Cells In Human Thymus Research Articles

CD90 Marks a Mesenchymal Program in Human Thymic Epithelial Cells In Vitro and In Vivo.

Thymic epithelium is critical for the structural integrity of the thymus and for T cell development. Within the fully formed thymus, large numbers of hematopoietic cells shape the thymic epithelium into a scaffold-like structure which bears little similarity to classical epithelial layers, such as those observed in the skin, intestine or pancreas. Here, we show that human thymic epithelial cells (TECs) possess an epithelial identity that also incorporates the expression of mesenchymal cell associated genes, whose expression levels vary between medullary and cortical TECs (m/cTECs). Using pluripotent stem cell (PSC) differentiation systems, we identified a unique population of cells that co-expressed the master TEC transcription factor FOXN1, as well as the epithelial associated marker EPCAM and the mesenchymal associated gene CD90. Using the same serum free culture conditions, we also observed co-expression of EPCAM and CD90 on cultured TECs derived from neonatal human thymus in vitro. Single cell RNA-sequencing revealed these cultured TECs possessed an immature mTEC phenotype and expressed epithelial and mesenchymal associated genes, such as EPCAM, CLDN4, CD90 and COL1A1. Importantly, flow cytometry and single cell RNA-sequencing analysis further confirmed the presence of an EPCAM+CD90+ population in the CD45- fraction of neonatal human thymic stromal cells in vivo. Using the human thymus cell atlas, we found that cTECs displayed more pronounced mesenchymal characteristics than mTECs during embryonic development. Collectively, these results suggest human TECs possess a hybrid gene expression program comprising both epithelial and mesenchymal elements, and provide a basis for the further exploration of thymus development from primary tissues and from the in vitro differentiation of PSCs.

Influence of AEDG and KE Peptides on Mitochondrial Staining and the Expression of Ribosomal Protein L7A with Aging of the Human Pineal Gland and Thymus Cell In Vitro

Influence of AEDG and KE Peptides on Mitochondrial Staining and the Expression of Ribosomal Protein L7A with Aging of the Human Pineal Gland and Thymus Cell In Vitro

Activated protein C targets immune cells and rheumatoid synovial fibroblasts to prevent inflammatory arthritis in mice.

To investigate whether activated protein C (APC), a physiological anticoagulant can inhibit the inflammatory/invasive properties of immune cells and rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and prevent inflammatory arthritis in murine antigen-induced arthritis (AIA) and CIA models. RASFs isolated from synovial tissues of patients with RA, human peripheral blood mononuclear cells (PBMCs) and mouse thymus cells were treated with APC or TNF-α/IL-17 and the following assays were performed: RASF proliferation and invasion by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell invasion assays, respectively; cytokines and signalling molecules using ELISA or western blot; Th1 and Th17 phenotypes in human PBMCs or mouse thymus cells by flow cytometry. The in vivo effect of APC was evaluated in AIA and CIA models. In vitro, APC inhibited IL-1β, IL-17 and TNF-α production, IL-17-stimulated cell proliferation and invasion and p21 and nuclear factor κB activation in RASFs. In mouse thymus cells and human PBMCs, APC suppressed Th1 and Th17 phenotypes. In vivo, APC inhibited pannus formation, cartilage destruction and arthritis incidence/severity in both CIA and AIA models. In CIA, serum levels of IL-1β, IL-6, IL-17, TNF-α and soluble endothelial protein C receptor were significantly reduced by APC treatment. Blocking endothelial protein C receptor, the specific receptor for APC, abolished the early or preventative effect of APC in AIA. APC prevents the onset and development of arthritis in CIA and AIA models via suppressing inflammation, Th1/Th17 phenotypes and RASF invasion, which is likely mediated via endothelial protein C receptor.

Noninvasive Imaging of Human Immune Responses in a Human Xenograft Model of Graft-Versus-Host Disease.

