Nicholas I. McCarthy

Differential Requirement for CCR4 and CCR7 during the Development of Innate and Adaptive αβT Cells in the Adult Thymus

αβT cell development depends upon serial migration of thymocyte precursors through cortical and medullary microenvironments, enabling specialized stromal cells to provide important signals at specific stages of their development. Although conventional αβT cells are subject to clonal deletion in the medulla, entry into the thymus medulla also fosters αβT cell differentiation. For example, during postnatal periods, the medulla is involved in the intrathymic generation of multiple αβT cell lineages, notably the induction of Foxp3+ regulatory T cell development and the completion of invariant NKT cell development. Although migration of conventional αβT cells to the medulla is mediated by the chemokine receptor CCR7, how other T cell subsets gain access to medullary areas during their normal development is not clear. In this study, we show that combining a panel of thymocyte maturation markers with cell surface analysis of CCR7 and CCR4 identifies distinct stages in the development of multiple αβT cell lineages in the thymus. Although Aire regulates expression of the CCR4 ligands CCL17 and CCL22, we show that CCR4 is dispensable for thymocyte migration and development in the adult thymus, demonstrating defective T cell development in Aire−/− mice is not because of a loss of CCR4-mediated migration. Moreover, we reveal that CCR7 controls the development of invariant NKT cells by enabling their access to IL-15 trans-presentation in the thymic medulla and influences the balance of early and late intrathymic stages of Foxp3+ regulatory T cell development. Collectively, our data identify novel roles for CCR7 during intrathymic T cell development, highlighting its importance in enabling multiple αβT cell lineages to access the thymic medulla.

Affinity for self antigen selects Treg cells with distinct functional properties

The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITRhiPD-1hiCD25hi (Triplehi) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITRloPD-1loCD25lo (Triplelo) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4+ Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triplehi-like and Triplelo-like CD4+ T cells zsuper> T cells infiltrated the skin, whereas Scurfy TripleloCD4+ T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.

CCR7 Controls Thymus Recirculation, but Not Production and Emigration, of Foxp3+ T Cells

Summary Current models of Foxp3+ regulatory T cell (Treg) development involve CCR7-mediated migration of thymocytes into the thymus medulla to enable essential interactions with medullary epithelium. However, increased Foxp3+ thymic Treg numbers in Ccr7−/− mice challenge this view, and the role of CCR7 in Treg development, emigration, and/or recirculation is unknown. Here, we have examined CCR7 and Rag2pGFP levels during Treg development and generated Rag2pGFPCcr7−/− mice to study its impact on the intrathymic Treg pool. We reveal surprising developmental heterogeneity in thymocytes described as Treg precursors, showing that they contain recirculating CCR6+CCR7−Rag2pGFP− T cells. Although CCR7 defines bona fide Rag2GFP+ Treg precursors, it is not required for Treg production and emigration. Rather, we show that lack of CCR7 renders the thymus more receptive to Treg thymus homing. Our study reveals a role for CCR7 in limiting Treg recirculation back to the thymus and enables separation of the mechanisms controlling Treg production and thymic recirculation.

Osteoprotegerin-Mediated Homeostasis of Rank+ Thymic Epithelial Cells Does Not Limit Foxp3+ Regulatory T Cell Development.

In the thymus, medullary thymic epithelial cells (mTEC) regulate T cell tolerance via negative selection and Foxp3+ regulatory T cell (Treg) development, and alterations in the mTEC compartment can lead to tolerance breakdown and autoimmunity. Both the receptor activator for NF-κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) axis and expression of the transcriptional regulator Aire are involved in the regulation of thymus medullary microenvironments. However, their impact on the mechanisms controlling mTEC homeostasis is poorly understood, as are the processes that enable the thymus medulla to support the balanced production of mTEC-dependent Foxp3+ Treg. In this study, we have investigated the control of mTEC homeostasis and examined how this process impacts the efficacy of Foxp3+ Treg development. Using newly generated RANK Venus reporter mice, we identify distinct RANK+ subsets that reside within both the mTEChi and mTEClo compartments and that represent direct targets of OPG-mediated control. Moreover, by mapping OPG expression to a subset of Aire+ mTEC, our data show how cis- and trans-acting mechanisms are able to control the thymus medulla by operating on multiple mTEC targets. Finally, we show that whereas the increase in mTEC availability in OPG-deficient (Tnfrsf11b−/−) mice impacts the intrathymic Foxp3+ Treg pool by enhancing peripheral Treg recirculation back to the thymus, it does not alter the number of de novo Rag2pGFP+Foxp3+ Treg that are generated. Collectively, our study defines patterns of RANK expression within the thymus medulla, and it shows that mTEC homeostasis is not a rate-limiting step in intrathymic Foxp3+ Treg production.

Mechanisms of Thymus Medulla Development and Function

The development of CD4(+) helper and CD8(+) cytotoxic T-cells expressing the αβ form of the T-cell receptor (αβTCR) takes place in the thymus, a primary lymphoid organ containing distinct cortical and medullary microenvironments. While the cortex represents a site of early T-cell precursor development, and the positive selection of CD4(+)8(+) thymocytes, the thymic medulla plays a key role in tolerance induction, ensuring that thymic emigrants are purged of autoreactive αβTCR specificities. In recent years, advances have been made in understanding the development and function of thymic medullary epithelial cells, most notably the subset defined by expression of the Autoimmune Regulator (Aire) gene. Here, we summarize current knowledge of the developmental mechanisms regulating thymus medulla development, and examine the role of the thymus medulla in recessive (negative selection) and dominant (T-regulatory cell) tolerance.

Natural Th17 cells are critically regulated by functional medullary thymic microenvironments.

