Therapeutic tolerance induced in lymphoreplete mice by short-term coreceptor and costimulation blockade is antigen-specific and spreads to naive T cells (infectious tolerance), yet the classical assay of the same regulatory process — adoptive transfer of natural regulatory T cells into lymphopenic recipients — suppresses rejection of tolerated-type and third-party grafts alike, with no detectable antigen specificity. Waldmann and Graca's 2020 synthesis posed the resulting mechanistic puzzle, which remains open: whether the discrepancy stems from homeostatic expansion during lymphopenia-driven reconstitution, a non-specific anti-inflammatory function of activated natural regulatory T cells, or a different cellular target of suppression (they proposed that Tregs act as gatekeepers on inflamed host endothelium in lymphopenic conditions, versus on dendritic cells in lymphoreplete hosts); at which checkpoint suppression actually operates; which regulatory population dominates in each setting; and whether the endothelial gatekeeping model is correct. No published study resolves any component of this puzzle. The problem is open.

Background

Regulatory T cells (Tregs) are CD4+ T lymphocytes that suppress other immune cells; most are defined by the transcription factor Foxp3. Thymus-derived (“natural”) Tregs develop in the thymus, whereas peripherally induced Tregs arise in the periphery from conventional T cells. “Infectious tolerance” denotes the observation, first made by Qin et al. in 1993, that a tolerance state induced by a short course of antibody therapy can be transmitted to incoming naive T cells, which themselves become tolerant and eventually suppressive, so that regulation spreads and persists beyond the initially regulated cells. In mice, short-term blockade of the CD4 coreceptor and of costimulatory receptors such as CD154 induces long-lasting tolerance to allogeneic skin grafts (grafts genetically mismatched at the major histocompatibility complex, MHC). This therapeutic tolerance is antigen-specific in a characteristic way: mice tolerized to graft A reject an unrelated third-party graft B, accept an (A×B)F1 graft through “linked suppression”, and later extend tolerance to the linked B antigen (Graca et al. 2000). Foxp3+ T cells colonize tolerated grafts (Graca et al. 2002), sustained suppression by Foxp3+ cells is required to maintain the tolerant state (Kendal et al. 2011), and peripherally induced Foxp3+ cells and signaling into T cells by the cytokine transforming growth factor-β (TGF-β) are required to establish it. This pattern is usually read as suppression acting antigen-specifically at the level of antigen presentation, presumably on dendritic cells, the antigen-presenting cells that prime T cells.

A paradox appears when regulation is measured by the classical adoptive-transfer assay: small numbers of purified CD4+CD25+ natural Tregs (CD25 being the alpha chain of the interleukin-2 receptor, a surface marker of Tregs) are mixed with naive responder T cells and injected into lymphopenic (lymphocyte-deficient) recipients carrying skin grafts. There the transferred Tregs suppress rejection not only of the graft type to which their donors were tolerized but also of third-party grafts — no specificity for the donor's transplantation antigens (alloantigens) is detectable (Graca et al. 2004). Candidate explanations date to the early 2000s: regulation may be “a side effect of homeostasis and competition”, since lymphopenic hosts drive homeostatic expansion of the transferred cells and Tregs may win the ensuing competition non-specifically (Barthlott et al. 2003); or activated natural Tregs may exert a broadly anti-inflammatory, non-specific function; or suppression may simply target different cells in the two settings. Relevant to the last option, endothelial cells — the inner lining of blood vessels, which leukocytes must cross by transmigration to enter a graft — can, when inflamed, be induced to express MHC class II and present peptides: vascular endothelium can activate Tregs, recognition of endothelium-expressed self-antigen enables Treg recruitment into tissues (Fu et al. 2014), and Treg transmigration across endothelium is regulated mechanistically distinctly from their intravascular crawling behavior (Snelgrove et al. 2019). Tregs additionally express MS4A4B, a membrane adaptor that increases the sensitivity of the T-cell receptor (TCR) to antigen (Howie et al. 2009), which could let them respond to the minute amounts of inducible self-antigen presented by inflamed endothelium.

In their 2020 perspective “Infectious tolerance. What are we missing?”, Herman Waldmann and Luis Graca assembled these threads into an explicit endothelial gatekeeping hypothesis: in lymphoreplete hosts, Tregs suppress antigen-specifically by acting on dendritic cells, whereas in lymphopenic conditions they act as gatekeepers on inflamed host endothelium presenting self-derived peptides, “decommissioning” vessels and blocking destructive leukocyte entry before tissue damage — which would explain protection of third-party grafts without alloantigen-specific recognition. They stated that this model remained a hypothesis rather than a demonstrated mechanism, and they posed the unresolved questions that define this problem: why antigen specificity is lost in lymphopenic transfer, at which checkpoints suppression operates, which regulatory populations dominate, and how the roles of dendritic cells, endothelium, and local inflammatory cues are partitioned between the two settings.

Problem Statement

The problem, as posed by Waldmann and Graca (2020), is to give a causal mechanistic account of Treg-mediated infectious tolerance that reconciles lymphopenic adoptive-transfer assays with lymphoreplete therapeutic tolerance, and to address the following unresolved questions:

  1. Antigen-specificity discrepancy. Why do lymphopenic adoptive-transfer experiments fail to display the antigen specificity that is evident in therapeutic tolerance in lymphoreplete hosts? Evaluate the candidate explanations — homeostatic expansion and subset competition during lymphopenia-driven immune reconstitution, a non-specific anti-inflammatory function of activated natural regulatory T cells, and a different cellular target of suppression in the two settings — and determine which applies, and whether they operate alone or in combination.

  2. Checkpoints of suppression. Identify the precise checkpoint(s) at which regulation operates in lymphopenic conditions: during T-cell expansion, at endothelial transmigration into the graft, or within the graft tissue itself.

  3. Dominant regulatory populations and target partitioning. Establish which regulatory population — natural (thymus-derived) Tregs, peripherally induced Foxp3+ T cells, or other induced regulatory populations — dominates suppression in each context, or whether suppression instead reflects joint contributions of multiple populations, and how the relative roles of dendritic cells, endothelium, and local inflammatory cues are partitioned between lymphoreplete and lymphopenic settings.

  4. The endothelial gatekeeping model. Directly demonstrate or refute the proposal that in lymphopenic conditions Tregs act as gatekeepers on inflamed host endothelium (e.g., on inflammation-induced endothelial MHC-class-II presentation of self-peptides, decommissioning vessels and blocking leukocyte entry).

These questions are interrelated: they concern where Treg suppression acts and why its apparent antigen specificity differs between the two standard assay settings. All four questions must be addressed, and the general mechanistic account in the first clause is not a substitute for them.

References

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