The immune system plays a crucial role in many diseases. Activation or suppression of immunity is often related to clinical outcome. Methods to explore the dynamics of immune responses are important to elucidate their role in conditions characterized by inflammation, such as infectious disease, cancer, or autoimmunity. Immuno-PET is a noninvasive method by which disease and immune cell infiltration can be explored simultaneously. Using radiolabeled antibodies or fragments derived from them, it is possible to image disease-specific antigens and immune cell subsets. Methods: We developed a method to noninvasively image human immune responses in a relevant humanized mouse model. We generated a camelid-derived single-domain antibody specific for human class II major histocompatibility complex products and used it to noninvasively image human immune cell reconstitution in nonobese diabetic severe combined immune deficiency γ-/- mice reconstituted with human fetal thymus, liver, and liver-derived hematopoietic stem cells (BLT mice). Results: We showed imaging of infiltrating immunocytes in BLT mice that spontaneously developed a graft-versus-host-like condition, characterized by alopecia and blepharitis. In diseased animals, we showed an increased PET signal in the liver, attributable to infiltration of activated class II major histocompatibility complex+ T cells. Conclusion: Noninvasive imaging of immune infiltration and activation could thus be of importance for diagnosis and evaluation of treatment of graft-versus-host disease and holds promise for other diseases characterized by inflammation.

The Checkpoint for Agonist Selection Precedes Conventional Selection in Human Thymus

The thymus plays a central role in self-tolerance by preventing strongly self-reactive thymocytes from accumulating as naïve T cell receptor (TCR) αβ+ T cells in the periphery. The elimination of auto-reactive T cells from the naïve pool is in part mediated by deletion during conventional negative selection. Alternatively, self-reactive thymocytes can also be positively selected in response to strong TCR signals during agonist selection and functionally differentiate to innate TCRαβ+ T cells such as the CD8αα+ double negative (DN) T cells. How thymocytes discriminate between these opposite outcomes remains unclear.We identified a novel agonist-selected PD-1+ CD8αα+ subset of mature CD8+ T cells in human thymus. Using the same markers a similar population was also identified in cord blood at about the same frequency as TCRγδ+ cells. This population expresses high levels of Helios, indicative of strong TCR engagement, and displays an effector phenotype associated with agonist selection. Indeed, PD-1+CD8αα+ T cells exhibit innate production of IFN-γ and an elevated T-bet to Eomes ratio typical of effector CD8 T cells. These cells are CD62L-, CXCR3+ and Hobit high suggesting that these cells leave the thymus and home to the tissues. Interestingly, in vitro CD3/TCR stimulation of sorted early post-β-selection thymocyte blasts uniquely gives rise to this innate subset, whereas small CD4+CD8+ double positive precursors fail to survive strong TCR signals. The generation of the innate subset seems to arise also in vivo from early post-β-selection thymocyte blasts as these two populations have an identical TCRα repertoire: ex vivo isolated PD-1+CD8αα+ thymocytes are skewed for early 3’ TRAV and 5’ TRAJ rearrangements compared to conventional CD8 T cells. A similar skewing was found in early post-β-selection thymocyte blasts. As TCRα rearrangements are terminated by TCR engagement of agonist selection, this is strong evidence for a precursor progeny relationship.Together, we conclude that human CD8αα+ T cells are preferentially selected by strong TCR engagement on a subset of progenitors that express a full TCRαβ early on, leading to the generation of a post-selection T cell population with innate functional capacity and a markedly distinct TCR repertoire. These findings uncover the heterogeneity among DP precursors in their potential to survive strong selection signals and suggests that the decision making in the thymus to divert immature thymocytes to the agonist selection pathway occurs early before conventional selection of DP cells. We propose that progression through the immature thymic developmental program influences the outcome of TCR engagement with early post-β-selection thymocytes triggered by strong TCR signals preferentially giving rise to innate CD8αα+ T cells in humans. DisclosuresNo relevant conflicts of interest to declare.

Humanized mouse model of mast cell–mediated passive cutaneous anaphylaxis and passive systemic anaphylaxis

Mast cells are a critical component of allergic responses in humans, and animal models that allow the invivo investigation of their contribution to allergy and evaluation of new human-specific therapeutics are urgently needed. To develop a new humanized mouse model that supports human mast cell engraftment and human IgE-dependent allergic responses. This model is based on the NOD-scid IL2rg(null)SCF/GM-CSF/IL3 (NSG-SGM3) strain of mice engrafted with human thymus, liver, and hematopoietic stem cells (termed Bone marrow, Liver, Thymus [BLT]). Large numbers of human mast cells develop in NSG-SGM3 BLT mice and populate the immune system, peritoneal cavity, and peripheral tissues. The human mast cells in NSG-SGM3 BLT mice are phenotypically similar to primary human mast cells and express CD117, tryptase, and FcεRI. These mast cells undergo degranulation in an IgE-dependent and -independent manner, and can be readily cultured invitro for additional studies. Intradermal priming of engrafted NSG-SGM3 mice with a chimeric IgE containing human constant regions resulted in the development of a robust passive cutaneous anaphylaxis response. Moreover, we describe the first report of a human mast cell antigen-dependent passive systemic anaphylaxis response in primed mice. NSG-SGM3 BLT mice provide a readily available source of human mast cells for investigation of mast cell biology and a preclinical model of passive cutaneous anaphylaxis and passive systemic anaphylaxis that can be used to investigate the pathogenesis of human allergic responses and to test new therapeutics before their advancement to the clinic.