The thymic medulla is critical for the enforcement of central tolerance. In addition to deletion of auto-reactive T-cells, the thymic medulla supports the maturation of heterogeneous natural αβT-cells linked to tolerance mechanisms. Natural IL-17-secreting CD4+αβT-cells (nTh17) represent recently described natural αβT-cells that mature and undergo functional priming intrathymically. Despite a proposed potential to impact upon either protective or pathological inflammatory responses, the intrathymic mechanisms regulating the balance of nTh17 development are unclear. Here we compare the development of distinct natural αβT-cells in the thymus. We reveal that thymic stromal MHC class II expression and RelB-dependent medullary thymic epithelial cells (mTEC), including Aire+ mTEC, are an essential requirement for nTh17 development. nTh17 demonstrate a partial, non-redundant requirement for both ICOS-ligand and CD80/86 costimulation, with a dispensable role for CD80/86 expression by thymic epithelial cells. Although mTEC constitutively expressed inducible nitric oxide synthase (iNOS), a critical negative regulator of conventional Th17 differentiation, iNOS was not essential to constrain thymic nTh17. These findings highlight the critical role of the thymic medulla in the differential regulation of novel natural αβT-cell subsets, and reveal additional layers of thymic medullary regulation of T-cell driven autoimmunity and inflammation.

Control of the thymic medulla and its influence on αβT-cell development.

The thymus is a primary lymphoid tissue that supports the generation of αβT cells. In this review, we describe the processes that give rise to the thymus medulla, a site that nurtures self‐tolerant T‐cell generation following positive selection events that take place in the cortex. To summarize the developmental pathways that generate medullary thymic epithelial cells (mTEC) from their immature progenitors, we describe work on both the initial emergence of the medulla during embryogenesis, and the maintenance of the medulla during postnatal stages. We also investigate the varying roles that receptors belonging to the tumor necrosis factor receptor superfamily have on thymus medulla development and formation, and highlight the impact that T‐cell development has on thymus medulla formation. Finally, we examine the evidence that the thymic medulla plays an important role during the intrathymic generation of distinct αβT‐cell subtypes. Collectively, these studies provide new insight into the development and functional importance of medullary microenvironments during self‐tolerant T‐cell production in the thymus.

Prdm1 Regulates Thymic Epithelial Function To Prevent Autoimmunity

Autoimmunity is largely prevented by medullary thymic epithelial cells (TECs) through their expression and presentation of tissue-specific Ags to developing thymocytes, resulting in deletion of self-reactive T cells and supporting regulatory T cell development. The transcription factor Prdm1 has been implicated in autoimmune diseases in humans through genome-wide association studies and in mice using cell type–specific deletion of Prdm1 in T and dendritic cells. In this article, we demonstrate that Prdm1 functions in TECs to prevent autoimmunity in mice. Prdm1 is expressed by a subset of mouse TECs, and conditional deletion of Prdm1 in either Keratin 14– or Foxn1-expressing cells in mice resulted in multisymptom autoimmune pathology. Notably, the development of Foxp3+ regulatory T cells occurs normally in the absence of Blimp1. Importantly, nude mice developed anti-nuclear Abs when transplanted with Prdm1 null TECs, but not wild-type TECs, indicating that Prdm1 functions in TECs to regulate autoantibody production. We show that Prdm1 acts independently of Aire, a crucial transcription factor implicated in medullary TEC function. Collectively, our data highlight a previously unrecognized role for Prdm1 in regulating thymic epithelial function.

Aire controls the recirculation of murine Foxp3+ regulatory T-cells back to the thymus

In the thymus, medullary thymic epithelial cells (mTEC) determine the fate of newly selected CD4+ and CD8+ single positive (SP) thymocytes. For example, mTEC expression of Aire controls intrathymic self‐antigen availability for negative selection. Interestingly, alterations in both Foxp3+ Regulatory T‐cells (T‐Reg) and conventional SP thymocytes in Aire−/− mice suggest additional, yet poorly understood, roles for Aire during intrathymic T‐cell development. To examine this, we analysed thymocytes from Aire−/− mice using Rag2GFP and Foxp3 expression, and a recently described CD69/MHCI subset definition of post‐selection CD4+ conventional thymocytes. We show that while Aire is dispensable for de novo generation of conventional αβT‐cells, it plays a key role in controlling the intrathymic T‐Reg pool. Surprisingly, a decline in intrathymic T‐Reg in Aire−/− mice maps to a reduction in mature recirculating Rag2GFP− T‐Reg that express CCR6 and re‐enter the thymus from the periphery. Furthermore, we show mTEC expression of the CCR6 ligand CCL20 is reduced in Aire−/− mice, and that CCR6 is required for T‐Reg recirculation back to the thymus. Collectively, our study re‐defines requirements for late stage intrathymic αβT‐cell development, and demonstrates that Aire controls a CCR6‐CCL20 axis that determines the developmental makeup of the intrathymic T‐Reg pool.

Thymic Microenvironments: Development, Organization, and Function

The thymus is a primary lymphoid tissue that is unique in its ability to support a full program of T cell development. Thymic microenvironments consist of anatomically distinct cortical and medullary areas that shape the developing T cell repertoire in a step-wise fashion to become both self-tolerant and responsive to foreign antigens. Specialized epithelial cells in the cortex and medulla, together with mesenchyme populations that are becoming increasingly well defined, are key in providing the signals that control T cell development. Here, we outline our current understanding of the developmental pathways that give rise to distinct cortical and medullary epithelial cell lineages in the thymus. We also summarize knowledge of the functional heterogeneity of thymic stromal cells in relation to their roles in αβT cell development.