Thymic Epithelium Research Articles

Dynamics of thymus organogenesis and colonization in early human development.

The thymus is the central site of T-cell development and thus is of fundamental importance to the immune system, but little information exists regarding molecular regulation of thymus development in humans. Here we demonstrate, via spatial and temporal expression analyses, that the genetic mechanisms known to regulate mouse thymus organogenesis are conserved in humans. In addition, we provide molecular evidence that the human thymic epithelium derives solely from the third pharyngeal pouch, as in the mouse, in contrast to previous suggestions. Finally, we define the timing of onset of hematopoietic cell colonization and epithelial cell differentiation in the human thymic primordium, showing, unexpectedly, that the first colonizing hematopoietic cells are CD45+CD34int/-. Collectively, our data provide essential information for translation of principles established in the mouse to the human, and are of particular relevance to development of improved strategies for enhancing immune reconstitution in patients.

DH consults on setting up list of managers barred from NHS

It was found that donor's serum and serum of patients with rheumatic fever, erysipelas and myasthenia gravis contained autoantibodies to the cytoplasmic antigens and to the antigen of perinuclear zone of differentiated layers cells of human epidermis. Using the serum with different level of autoantibodies to these epidermal antigens it's localization in the human thymus epithelium was determined. It was shown that perinuclear antigen of differentiated epidermis cells is localized in the cytoplasm of cortical and medullar thymus epithelial cells and in the perinuclear zone of some cells in Hassall's corpuscles. The cytoplasmic antigen of differentiated epidermal cells is detected only in the cytoplasm of the Hassall's corpuscle cells. The ability of many tissues (possibly all of them) and in the first turn of epidermis to produce lymphokine along with immunomodulating properties of thymus hetero-organic antigens enables to consider these thymus antigens as a complex of immunomodulating factor proper for other tissues. These factors may provide the competence of certain T-cell subpopulations to different organ's tissues necessary for proceedings them the immunological survey both in normal and pathological conditions.

Ephrin-B–Dependent Thymic Epithelial Cell–Thymocyte Interactions Are Necessary for Correct T Cell Differentiation and Thymus Histology Organization: Relevance for Thymic Cortex Development

Previous analysis on the thymus of erythropoietin-producing hepatocyte kinases (Eph) B knockout mice and chimeras revealed that Eph-Eph receptor-interacting proteins (ephrins) are expressed both on T cells and thymic epithelial cells (TECs) and play a role in defining the thymus microenvironments. In the current study, we have used the Cre-LoxP system to selectively delete ephrin-B1 and/or ephrin-B2 in either thymocytes (EfnB1(thy/thy), EfnB2(thy/thy), and EfnB1(thy/thy)EfnB2(thy/thy) mice) or TECs (EfnB1(tec/tec), EfnB2(tec/tec), and EfnB1(tec/tec)EfnB2(tec/tec) mice) and determine the relevance of these Eph ligands in T cell differentiation and thymus histology. Our results indicate that ephrin-B1 and ephrin-B2 expressed on thymocytes play an autonomous role in T cell development and, expressed on TECs, their nonautonomous roles are partially overlapping. The effects of the lack of ephrin-B1 and/or ephrin-B2 on either thymocytes or TECs are more severe and specific on thymic epithelium, contribute to the cell intermingling necessary for thymus organization, and affect cortical TEC subpopulation phenotype and location. Moreover, ephrin-B1 and ephrin-B2 seem to be involved in the temporal appearance of distinct cortical TECs subsets defined by different Ly51 levels of expression on the ontogeny.

Analysis of APC Types Involved in CD4 Tolerance and Regulatory T Cell Generation Using Reaggregated Thymic Organ Cultures