Development of humanized mouse models to study human immune system-tumor interactions (TUM10P.1055)

Abstract Cancer progression involves interplay between tumor cells and non-malignant stoma, including immune cells. Our understanding of the interactions between immune systems and tumors is based mostly on mouse models. However many differences exist between murine and human biology, and translation of findings from mice to clinical application is challenging. The study of human immune systems and tumors is limited by logistical and ethical restrictions of working with primary specimens from patients. Here we describe a humanized mouse model to study the in vivo interactions between human immune systems and human tumors. In these studies NOD-scid IL2rgnull (NSG) mice expressing human SCF, GM-CSF and IL-3 (NSG-SGM3) were engrafted with human fetal thymus and fetal liver-derived hematopoietic stem cells (BLT). NSG-SGM3-BLT mice support high levels of human cell chimerism, including development of human myeloid cells, T cells and B cells. Engrafted NSG-SGM3-BLT mice were implanted with patient melanoma cells and monitored for tumor growth and immune system homeostasis. The human melanoma grew with similar kinetics in NSG-SGM3-BLT mice as compared to control NSG-SGM3 mice. Human immune cells, including human macrophage and T cells, infiltrated the melanoma. The T cell infiltrate was dominated by human FOXP3+ Treg and by PD1+ CD4 and CD8 T cells. These data show that NSG-SGM3-BLT mice, are a promising model to study human immune system-tumor interactions and to test novel therapies.

Humanized mouse model of mast cell-mediated passive cutaneous anaphylaxis and passive systemic anaphylaxis (HYP4P.302)

Abstract Mast cells are critical components of allergic responses in humans, and animal models that allow the in vivo investigation of human allergic responses and the evaluation of new human-specific therapeutics are urgently needed. Here we have developed a new humanized mouse model for human mast cell engraftment and human IgE-dependent allergic responses. This model is based on the NOD-scid IL2rgnull IL3/GM-CSF/SCF (NSG-SGM3) strain of mouse engrafted with human fetal thymus, liver and hematopoietic stem cells (termed the BLT model). Large numbers of human mast cells develop in NSG-SGM3 BLT mice and populate peripheral lymphoid tissues, mucosal tissues and the peritoneal cavity. The human mast cells in NSG-SGM3 BLT mice express CD117, tryptase, and IgE-receptor, undergo a calcium flux and degranulate in an IgE-dependent antigen-specific manner, and can readily be cultured and expanded in vitro. The NSG-SGM3-BLT mice develop a human mast cell dependent, antigen-specific IgE-mediated passive cutaneous anaphylaxis (PCA) response upon intradermal challenge. Moreover, we describe the first report of a human mast cell antigen-dependent passive systemic anaphylaxis (PSA) response in humanized mice following intravenous challenge with antigen. The NSG-SGM3 BLT mouse provides a readily available source of human mast cells and is a promising pre-clinical model to investigate the pathogenesis of human allergic responses and to testing new therapeutics.

Engrafted human cells generate adaptive immune responses to Mycobacterium bovis BCG infection in humanized mice

