FOXN1 (GFP/w) reporter hESCs enable identification of integrin-ß4, HLA-DR, and EpCAM as markers of human PSC-derived FOXN1(+) thymic epithelial progenitors.

Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1 (GFP/w) line as a readout, we developed a reproducible protocol for generating FOXN1-GFP(+) thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-ß4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1(+) TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1(+) TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine.

Double-positive thymocytes select mucosal-associated invariant T cells.

NKT and mucosal-associated invariant T (MAIT) cells express semi-invariant TCR and restriction by nonclassical MHC class Ib molecules. Despite common features, the respective development of NKT and MAIT subsets is distinct. NKTs proliferate extensively and acquire effector properties prior to thymic export. MAIT cells exit the thymus as naive cells and acquire an effector/memory phenotype in a process requiring both commensal flora and B cells. During thymic development, NKTs are selected by CD1d-expressing cortical thymocytes; however, the hematopoietic cell type responsible for MAIT cell selection remains unresolved. Using reaggregated thymic organ culture and bone marrow chimeras, we demonstrate that positive selection of mouse iVα19 transgenic and Vß6 transgenic MAIT cell progenitors requires MHC-related 1-expressing CD4(+)CD8(+) double positive thymocytes, whereas thymic B cells, macrophages, and dendritic cell subsets are dispensable. Preincubation of double positive thymocytes with exogenous bacterial ligand increases MHC-related 1 surface expression and enhances mature MAIT cell activation in the in vitro cocultures. The revelation of a common cell type for the selection of both NKT and MAIT subsets raises questions about the mechanisms underlying acquisition of their specific features.

Upregulation of RCAN1 causes Down syndrome-like immune dysfunction.

BACKGROUND: People with Down syndrome (DS) are more susceptible to infections and autoimmune disease, but the molecular genetic basis for these immune defects remains undetermined. In this study, we tested whether increased expression of the chromosome 21 gene RCAN1 contributes to immune dysregulation. METHODS: We investigated the immune phenotype of a mouse model that overexpresses RCAN1. RCAN1 transgenic (TG) mice exhibit T cell abnormalities that bear a striking similarity to the abnormalities described in individuals with DS. RESULTS: RCAN1-TG mice display T cell developmental defects in the thymus and peripheral immune tissues. Thymic cellularity is reduced by substantial losses of mature CD4 and CD8 thymocytes and medullary epithelium. In peripheral immune organs T lymphocytes are reduced in number and exhibit reduced proliferative capacity and aberrant cytokine production. These T cell defects are stem cell intrinsic in that transfer of wild type bone marrow into RCAN1-TG recipients restored medullary thymic epithelium and T cell numbers in the thymus, spleen and lymph nodes. However, bone marrow transplantation failed to improve T cell function, suggesting an additional role for RCAN1 in the non-haemopoietic compartment. CONCLUSIONS: RCAN1 therefore facilitates T cell development and function, and when overexpressed, may contribute to immune dysfunction in DS.

Isolation, characterization, and reaggregate culture of thymic epithelial cells.

The thymus organ is composed of a three-dimensional (3D) network of adjoining epithelium and stromal cells. Bone marrow-derived T cell precursors, upon entering the thymus, interact with and migrate through this cellular network as they differentiate and mature. An essential component of the stroma is the thymic epithelial cells (TEC), which play a vital role in T cell development and induction of self-tolerance for adaptive immunity. TEC can be isolated from the embryonic and adult thymus by a series of gentle enzymatic digestions and characterized into discrete subpopulations based on their expression of surface markers by flow cytometry. Enrichment of adult TEC can be achieved by depletion of hematopoietic cells, allowing sufficient numbers to be purified for subsequent functional and molecular analysis. Although monolayer cultures have been used to study TEC phenotype and T cell interaction, methods that mimic the 3D thymic microenvironment, such as fetal and reaggregate thymic organ cultures, are more accurate for the analysis of TEC function and support more complete T cell development. Herein, we describe methods for the efficient isolation and enrichment of TEC for downstream analyses as well as the reaggregation of embryonic progenitor epithelium to form a functional thymus graft under the kidney capsule.