Tolerance to self-Ags is generated in the thymus. Both epithelial and hematopoietic thymic stromal cells play an active and essential role in this process. However, the role of each of the various stromal cell types remains unresolved. To our knowledge, we describe the first comparative analysis of several types of thymic hematopoietic stromal cells (THSCs) for their ability to induce CD4 tolerance to self, in parallel with the thymic epithelium. The THSCs--two types of conventional dendritic cells (cDCs), plasmacytoid dendritic cells, macrophages (MΦs), B lymphocytes, and eosinophils--were first characterized and quantified in adult mouse thymus. They were then examined in reaggregated thymic organ cultures containing mixtures of monoclonal and polyclonal thymocytes. This thymocyte mixture allows for the analysis of Ag-specific events while avoiding the extreme skewing frequently seen in purely monoclonal systems. Our data indicate that thymic epithelium alone is capable of promoting self-tolerance by eliminating autoreactive CD4 single-positive thymocytes and by supporting regulatory T cell (Treg) development. We also show that both non-Treg CD4 single-positive thymocytes and Tregs are efficiently deleted by the two populations of cDCs present in the thymus, as well as to a lesser extent by MΦs. Plasmacytoid dendritic cells, B lymphocytes, and eosinophils were not able to do so. Finally, cDCs were also the most efficient THSCs at supporting Treg development in the thymus, suggesting that although they may share some characteristics required for negative selection with MΦs, they do not share those required for the support of Treg development, making cDCs a unique cell subset in the thymus.

Cbx4 regulates the proliferation of thymic epithelial cells and thymus function

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.

Cbx4 regulates the proliferation of thymic epithelial cells and thymus function

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.

Plxnd1 Expression in Thymocytes Regulates Their Intrathymic Migration While That in Thymic Endothelium Impacts Medullary Topology

An important role for plexinD1 in thymic development is inferred from studies of germline Plxnd1 knockout (KO) mice where mislocalized CD69+ thymocytes as well as ectopic thymic subcapsular medullary structures were observed. Given embryonic lethality of the Plxnd1−/− genotype, fetal liver transplantation was employed in these prior analyses. Such embryonic hematopoietic reconstitution may have transferred Plxnd1 KO endothelial and/or epithelial stem cells in addition to Plxnd1 KO lymphoid progenitors, thereby contributing to that phenotype. Here we use Plxnd1flox/flox mice crossed to pLck-Cre, pKeratin14-Cre, or pTek-Cre transgenic animals to create cell-type specific conditional knockout (CKO) lines involving thymocytes (D1ThyCKO), thymic epithelium (D1EpCKO), and thymic endothelium (D1EnCKO), respectively. These CKOs allowed us to directly assess the role of plexinD1 in each lineage. Loss of plexinD1 expression on double positive (DP) thymocytes leads to their aberrant migration and cortical retention after TCR-mediated positive selection. In contrast, ectopic medulla formation is a consequence of loss of plexinD1 expression on endothelial cells, in turn linked to dysregulation of thymic angiogenesis. D1EpCKO thymi manifest neither abnormality. Collectively, our findings underscore the non-redundant roles for plexinD1 on thymocytes and endothelium, including the dynamic nature of medulla formation resulting from crosstalk between these thymic cellular components.

FOXN1: A Master Regulator Gene of Thymic Epithelial Development Program

T cell ontogeny is a sophisticated process, which takes place within the thymus through a series of well-defined discrete stages. The process requires a proper lympho-stromal interaction. In particular, cortical and medullary thymic epithelial cells (cTECs, mTECs) drive T cell differentiation, education, and selection processes, while the thymocyte-dependent signals allow thymic epithelial cells (TECs) to maturate and provide an appropriate thymic microenvironment. Alterations in genes implicated in thymus organogenesis, including Tbx1, Pax1, Pax3, Pax9, Hoxa3, Eya1, and Six1, affect this well-orchestrated process, leading to disruption of thymic architecture. Of note, in both human and mice, the primordial TECs are yet unable to fully support T cell development and only after the transcriptional activation of the Forkhead-box n1 (FOXN1) gene in the thymic epithelium this essential function is acquired. FOXN1 is a master regulator in the TEC lineage specification in that it down-stream promotes transcription of genes, which, in turn, regulate TECs differentiation. In particular, FOXN1 mainly regulates TEC patterning in the fetal stage and TEC homeostasis in the post-natal thymus. An inborn null mutation in FOXN1 leads to Nude/severe combined immunodeficiency (SCID) phenotype in mouse, rat, and humans. In Foxn1−/− nude animals, initial formation of the primordial organ is arrested and the primordium is not colonized by hematopoietic precursors, causing a severe primary T cell immunodeficiency. In humans, the Nude/SCID phenotype is characterized by congenital alopecia of the scalp, eyebrows, and eyelashes, nail dystrophy, and a severe T cell immunodeficiency, inherited as an autosomal recessive disorder. Aim of this review is to summarize all the scientific information so far available to better characterize the pivotal role of the master regulator FOXN1 transcription factor in the TEC lineage specifications and functionality.

