Frederico S. Regateiro

Sustained suppression by Foxp3+ regulatory T cells is vital for infectious transplantation tolerance.

A new genetic mouse model demonstrates the necessity of Foxp3+ T reg cells for infectious tolerance.

Generation of anti‐inflammatory adenosine byleukocytes is regulated by TGF‐β

Levels of anti‐inflammatory extracellular adenosine are controlled by the sequential action of the ectonucleotidases CD39 and CD73, whose expression in CD4+ T cells has been associated with natural regulatory T cells (nTregs). We here show that CD73 expression on activated murine CD4+ T cells is induced by TGF‐β independently of Foxp3 expression, operates at the transcriptional level and translates into gain of functional capacity to generate adenosine. In the presence of AMP, CD73 induced by TGF‐β generates adenosine able to suppress proliferation of activated CD4+ T cells in vitro. These effects are contextual and opposed by proinflammatory cytokines. CD73 is also upregulated by TGF‐β in CD8+ T cells, DCs and macrophages, so providing an amplification mechanism for adenosine generation in tissue microenvironments. Together, these findings expose a novel anti‐inflammatory role for TGF‐β.

CD73 and adenosine generation in the creation of regulatory microenvironments.

Extracellular adenosine 5′‐triphosphate (ATP) acts on many immune cells to promote inflammation. Conversely, the ATP metabolite adenosine is mainly an anti‐inflammatory molecule. The ecto‐enzymes CD39 and CD73 can dephosphorylate extracellular ATP to adenosine, thereby controlling this important pathway of immune modulation. Despite their established roles in the immune system, little is known of how CD39 and CD73 are themselves regulated. Recent data have shown that CD73 expression and adenosine generation are up‐regulated by transforming growth factor‐β, depending on the cytokine content of the local microenvironment. We review here these recent findings and discuss their implications in disease.

Connecting the mechanisms of T‐cell regulation: dendritic cells as the missing link

Summary:  A variety of different molecular mechanisms have been proposed to explain the suppressive action of regulatory T cells, including the production of anti‐inflammatory cytokines, negative costimulatory ligands, indoleamine 2,3‐dioxygenase‐mediated tryptophan catabolism, CD73‐mediated adenosine generation, and downregulation of antigen‐presenting cells. Until now it has been unclear how important each of these different mechanisms might be and how they are coordinated. In this review, we examine the hypothesis that it is the interaction between regulatory T cells and dendritic cells that creates a local microenvironment depleted of essential amino acids and rich in adenosine that leads to the amplification of a range of different tolerogenic signals. These signals are all eventually integrated by mammalian target of rapamycin inhibition, which enables the induction of new forkhead box protein 3‐expressing Tregs. If correct, this provides a molecular explanation for the in vivo phenomena of linked suppression and infectious tolerance.

TGF-β in transplantation tolerance

TGF-β is a cytokine required for the induction and maintenance of transplantation tolerance in animal models. TGF-β mediates anti-inflammatory effects by acting on many immune cell-types. Central for transplantation tolerance is the role for TGF-β in the induction of Foxp3 and regulatory capacity in CD4(+) T cells. Recently, however, the general anti-inflammatory role of TGF-β in CD4(+) T cell polarization was questioned by the discovery that, in the presence of inflammatory cytokines such as IL-6 or IL-1, TGF-β drives the differentiation of Th17 cells associated with transplant rejection. A better understanding of the factors determining TGF-β production and activation, Foxp3 induction and Treg stability is vital for the development of tolerogenic strategies in transplantation.

Foxp3 Expression Is Required for the Induction of Therapeutic Tissue Tolerance

CD4+Foxp3+ regulatory T cells (Treg) are essential for immune homeostasis and maintenance of self-tolerance. They are produced in the thymus and also generated de novo in the periphery in a TGF-β–dependent manner. Foxp3+ Treg are also required to achieve tolerance to transplanted tissues when induced by coreceptor or costimulation blockade. Using TCR-transgenic mice to avoid issues of autoimmune pathology, we show that Foxp3 expression is both necessary and sufficient for tissue tolerance by coreceptor blockade. Moreover, the known need in tolerance induction for TGF-β signaling to T cells can wholly be explained by its role in induction of Foxp3, as such signaling proved dispensable for the suppressive process. We analyzed the relative contribution of TGF-β and Foxp3 to the transcriptome of TGF-β–induced Treg and showed that TGF-β elicited a large set of downregulated signature genes. The number of genes uniquely modulated due to the influence of Foxp3 alone was surprisingly limited. Retroviral-mediated conditional nuclear expression of Foxp3 proved sufficient to confer transplant-suppressive potency on CD4+ T cells and was lost once nuclear Foxp3 expression was extinguished. These data support a dual role for TGF-β and Foxp3 in induced tolerance, in which TGF-β stimulates Foxp3 expression, for which sustained expression is then associated with acquisition of tolerance.

Th17 Cells Induce a Distinct Graft Rejection Response That Does Not Require IL-17A

IL‐17A‐producing helper T (Th17) cells have been implicated in the pathogenesis of autoimmune disease, inflammatory bowel disease and graft rejection, however the mechanisms by which they cause tissue damage remain ill‐defined. We examined what damage Th17 cell lines could inflict on allogeneic skin grafts in the absence of other adaptive lymphocytes. CD4+ Th17 cell lines were generated from two TCR transgenic mouse strains, A1(M).RAG1−/− and Marilyn, each monospecific for the male antigen Dby. After prolonged in vitro culture in polarizing conditions, Th17 lines produced high levels of IL‐17A with inherently variable levels of interferon gamma (IFNγ) and these cells were able to maintain IL‐17A expression following adoptive transfer into lymphopenic mice. When transferred into lymphopenic recipients of male skin grafts, Th17 lines elicited a damaging reaction within the graft associated with pathological findings of epidermal hyperplasia and neutrophil infiltration. Th17 cells could be found in the grafted skins and spleens of recipients and maintained their polarized phenotype both in vivo and after ex vivo restimulation. Antibody‐mediated neutralization of IL‐17A or IFNγ did not interfere with Th17‐induced pathology, nor did it prevent neutrophil infiltration. In conclusion, tissue damage by Th17 cells does not require IL‐17A.

Activation rather than Foxp3 expression determines that TGF-β-induced regulatory T cells out-compete naïve T cells in dendritic cell clustering.

Regulatory T (Treg) cells are critically important for the maintenance of immunological tolerance. Both centrally arising natural nTreg cells and those emerging in the periphery in response to TGF‐β, iTreg cells, play a role in the control of unwanted immune responses. Treg cells adopt multiple mechanisms to inhibit effector T cells, yet it is unclear whether these mechanisms are shared by nTreg cells and iTreg cells alike. Here, we show that iTreg cells, like nTreg cells, are able to out‐compete naïve T cells in clustering around dendritic cells (DCs). However, using both a tamoxifen‐responsive inducible Foxp3 retroviral construct and TGF‐β‐induced iTreg cells from hCD2‐Foxp3 knock in reporter mice, we show that it is prior antigen‐induced activation rather than Foxp3 expression per se that determines the ability of iTreg cells to competitively cluster around DCs. We found no difference in the capacity of iTreg cells to displace naïve T cells around DCs to that of Tr1, Th1, Th2, or Th9 cells. An important difference was, however, that clustering of iTreg cells around DCs, just as for naïve T cells, did not effectively activate DCs.