CCX-CKR deficiency alters thymic stroma impairing thymocyte development and promoting autoimmunity.

The atypical chemokine receptor CCX-CKR regulates bioavailability of CCL19, CCL21, and CCL25, homeostatic chemokines that play crucial roles in thymic lymphopoiesis. Deletion of CCX-CKR results in accelerated experimental autoimmunity induced by immunization. Here we show that CCX-CKR deletion also increases incidence of a spontaneous Sjögren's syndrome-like pathology, characterized by lymphocytic infiltrates in salivary glands and liver of CCX-CKR(-/-) mice, suggestive of a defect in self-tolerance when CCX-CKR is deleted. This prompted detailed examination of the thymus in CCX-CKR(-/-) mice. Negatively selected mature SP cells were less abundant in CCX-CKR(-/-) thymi, yet expansion of both DP and immature SP cells was apparent. Deletion of CCX-CKR also profoundly reduced proportions of DN3 thymocyte precursors and caused DN2 cells to accumulate within the medulla. These effects are likely driven by alterations in thymic stroma as CCX-CKR(-/-) mice have fewer cTECs per thymocyte, and cTECs express the highest level of CCX-CKR in the thymus. A profound decrease in CCL25 within the thymic cortex was observed in CCX-CKR(-/-) thymi, likely accounting for their defects in thymocyte distribution and frequency. These findings identify a novel role for CCX-CKR in regulating cTEC biology, which promotes optimal thymocyte development and selection important for self-tolerant adaptive immunity.

Purified enzymes improve isolation and characterization of the adult thymic epithelium.

The reproducible isolation and accurate characterization of thymic epithelial cell (TEC) subsets is of critical importance to the ongoing study of thymopoiesis and its functional decline with age. The study of adult TEC, however, is significantly hampered due to the severely low stromal to hematopoietic cell ratio. Non-biased digestion and enrichment protocols are thus essential to ensure optimal cell yield and accurate representation of stromal subsets, as close as possible to their in vivo representation. Current digestion protocols predominantly involve diverse, relatively impure enzymatic variants of crude collagenase and collagenase/dispase (col/disp) preparations, which have variable efficacy and are often suboptimal in their ability to mediate complete digestion of thymus tissue. To address these issues we compared traditional col/disp preparations with the latest panel of Liberase products that contain a blend of highly purified collagenase and neutral protease enzymes. Liberase enzymes revealed a more rapid, complete dissociation of thymus tissue; minimizing loss of viability and increasing recovery of thymic stromal cell (TSC) elements. In particular, the recovery and viability of TEC, notably the rare cortical subsets, were significantly enhanced with Liberase products containing medium to high levels of thermolysin. The improved stromal dissociation led to numerically increased TEC yield and total TEC RNA isolated from pooled digests of adult thymus. Furthermore, the increased recovery of TEC enhanced resolution and quantification of TEC subsets in both adult and aged mice, facilitating flow cytometric analysis on a per thymus basis. We further refined the adult TEC phenotype by correlating surface expression of known TEC markers, with expression of intracellular epithelial lineage markers, Keratin 5 and Keratin 8. The data reveal more extensive expression of K8 than previously recognized and indicates considerable heterogeneity still exists within currently defined adult TEC subsets.

Thymic gene transfer of myelin oligodendrocyte glycoprotein ameliorates the onset but not the progression of autoimmune demyelination.