Invasive thymoma metastatic to the cavernous sinus

Background:Thymomas are typically benign tumors of thymic epithelium. Metastases to distal sites, particularly intracranial locations, are extremely rare. Herein, we present the third case of thymoma and the second invasive thymoma to metastasize to the cavernous sinus, adjacent to the pituitary.Case Description:A 41-year-old female patient presented with headaches, stuffy nose, and drooping of the right face. A magnetic resonance imaging scan revealed a complex, multilobulated mass centered upon the right cavernous sinus. The mass was removed via transsphenoidal surgery, and histopathological investigation confirmed the diagnosis of metastatic thymoma. A positron emission tomography-computed tomography scan demonstrated a large anterior mediastinal mass. A biopsy confirmed the diagnosis of invasive thymoma morphologically identical to the World Health Organization type B2 sellar region metastasis.Conclusion:Although rare, thymomas can metastasize to the central nervous system. Our case is the second invasive thymoma to metastasize to the cavernous sinus, adjacent to the pituitary.

Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.

A Phase II Trial of Peri-Transplant Palifermin with Busulfan, Melphalan and Fludarabine Followed by T-Cell Depleted Hematopoietic Stem Cell Transplants in Patients with Advanced Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) Evolved From MDS

AbstractAbstract 1935 Introduction:The success of allogeneic HSCT in the treatment of advanced MDS has been limited by high incidence of relapse and transplant-related mortality (TRM). The main complications contributing to TRM are graft-vs-host disease (GvHD) and infections. Although T cell depletion has successfully reduced the incidence of GvHD, slow immune reconstitution and high rate of infections have compromised its full potential. T cell reconstitution after transplant requires a functional thymic epithelium. Transplant conditioning regimens damage the thymus and impair the production de novo of T cells. Keratinocyte growth factor (KGF) has an important role in healing the epithelium after injury. Palifermin, a human recombinant form of KGF, decreases the incidence and duration of mucositis after total body irradiation and has been approved by the FDA for this purpose in autologous and allogeneic HSCT. In murine models, KGF given before allogeneic transplant has a protective effect on the thymus and accelerates T cell reconstitution.The aim of this study was to assess whether administration of palifermin peri-transplant decreases TRM and improves the overall and disease-free survival (OS and DFS). This study was designed to reduce one- year TRM from 30% and 35% to 10% in HLA matched and mismatched arms, respectively. Patients:Between 11/2009–05/2012, 42 patients (advanced MDS: 26 or AML evolved from MDS: 16) enrolled on this trial. At transplant, 23 were in CR1, 3 were in second refractory cytopenia phase, and 16 had limited disease (<5% circulating blasts and <9% marrow blasts). The median age was 57.5 years (1–65), with 22 males and 20 females. Conditioning consisted of busulfan (12 doses over three days of 0.8 mg/kg IV for patients > 4 years old or 1.0 mg/kg IV for patients < 4 years old), melphalan (70 mg/m2 IV × 2 days), fludarabine (25 mg/m2 IV × 5 days), and rabbit ATG pre-transplant (2 doses and 3 doses for HLA matched and mismatched recipients, respectively). Palifermin was given according to the approved dose for mucositis prevention: 60 mcg/kg/day IV for 3 consecutive days before the preparative regimen, and 3 doses post transplant (day 0, +24hours, +48hours). Donors were HLA matched (31; 13 related and 18 unrelated) or unrelated mismatched (11). G-CSF mobilized donor peripheral blood stem cells underwent CD34+ selection and depletion of T cells using CliniMACS immunomagnetic selection columns (Milteny Biotec). Results:All 42 patients engrafted, 1 patient developed secondary graft failure. The cumulative incidence of grade III-IV aGvHD at day +100 and 1 year were 4.8% and 10.5%, respectively.The latter increase was due to late-onset acute GvHD. Only 1 of 33 patients at risk developed moderate chronic GvHD. The 2-year OS and DFS were 77% and 65%, respectively, and similar in the two arms (HLA matched and mismatched). The CI of relapse at 1-year was 12 % (similar in the two arms). The 1-year TRM was 20%; 18.4% in the HLA matched group (accrual met) and 22% in the mismatched arm (accrual ongoing). Causes of death were: infections (N=5; 3 HLA matched, 2 HLA mismatched), regimen related toxicity (N=2, liver VOD) and relapse (N=1). The frequency of infections was similar to historical controls; at 3 months post transplant, 30% of patients developed active CMV, EBV, or adenovirus infections, at 6 months 16.6%, and at 12 months 7.1%. The 6-month CI of CMV viremia in CMV seropositive patients was 75% and the 6-month CI of EBV viremia was 31%. Immune reconstitution as measured by CD4 count was slow, similar to historical controls, with median absolute CD4 count at 3 months of 77 cells/μl and 200 cells/μl by 1 year posttransplant. Other parameters to assess safety of this regimen, namely duration of narcotics use and days on TPN to measure severity of mucositis were similar to historical controls (same preparative regimen without palifermin). Conclusion:In this ongoing phase II trial the addition of peri transplant palifermin to a chemotherapy only myeloablative conditioning regimen in recipients of TCD HSCT for advanced MDS decreased the TRM, although did not meet the primary objective of this study (reduction to 10%). Also, there was no reduction in the incidence of viral infections and no improvement in immune reconstitution. The reduction in TRM resulted from early detection of infection and improved treatment options. [Display omitted] Disclosures:Perales:SOBI Biovitrum, pharma company: SOBI Biovitrum, pharma company Other. Goldberg:SOBI Biovitrum: Research Funding.

