Oral tolerance to a contact sensitizer proceeds in two sequential steps established by Dubois et al. (Gastroenterology 2009): orally delivered antigen triggers plasmacytoid dendritic cell (pDC)-mediated deletion of antigen-specific CD8+ T cells in gut-associated tissues, and the residual CD8+ T cells that escape deletion are then suppressed by activated CD4+CD25+Foxp3+ regulatory T cells (Tregs) at systemic sensitization. The paper left open the underlying molecular mechanisms. Later work has resolved the Treg-induction arm: CD103+ intestinal dendritic cells (DCs) and pDCs drive de novo Foxp3+ Treg generation and expansion, and gut macrophage-derived IL-10 expands lamina propria Tregs. What remains unresolved is the effector side of the model: the molecular changes that oral antigen exposure imprints on residual CD8+ effector T cells that render them susceptible to Treg-mediated inhibition of their differentiation into IFN-gamma-producing, tissue-damaging effectors — with PD-1/PD-L co-inhibitory interactions and altered TGF-beta receptor signaling as untested candidates — together with the role and cellular source of environmental IL-10 in this conditioning. Identifying this conditioning mechanism would complete the mechanistic account of the sequential deletion-then-suppression model and enable the mucosal biotherapies it motivates, namely interventions that raise the susceptibility of pathogenic effector T cells to Treg suppression.

Background

Oral tolerance is the state of systemic immunological unresponsiveness induced when antigen is delivered through the gastrointestinal route; it is the default response to food and commensal antigens and operates by clonal deletion or anergy of antigen-reactive T cells at high antigen dose and by active suppression mediated by regulatory T cells at low dose. CD4+CD25+Foxp3+ regulatory T cells (Tregs) are a thymus-derived and peripherally induced T-cell lineage that suppresses other immune cells; Foxp3 is the lineage-defining transcription factor. Plasmacytoid dendritic cells (pDCs) are a dendritic-cell subset specialized for type I interferon production that can also act as tolerogenic antigen-presenting cells. CD103 (alphaE integrin)-positive gut dendritic cells are a conventional DC subset that, conditioned by gut epithelial factors, converts naive CD4+ T cells into Foxp3+ Tregs using transforming growth factor beta (TGF-beta) and retinoic acid, the vitamin A metabolite that also imprints gut-homing receptors on lymphocytes. IL-10 is an anti-inflammatory cytokine produced by macrophages, DCs, and T cells. Throughout this task, environmental IL-10 denotes the IL-10 present in the tissue milieu that is contributed by sources other than the effector CD8+ T cell itself (for example, macrophages, dendritic cells, or other non-effector cells), as distinct from IL-10 produced by the effector CD8+ T cell; whether it acts during antigen feeding, at systemic sensitization, or both is left open. PD-1 is an inhibitory receptor on activated T cells whose ligands (PD-L1/PD-L2) dampen T-cell activation.

The source work studied oral tolerance in a mouse model of allergic contact dermatitis (ACD), a T-cell-mediated skin inflammation. Feeding the hapten 2,4-dinitrofluorobenzene (DNFB) before skin sensitization prevented the contact hypersensitivity (CHS) effector response, which in this model is mediated by cytolytic CD8+ T cells. The same group had earlier shown that naturally occurring CD4+CD25+ Tregs are required for this oral tolerance (Dubois et al., Blood 2003) and that pDCs mediate oral tolerance through anergy/deletion of antigen-reactive T cells (Goubier et al., Immunity 2008). The 2009 Gastroenterology paper integrated these findings into a sequential two-step model: (i) a mucosal step in mesenteric lymph nodes and liver in which pDCs delete a large fraction of antigen-specific CD8+ T cells — an event independent of Tregs and of IL-10 — and (ii) a systemic step at the time of skin sensitization in which activated Tregs suppress priming of the residual, non-deleted CD8+ T cells. Two quantitative observations defined the mechanistic puzzle: CD8+ T cells from antigen-fed donors were far more effectively inhibited by Tregs from fed donors (about 87% versus about 25% suppression of IFN-gamma-producing effectors in adoptive-transfer assays, in which isolated cell populations are transferred into lymphocyte-deficient recipient mice to test their function in vivo) than CD8+ T cells from control donors, and Tregs isolated after antigen feeding were more suppressive than Tregs from control mice. Hence oral antigen exposure conditions both the effector and the regulatory arm.