Tolerance induction, and thus prevention of autoimmunity, is linked with the amount of self-antigen presented on thymic stroma. We describe that intrathymic (i.t.) delivery of the autoantigen, myelin oligodendrocyte glycoprotein (MOG), via a lentiviral vector (LV), led to tolerance induction and prevented mice from developing fulminant experimental autoimmune encephalomyelitis (EAE). This protective effect was associated with the long-term expression of antigen in transduced stromal cells, which resulted in the negative selection of MOG-specific T cells and the generation of regulatory T cells (Tregs). These selection events were effective at decreasing T-cell proliferative responses and reduced Th1 and Th17 cytokines. In vivo, this translated to a reduction in inflammation and demyelination with minimal, or no axonal loss in the spinal cords of treated animals. Significantly intrathymic delivery of MOG to mice during the priming phase of the disease failed to suppress clinical symptoms despite mice being previously treated with a clearing anti-CD4 antibody. These results indicate that targeting autoantigens to the thymic stroma might offer an alternative means to induce the de novo production of tolerant, antigen-specific T cells; however, methods that control the number and or the activation of residual autoreactive cells in the periphery are required to successfully treat autoimmune neuroinflammation.

Sex steroid ablation enhances immune reconstitution following cytotoxic antineoplastic therapy in young mice.

Cytotoxic antineoplastic therapy is used to treat malignant disease but results in long-term immunosuppression in postpubertal and adult individuals, leading to increased incidence and severity of opportunistic infections. We have previously shown that sex steroid ablation (SSA) reverses immunodeficiencies associated with age and hematopoietic stem cell transplantation in both autologous and allogeneic settings. In this study, we have assessed the effects of SSA by surgical castration on T cell recovery of young male mice following cyclophosphamide treatment as a model for the impact of chemotherapy. SSA increased thymic cellularity, involving all of the thymocyte subsets and early T lineage progenitors. It also induced early repair of damage to the thymic stromal microenvironment, which is crucial to the recovery of a fully functional T cell-based immune system. These functional changes in thymic stromal subsets included enhanced production of growth factors and chemokines important for thymopoiesis, which preceded increases in both thymocyte and stromal cellularity. These effects collectively translated to an increase in peripheral and splenic naive T cells. In conclusion, SSA enhances T cell recovery following cyclophosphamide treatment of mice, at the level of the thymocytes and their stromal niches. This provides a new approach to immune reconstitution following antineoplastic therapy.

Vascularized tissue engineering mouse chamber model supports thymopoiesis of ectopic thymus tissue grafts.

We have previously established a chamber model of tissue engineering that promotes de novo angiogenesis and vascularization of engrafted cells and tissues when combined with an extracellular matrix. Here we demonstrate that the mouse chamber (MC) model can sustain ectopic grafts of murine fetal thymus lobes and, to a limited degree, human pediatric thymus tissue, resulting in de novo T-cell production. Silicone chambers containing Matrigel((R)) and thymus tissues were placed around exposed epigastric vessels and the ends sealed with bone wax, before implantation into the inguinal fat pad of athymic Balb/c(nu/nu) (nude) mice. Murine, embryonic day 15 (E15) thymus grafts were found to be well vascularized and viable within the MC upon harvest at week 11. In contrast, engraftment of both adult murine and pediatric human thymus tissue was limited, with only one out of the seven human thymus grafts sustaining mature, murine-derived T-cell development. Increased CD4(+) and CD8(+) T-cell numbers were observed in the peripheral blood of nude mice within 2 weeks after E15 thymus-MC grafts (n = 8), compared with nude control mice. Peripheral blood T-cell percentage and subset distribution were comparable to mice receiving conventional thymus kidney capsule grafts. T-cell function of both kidney capsule- and MC-E15 thymus grafts was established via successful rejection of major histocompatibility complex (MHC)-mismatched skin grafts. Sustained growth of fetal thymus tissue in the MC provides an alternative model for the study of thymopoiesis and related applications of T-cell-mediated immunity.

Ablation and regeneration of tolerance-inducing medullary thymic epithelial cells after cyclosporine, cyclophosphamide, and dexamethasone treatment.