Generation and characterization of Tbx1-AmCyan1 transgenic reporter mouse line that selectively labels developing thymus primordium

Thymus development is a complicated process that includes highly dynamic morphological changes and reciprocal tissue interactions between endoderm-derived epithelial cells of the anterior foregut and neural crest-derived mesenchymal cells. We generated and characterized a Tbx1-AmCyan1 reporter transgenic mouse to visualize thymus precursor cells during early embryonic development. In transgenic embryos, AmCyan1 fluorescence was specifically detected in the endoderm of the developing 3rd and 4th pharyngeal pouches and later in thymus epithelium until E14.5. Cells expressing AmCyan1 that were isolated based on AmCyan1 fluorescence expressed endodermal, thymic, and parathyroid markers, but they did not express neural crest or endothelial markers; these findings indicated that this transgenic mouse strain could be used to collect thymic or parathyroid precursor cells or both. We also showed that in nude mice, which exhibit defects in thymus development, the thymus precursors were clearly labeled with AmCyan1. In summary, these AmCyan1-fluorescent transgenic mice are useful for investigating early thymus development.

Establishment and characterization of a novel cell line derived from human thymoma AB tumor

Thymomas are low-grade epithelial tumors of the anterior mediastinum. The complexity of the disease and the lack of in vitro and in vivo models hamper the development of better therapeutics. In this study, we report a novel cell line, designated as IU-TAB-1, which was established from a patient with stage II thymoma (World Health Organization-type AB). The IU-TAB-1 cell line was established in vitro and characterized using histological and immunohistochemical staining, fluorescence-activated cell sorting, cytogenetic analyses and functional assays including in vitro and a NOD/SCID xenograft model. A whole-genome gene expression analysis (Illumina) was performed on the IU-TAB-1 cell line and 34 thymomas to determine the clinical relevance of the cell line. The IU-TAB-1 cell line was positive for epithelial markers (pan-cytokeratin and EpCAM/CD326) including thymic epithelial (TE) surface markers (such as CD29, CD9, CD54/ICAM-1, CD58 and CD24) and p63, and negative for B- and T-cell lineage markers. Gene expression profiling demonstrated overlapping and distinct genes between IU-TAB-1 and primary thymomas including the primary tumor (from which the cell line was derived). IU-TAB-1 cells are tumorigenic when implanted in immunodeficient mice with tumors reaching a volume of 1000 mm3 at around 130 days. The established cell line represents a biologically relevant new tool to investigate the molecular pathology of thymic malignancies and to evaluate the efficacy of novel therapeutics both in vitro and in vivo.

