cells (Tregs), CD49b+LAG3+ 'type 1 regulatory (Tr1) T cells, and IL-10-producing B cells and are less T cell stimulatory compared to cultures with matured DCs. Factor VIII (FVIII) and tetanus toxoid (TT) were used as model antigens to study ItolDC antigen-loading. ItolDCs can take up FVIII, process, and present FVIII peptides on HLA-DR. By loading both ItolDCs and mDCs with TT, antigen-specific T cell proliferation was observed. Cryo-preserved ItolDCs showed a stable tolerogenic phenotype that was maintained after stimulation with LPS, CD40L, or a pro-inflammatory cocktail. Moreover, exposure to other immune cells did not negatively impact ItolDCs' expression of tolerogenic markers. In summary, a novel protocol was developed supporting the generation of a stable population of human DCs in vitro that exhibited a tolerogenic phenotype with an ability to increase proportions of induced regulatory T and B cells in mixed cultures. This protocol has the potential to constitute the base of a tolDC platform for inducing antigen-specific tolerance in disorders caused by undesired antigen-specific immune cell activation.

Deletion of IRF4 in Dendritic Cells Leads to Delayed Onset of T Cell-Dependent Colitis.

Classical dendritic cells (cDC) can be classified into two major subsets: Irf8-dependent cDC1 and Irf4-expressing cDC2. Although these subsets play distinct roles in intestinal immune homeostasis, their functions in T cell-driven colitis remain unknown. To assess the role of IRF4 expression in cDC2 in T cell-driven colitis, CD11c-Cre.Irf4 fl/fl and Irf4 fl/fl mice were backcrossed onto a Rag-1 -/- background and used as recipients of CD45RBhiCD4+ T cells. Colitis score and innate immune cell influx were reduced in Cre+ mice 4 wk posttransfer, and these changes were associated with reduced CD4+ T cell counts in both the mesenteric lymph nodes and colon. By 7 wk, colitis score and colon CD4+ T cell numbers were similar in Cre+ and Cre- mice despite a selective reduction in Th17 cells in the colon of Cre+ mice and a continued reduction in CD4+ T cell numbers in mesenteric lymph nodes. Cotransfer of CD25+CD45RBlo CD4+ T cells prevented CD45RBhiCD4+ T cell-driven colitis in both Cre+ and Cre- recipients, demonstrating that IRF4 expression by cDC is not required for CD4+ regulatory T cell-mediated control of colitis. Collectively these results suggest a role for IRF4 expression in cDC2 in the generation of colitogenic CD4+ T cells, which becomes redundant as colitis progresses.

IRF8 Transcription-Factor-Dependent Classical Dendritic Cells Are Essential for Intestinal T Cell Homeostasis.

The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 transcription-factor-dependent DCs had reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence of SI CD8αß(+) and CD4(+)CD8αα(+) T cells; the latter requiring ß8 integrin expression by migratory IRF8 dependent CD103(+)CD11b(-) DCs. SI homing receptor induction was impaired during T cell priming in mesenteric lymph nodes (MLN), which correlated with a reduction in aldehyde dehydrogenase activity by SI-derived MLN DCs, and inefficient T cell localization to the SI. These mice also lacked intestinal T helper 1 (Th1) cells, and failed to support Th1 cell differentiation in MLN and mount Th1 cell responses to Trichuris muris infection. Collectively these results highlight multiple non-redundant roles for IRF8 dependent DCs in the maintenance of intestinal T cell homeostasis.

IL-18Rα-deficient CD4(+) T cells induce intestinal inflammation in the CD45RB(hi) transfer model of colitis despite impaired innate responsiveness.

IL-18 has been implicated in inflammatory bowel disease (IBD), however its role in the regulation of intestinal CD4(+) T-cell function remains unclear. Here we show that murine intestinal CD4(+) T cells express high levels of IL-18Rα and provide evidence that IL-18Rα expression is induced on these cells subsequent to their entry into the intestinal mucosa. Using the CD45RB(hi) T-cell transfer colitis model, we show that IL-18Rα is expressed on IFN-γ(+) , IL-17(+) , and IL-17(+) IFN-γ(+) effector CD4(+) T cells in the inflamed colonic lamina propria (cLP) and mesenteric lymph node (MLN) and is required for the optimal generation and/or maintenance of IFN-γ-producing cells in the cLP. In the steady state and during colitis, TCR-independent cytokine-induced IFN-γ and IL-17 production by intestinal CD4(+) T cells was largely IL-18Rα-dependent. Despite these findings however, IL-18Rα-deficient CD4(+) T cells induced comparable intestinal pathology to WT CD4(+) T cells. These findings suggest that IL-18-dependent cytokine induced activation of CD4(+) T cells is not critical for the development of T-cell-mediated colitis.