Immunosuppressive drugs and cytotoxic chemotherapy agents are designed to kill or suppress autoreactive, alloaggressive, or hyperinflammatory T cells, or disseminated malignancies. However, they also cause severe immunological side effects ranging from interrupted thymopoiesis and general immunodeficiency to, paradoxically, autoimmunity. Consistent with the cross-talk between thymocytes and stromal cells, we now show that these common therapeutic agents have major effects on murine thymic epithelial cells (TEC), crucially required to rebuild immunity posttreatment. We show that the immunosuppressant cyclosporine A, which has been linked to a thymus-dependent autoimmune syndrome in some patients, causes extensive loss of autoimmune regulator (Aire(+)) tolerance-inducing MHC class II(high) medullary TEC (mTEC(high)). Post-cyclosporine A, Aire expression was restored within 7 days. Full recovery of the mTEC(high) subset occurred within 10 days and was linked to a decrease in a relatively resistant MHC class II(low) mTEC subset (mTEC(low)), consistent with a previously described precursor-product relationship. Cyclophosphamide and dexamethasone caused more extensive ablation of thymocytes and stromal cells but again severely depleted tolerance-inducing mTEC(high). Together, these data show that Aire(+) mTECs are highly sensitive to damage and that mTEC regeneration follows a conserved pattern regardless of the treatment regimen used.

Reduced thymic Aire expression and abnormal NF-kappa B2 signaling in a model of systemic autoimmunity.

The thymic stromal niche normally directs the production and export of a self-tolerant T cell repertoire. Many models of spontaneous autoimmunity, however, develop thymic architectural abnormalities before disease onset. Although this is suspected to affect central tolerance induction, creating an autoimmune predisposition, in-depth analysis of the microenvironment within these thymi is lacking, such that the mechanisms and likely direct effects on the T cell repertoire are unknown or speculative. Here we show that NZB mice, the first described model for systemic autoimmunity, demonstrate a complex thymic phenotype, including a lack of the autoimmune regulator (Aire), early defects in thymic epithelial cell (TEC) expansion, and evidence for altered NF-kappaB2 signaling. Analysis of medullary TEC revealed a numerical loss of the Aire-expressing MHC class II(high) (mTEC-high) subset as well reduced Aire protein and mRNA per cell. RelB expression was also reduced, while chemokines CCL19 and CCL21 were increased. Unexpectedly, the proportion of cortex and medulla in the NZB mice was normal from 36 wk, despite worsening architectural abnormalities. These data show that the NZB defect is more complex than previously appreciated, segregating into early numerical TEC deficiencies that correct with age, late degeneration of the niche architecture that does not affect TEC number, and a persistent reduction in Aire and RelB expression per cell acquired upon mTEC-high differentiation.

Strategies for reconstituting and boosting T cell-based immunity following haematopoietic stem cell transplantation: pre-clinical and clinical approaches.

Poor immune recovery is characteristic of bone marrow transplantation and leads to high levels of morbidity and mortality. The primary underlying cause is a compromised thymic function, resulting from age-induced atrophy and further compounded by the damaging effects of cytoablative conditioning regimes on thymic epithelial cells (TEC). Several strategies have been proposed to enhance T cell reconstitution. Some, such as the use of single biological agents, are currently being tested in clinical trials. However, a more rational approach to immune restoration will be to leverage the evolving repertoire of new technologies. Specifically, the combined targeting of TEC, thymocytes and peripheral T cells, together with the bone marrow niches, promises a more strategic clinical therapeutic platform.

The lymphotoxin pathway regulates Aire-independent expression of ectopic genes and chemokines in thymic stromal cells.