Genetic Basis of Altered Central Tolerance and Autoimmune Diseases: A Lesson from AIRE Mutations

The thymus is a specialized organ that provides an inductive environment for the development of T cells from multipotent hematopoietic progenitors. Self–nonself discrimination plays a key role in inducing a productive immunity and in preventing autoimmune reactions. Tolerance represents a state of immunologic nonresponsiveness in the presence of a particular antigen. The immune system becomes tolerant to self-antigens through the two main processes, central and peripheral tolerance. Central tolerance takes place within the thymus and represents the mechanism by which T cells binding with high avidity self-antigens, which are potentially autoreactive, are eliminated through so-called negative selection. This process is mostly mediated by medullary thymic epithelia cells (mTECs) and medullary dendritic cells (DCs). A remarkable event in the process is the expression of tissue-specific antigens (TSA) by mTECs driven by the transcription factor autoimmune regulator (AIRE). Mutations in this gene result in autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), a rare autosomal recessive disease (OMIM 240300). Thus far, this syndrome is the paradigm of a genetically determined failure of central tolerance and autoimmunty. Patients with APECED have a variable pattern of autoimmune reactions, involving different endocrine and nonendocrine organs. However, although APECED is a monogenic disorder, it is characterized by a wide variability of the clinical expression, thus implying a further role for disease-modifying genes and environmental factors in the pathogenesis. Studies on this polyreactive autoimmune syndrome contributed enormously to unraveling several issues of the molecular basis of autoimmunity. This review focuses on the developmental, functional, and molecular events governing central tolerance and on the clinical implication of its failure.

MicroRNAs Control the Maintenance of Thymic Epithelia and Their Competence for T Lineage Commitment and Thymocyte Selection

Thymic epithelial cells provide unique cues for the lifelong selection and differentiation of a repertoire of functionally diverse T cells. Rendered microRNA (miRNA) deficient, these stromal cells in the mouse lose their capacity to instruct the commitment of hematopoietic precursors to a T cell fate, to effect thymocyte positive selection, and to achieve promiscuous gene expression required for central tolerance induction. Over time, the microenvironment created by miRNA-deficient thymic epithelia assumes the cellular composition and structure of peripheral lymphoid tissue, where thympoiesis fails to be supported. These findings emphasize a global role for miRNA in the maintenance and function of the thymic epithelial cell scaffold and establish a novel mechanism how these cells control peripheral tissue Ag expression to prompt central immunological tolerance.

Genetic Labeling of Tas1r1 and Tas2r131 Taste Receptor Cells in Mice

Characterization of the peripheral taste system relies on the identification and visualization of the different taste bud cell types. So far, genetic strategies to label taste receptor cells are limited to sweet, sour, and salty detecting cells. To visualize Tas1r1 umami and Tas2r131 bitter sensing cells, we generated animals in which the Tas1r1 and Tas2r131 open reading frames are replaced by expression cassettes containing the fluorescent proteins mCherry or hrGFP, respectively. These animals enabled us to visualize and quantify the entire oral Tas1r1 and Tas2r131 cell populations. Tas1r1-mCherry cells were predominantly detected in fungiform papillae, whereas Tas2r131-hrGFP cells, which are ~4-fold more abundant, were mainly present in foliate and vallate papillae. In the palate, both cell types were similarly distributed. Mice carrying both recombinant alleles demonstrated completely segregated Tas1r1 and Tas2r131 cell populations. Only ~50% of the entire bitter cell population expressed hrGFP, indicating that bitter taste receptor cells express a subset of the bitter receptor repertoire. In extragustatory tissues, mCherry fluorescence was observed in testis and hrGFP fluorescence in testis, thymus, vomeronasal organ, and respiratory epithelium, suggesting that only few extraoral sites express Tas2r131 and Tas1r1 receptors at levels comparable to taste tissue.

MicroRNA-29 in the adaptive immune system: setting the threshold.