Subsequent work clarified the regulatory arm. Reviews of oral tolerance (Weiner et al., Immunological Reviews 2011) codified the CD103+ DC/TGF-beta/retinoic-acid pathway of Foxp3+ Treg induction; Hadis et al. (Immunity 2011) showed that oral tolerance requires Treg generation in gut-draining lymph nodes, homing to the lamina propria, and local expansion driven by IL-10 from CX3CR1+ gut macrophages (fractalkine-receptor-expressing resident macrophages of the intestinal wall); Esterhazy et al. (Nature Immunology 2016) showed classical DCs are the critical antigen-presenting cells for dietary-antigen-induced peripheral Tregs, with subset redundancy; and Uto et al. (JACI 2018) demonstrated directly that gut-associated pDCs preferentially generate CD4+Foxp3+ Tregs through an autocrine TGF-beta loop and that pDC deficiency abrogates the de novo Foxp3+ Treg generation that follows antigen feeding, impairing oral tolerance. In the same DNFB model, the authors' own laboratory showed that TLR4-dependent licensing of migratory CD103+CD11b+ retinaldehyde-dehydrogenase-positive intestinal DCs (retinaldehyde dehydrogenase being the vitamin A-processing enzyme that produces retinoic acid), a subset specialized in Foxp3+ Treg conversion, is required for oral tolerance (Hacini-Rachinel et al., JACI 2018). By contrast, the effector arm has not advanced: how oral antigen exposure changes residual CD8+ T cells at the molecular level so that they become susceptible to Treg-mediated inhibition is not known. The source paper could not detect PD-1 or CTLA-4 expression in its model and left PD-1/PD-L interactions, TGF-beta receptor modulation, and a role for environmental IL-10 as open possibilities.

Problem Statement

In the sequential two-step model of oral tolerance established by Dubois et al. (2009) — oral administration of antigen causing pDC-dependent deletion of antigen-specific CD8+ T cells in gut-associated lymphoid tissues and liver, followed at systemic sensitization by suppression of the residual antigen-specific CD8+ T cells by activated CD4+CD25+Foxp3+ regulatory T cells — determine the molecular mechanism by which oral antigen exposure conditions the residual, non-deleted effector CD8+ T cells so that they become susceptible to Treg-mediated inhibition of their differentiation into IFN-gamma-producing, tissue-damaging effectors. Specifically: (i) which molecular pathway(s) are necessary and/or sufficient for this heightened susceptibility — candidates include PD-1/PD-L co-inhibitory interactions, altered TGF-beta receptor expression or signaling, and other cell-intrinsic changes, none of which has direct evidence to date; and (ii) what is the role, and the cellular source, of environmental IL-10 in promoting the susceptibility of effector T cells to Treg-mediated suppression, or in otherwise enhancing Treg suppressive function, during oral tolerance. The question is answered when both enumerated parts are resolved: (i) the conditioning mechanism is identified and causally validated in an oral-tolerance system in which deletion versus conditioning is separated and CD8-intrinsic conditioning is distinguished from Treg-intrinsic or antigen-presenting-cell effects, and (ii) the role and cellular source of environmental IL-10 are determined, whether that IL-10 acts on the effector CD8+ T cell or enhances Treg suppressive function; a Treg-side IL-10 effect resolves (ii) but does not by itself resolve (i). The related questions of how oral feeding expands and induces Foxp3+ Tregs, and whether pDCs contribute to that induction, are already resolved and are not part of the open problem.

References

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