Medullary thymic epithelial cells (mTEC) play an important and unique role in central tolerance, expressing tissue-restricted Ags (TRA) which delete thymocytes autoreactive to peripheral organs. Since deficiencies in this cell type or activity can lead to devastating autoimmune diseases, it is important to understand the factors which regulate mTEC differentiation and function. Lymphotoxin (LT) ligands and the LTbetaR have been recently shown to be important regulators of mTEC biology; however, the precise role of this pathway in the thymus is not clear. In this study, we have investigated the impact of this signaling pathway in greater detail, focusing not only on mTEC but also on other thymic stromal cell subsets. LTbetaR expression was found in all TEC subsets, but the highest levels were detected in MTS-15(+) thymic fibroblasts. Rather than directing the expression of the autoimmune regulator Aire in mTEC, we found LTbetaR signals were important for TRA expression in a distinct population of mTEC characterized by low levels of MHC class II (mTEC(low)), as well as maintenance of MTS-15(+) fibroblasts. In addition, thymic stromal cell subsets from LT-deficient mice exhibit defects in chemokine production similar to that found in peripheral lymphoid organs of Lta(-/-) and Ltbr(-/-) mice. Thus, we propose a broader role for LTalpha1beta2-LTbetaR signaling in the maintenance of the thymic microenvironments, specifically by regulating TRA and chemokine expression in mTEC(low) for efficient induction of central tolerance.

Unbiased analysis, enrichment and purification of thymic stromal cells.

The microenvironment of the thymus consists of functionally discrete niches composed of distinct stromal cell subsets. Clinically relevant changes affecting T-cell differentiation occur within these niches with age and injury caused by irradiation and chemotherapy treatments. The study of thymic stromal cells has been hampered by the technical difficulty in isolating significant numbers of this important population. Here we present an improved protocol for enzymatic isolation of stromal cells that enables comparative flow cytometric analyses and their purification for downstream cellular or molecular analysis. Fractions analyzed throughout enzymatic digestion of the thymus revealed that various stromal subsets are isolated at characteristic intervals. This highlights the importance of pooling all cells isolated from the thymus for numerical and phenotypic analysis to avoid biased representation of subpopulations. We also describe refined magnetic bead separation techniques that yield almost pure preparations of CD45(-) stroma. Sorting of these suspensions using defined markers enabled purification of the major epithelial subsets, confirmed by keratin staining and PCR analysis. This three-step procedure represents a rapid, reproducible method for the unbiased purification of the stromal cells that direct thymic T-cell differentiation.

Impact of niche aging on thymic regeneration and immune reconstitution.

The immune system undergoes dramatic changes with age-the thymus involutes, particularly from puberty, with the gradual loss of newly produced naïve T cells resulting in a restricted T cell receptor repertoire, skewed towards memory cells. Coupled with a similar, though less dramatic age-linked decline in bone marrow function, this translates to a reduction in immune responsiveness and has important clinical implications particularly in immune reconstitution following cytoablation regimes for cancer treatment or following severe viral infections such as HIV. Given that long-term reconstitution of the immune system is dependent on the bi-directional interplay between primary lymphoid organ stromal cells and the progenitors whose downstream differentiation they direct, regeneration of the thymus is fundamental to developing new strategies for the clinical management of many major diseases of immunological origin. This review will discuss the impact of aging on primary lymphoid organ niches and current approaches for thymic regeneration and immune reconstitution.

Thymic generation and regeneration: a new paradigm for establishing clinical tolerance of stem cell-based therapies.

Tolerance to tissue-engineering products is a major obstacle hindering the clinical application of this rapidly advancing technology. Manipulation of central tolerance, by establishing thymus chimerism of both donor and host-derived haemopoietic cells (haemopoietic stem cell transplant--HSCT), should purge any T cells reactive to potential donor organ or tissue transplant. A functional thymus, however, is required to induce chimerism and repopulate the peripheral T cell pool, but age-related thymic atrophy and damage caused by ablative conditioning regimes significantly reduce thymic function and increase incident of infection-dependent morbidity and mortality. Thus rejuvenation of the thymus alongside HSCT may potentiate the use of this strategy in the clinic. In addition, the use of thymic epithelial progenitor cell technology may allow growth of ex vivo thymic tissue for use in clinical situations of immunodeficiency as well as in establishing tolerance to tissue/organ products derived from the same source.