Recent research into the role of microRNA (miR) in the immune system has identified the miR-29 family as critical regulators of key processes in adaptive immunity. The miR-29 family consists of four members with shared regulatory capacity, namely miR-29a, miR-29b-1, miR-29b-2 and miR-29c. Being expressed in both T and B cells, as well as the main accessory cell types of thymic epithelium and dendritic cells, the miR-29 family has been identified as a putative regulator of immunity due to the predicted suppression of key immunological pathways. The generation of a series of in vivo molecular tools targeting the miR-29 family has identified the critical role of these miR in setting the molecular threshold for three central events in adaptive immunity: (1) control over thymic production of T cells by modulating the threshold for infection-associated thymic involution, (2) creating a neutral threshold for T cell polarization following activation, and (3) setting the threshold for B cell oncogenic transformation. These results identify the miR-29 family as potent immune modulators which have already been exploited through the evolution of a viral mimic and could potentially be exploited further for therapeutic intervention.

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.

De novo Generation of Cells within Human Nurse Macrophages and Consequences following HIV-1 Infection

Nurse cells are defined as those that provide for the development of other cells. We report here, that in vitro, human monocyte-derived macrophages can behave as nurse cells with functional capabilities that include de novo generation of CD4+ T-lymphocytes and a previously unknown small cell with monocytoid characteristics. We named these novel cells “self-renewing monocytoid cells” (SRMC), because they could develop into nurse macrophages that produced another generation of SRMC. SRMC were not detectable in blood. Their transition to nurse behavior was characterized by expression of CD10, a marker of thymic epithelium and bone marrow stroma, typically absent on macrophages. Bromodeoxyuridine labeling and immunostaining for cdc6 expression confirmed DNA synthesis within nurse macrophages. T-cell excision circles were detected in macrophages, along with expression of pre-T-cell receptor alpha and recombination activating gene 1, suggesting that genetic recombination events associated with generation of the T-cell receptor were occurring in these cells. SRMC expressed CCR5, the coreceptor for R5 HIV-1 isolates, and were highly susceptible to HIV-1 entry leading to productive infection. While expressing HIV-1, SRMC could differentiate into nurse macrophages that produced another generation of HIV-1-expressing SRMC. The infected nurse macrophage/SRMC cycle could continue in vitro for multiple generations, suggesting it might represent a mechanism whereby HIV-1 can maintain persistence in vivo. HIV-1 infection of nurse macrophages led to a decline in CD4+ T-cell production. There was severe, preferential loss of the CCR5+ CD4+ T-cell subpopulation. Confocal microscopy revealed individual HIV-1-expressing nurse macrophages simultaneously producing both HIV-1-expressing SRMC and non-expressing CD3+ cells, suggesting that nurse macrophages might be a source of latently infected CD4+ T-cells. Real-time PCR experiments confirmed this by demonstrating 10-fold more HIV-1-genome-harboring T-cells, than virus-expressing ones. These phenomena have far-reaching implications, and elicit new perspectives regarding HIV pathogenesis and T-cell and hematopoietic cell development.

Thymocytes may persist and differentiate without any input from bone marrow progenitors

Thymus transplants can correct deficiencies of the thymus epithelium caused by the complete DiGeorge syndrome or FOXN1 mutations. However, thymus transplants were never used to correct T cell-intrinsic deficiencies because it is generally believed that thymocytes have short intrinsic lifespans. This notion is based on thymus transplantation experiments where it was shown that thymus-resident cells were rapidly replaced by progenitors originating in the bone marrow. In contrast, here we show that neonatal thymi transplanted into interleukin 7 receptor-deficient hosts harbor populations with extensive capacity to self-renew, and maintain continuous thymocyte generation and export. These thymus transplants reconstitute the full diversity of peripheral T cell repertoires one month after surgery, which is the earliest time point studied. Moreover, transplantation experiments performed across major histocompatibility barriers show that allogeneic transplanted thymi are not rejected, and allogeneic cells do not induce graft-versus-host disease; transplants induced partial or total protection to infection. These results challenge the current dogma that thymocytes cannot self-renew, and indicate a potential use of neonatal thymus transplants to correct T cell-intrinsic deficiencies. Finally, as found with mature T cells, they show that thymocyte survival is determined by the competition between incoming progenitors and resident cells.