A unique thymic fibroblast population revealed by the monoclonal antibody MTS-15.

T cell differentiation in the thymus is dependent upon signals from thymic stromal cells. Most studies into the nature of these signals have focused only on the support provided by the thymic epithelium, but there is an emerging view that other stromal cells such as mesenchymal fibroblasts may also be involved. Study of the latter has been hindered by a lack of appropriate markers, particularly those allowing their isolation. In this study, we describe a new surface marker of thymic stroma, MTS-15, and demonstrate its specificity for fibroblasts and a subset of endothelial cells. Coculture experiments showed that the determinant could be transferred between cells. Extensive biochemical analysis demonstrated that the Ag bound by MTS-15 was the glycosphingolipid Forssman determinant, consistent with the distribution observed. Transcriptional analysis of purified MTS-15(+) thymic fibroblasts revealed a unique expression profile for a number of chemokines and growth factors important to thymocyte and epithelial cell development. In a model of cyclophosphamide-induced thymic involution and regeneration, fibroblasts were found to expand extensively and express growth factors important to epithelial proliferation and increased T cell production just before thymic regeneration. Overall, this study identifies a useful marker of thymic fibroblasts and highlights this subpopulation as a key player in thymic function by virtue of their support of both thymocytes and epithelial cells.

Developmental kinetics, turnover, and stimulatory capacity of thymic epithelial cells.

Despite the importance of thymic stromal cells to T-cell development, relatively little is known about their biology. Here, we use single-cell analysis of stromal cells to analyze extensive changes in the number and composition of thymic stroma throughout life, revealing a surprisingly dynamic population. Phenotypic progression of thymic epithelial subsets was assessed at high resolution in young mice to provide a developmental framework. The cellular and molecular requirements of adult epithelium were studied, using various mutant mice to demonstrate new cross talk checkpoints dependent on RelB in the cortex and CD40 in the medulla. With the use of Ki67 and BrdU labeling, the turnover of thymic epithelium was found to be rapid, but then diminished on thymic involution. The various defects in stromal turnover and composition that accompanied involution were rapidly reversed following sex steroid ablation. Unexpectedly, mature cortical and medullary epithelium showed a potent capacity to stimulate naive T cells, comparable to that of thymic dendritic cells. Overall, these studies show that the thymic stroma is a surprisingly dynamic population and may have a more direct role in negative selection than previously thought.

CCR7-dependent cortex-to-medulla migration of positively selected thymocytes is essential for establishing central tolerance.

Immature CD4+CD8+ thymocytes, which are generated in the thymic cortex, are induced upon positive selection to differentiate into mature T lymphocytes and relocate to the thymic medulla. It was recently shown that a chemokine signal via CCR7 is essential for the cortex-to-medulla migration of positively selected thymocytes in the thymus. However, the role of the cortex-to-medulla migration in T cell development and selection has remained unclear. The present study shows that the developmental kinetics and the thymic export of mature thymocytes were undisturbed in adult mice lacking CCR7 or its ligands (CCR7L). The inhibition of sphingosine-1-phosphate-mediated lymphocyte egress from the thymus led to the accumulation of mature thymocytes in the cortex of CCR7- or CCR7L-deficient mice, unlike the accumulation in the medulla of normal mice, thereby suggesting that mature thymocytes may be exported directly from the cortex in the absence of CCR7 signals. However, the thymocytes that were generated in the absence of CCR7 or CCR7L were potent in causing autoimmune dacryoadenitis and sialadenitis in mice and were thus incapable of establishing central tolerance to organ-specific antigens. These results indicate that CCR7-mediated cortex-to-medulla migration of thymocytes is essential for establishing central tolerance rather than for supporting the maturation or export of thymocytes.