BackgroundCurrently used mouse models fail to fully reflect human immunity to tuberculosis (TB), which hampers progress in research and vaccine development. Bone marrow-liver-thymus (BLT) mice, generated by engrafting human fetal liver, thymus, and hematopoietic stem cells in severely immunodeficient NOD/SCID/IL-2Rγ-/- (NSG) mice, have shown potential to model human immunity to infection. We engrafted HLA-A2-positive fetal tissues into NSG mice transgenically expressing human leukocyte antigen (HLA)-A2.1 (NSG-A2) to generate NSG-A2-BLT mice and characterized their human immune response to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection to assess the utility of this model for investigating human TB.ResultsNSG-A2-BLT mice were infected intravenously with BCG and the immune response of engrafted human immune cells was characterized. After ex vivo antigenic stimulation of splenocytes, interferon (IFN)-γ-producing cells were detected by ELISPOT from infected, but not uninfected NSG-A2-BLT mice. However, the levels of secreted IFN-γ, determined by ELISA, were not significantly elevated by antigenic stimulation. NSG-A2-BLT mice were susceptible to BCG infection as determined by higher lung bacillary load than the non-engrafted control NSG-A2 mice. BCG-infected NSG-A2-BLT mice developed lung lesions composed mostly of human macrophages and few human CD4+ or CD8+ T cells. The lesions did not resemble granulomas typical of human TB.ConclusionsEngrafted human immune cells in NSG-A2-BLT mice showed partial function of innate and adaptive immune systems culminating in antigen-specific T cell responses to mycobacterial infection. The lack of protection was associated with low IFN-γ levels and limited numbers of T cells recruited to the lesions. The NSG-A2-BLT mouse is capable of mounting a human immune response to M. tuberculosis in vivo but a quantitatively and possibly qualitatively enhanced effector response will be needed to improve the utility of this model for TB research.

Immunomodulating Effects of Vilon and Its Analogue in the Culture of Human and Animal Thymus Cells

We studied molecular mechanisms of immunoprotective effects of two dipeptides, AB-O and R-1, on cultured human and rat thymic cells. Both dipeptides were shown to increase the expression of lymphocyte differentiation marker CD5 in thymic cells. Dipeptide AB-O induced T-cells precursor differentiation towards CD4(+)T-helpers and its effect was weaker than that of dipeptide R-1. Dipeptide R-1 stimulates differentiation of CD5(+) cells to mature T-helpers and cytotoxic CD8(+) T cells and hence can be considered as a bioactive substance possessing immunomodulator and antiallergic activity.

Lack of iGb3 and Isoglobo-Series Glycosphingolipids in Pig Organs Used for Xenotransplantation: Implications for Natural Killer T-Cell Biology

α-1,3-Terminated galactose residues on glycoproteins and glycosphingolipids are recognized by natural anti-α-1,3-galactose antibodies in human serum and cause hyperacute rejection in pig-to-human xenotransplantation. Genetic depletion of α-1,3-galactosyl- transferase-1 in pigs abolishes the hyperacute rejection reaction. However, the isoglo- botriosylceramide (iGb3) synthase in pigs may produce additional α-1,3-terminated galactose residues on glycosphingolipids. In both α-1,3-galactosyltranserase-1 knockout mice and pigs, cytotoxic anti-α-1,3-galactose antibodies could be induced; thus, a paradox exists that anti-α-1,3-galactose antibodies are present in animals with functional iGb3 synthases. Furthermore, iGb3 has been found to be an endogenous antigen for natural killer T (NKT) cells, an innate type of lymphocyte that may initiate the adaptive immune responses. It has been reasoned that iGb3 may trigger the activation of NKT cells and cause the rejection of α-1,3-galactosyltransferase-1-deficient organs through the potent stimulatory effects of NKT cells on adaptive immune cells (see ref.[20]). In this study, we examined the expression of iGb3 and the isoglobo-series glycosphingolipids in pig organs, including the heart, liver, pancreas, and kidney, by ion-trap mass spectrometry, which has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 5 μg/mL of the total iGb3/Gb3 mixture is present (see ref.[35]). We did not detect iGb3 or other isoglobo-series glycosphingolipids in any of these organs, although they were readily detected in mouse and human thymus and dendritic cells. The lack of iGb3 and isoglobo-series glycosphingolipids in pig organs indicates that iGb3 is unlikely to be a relevant immune epitope in xenotransplantation.

Modification of immune activation in HIV-1-infected humanized mouse model using TLR7/9 antagonists (113.19)

Abstract Background: T cell immune activation is a strong predictor of HIV-1 disease progression, and IFNα production following TLR7 stimulation has been associated with elevated CD8+ T cell activation. We hypothesize that modulation of TLR7 stimulation could be used to manipulate IFNα production and subsequently reduce HIV-1-associated immune activation. Methods: Humanized BLT mice were generated by transplanting irradiated NOD/SCID/IL2Rγc-/- mice with human fetal thymus, liver tissue and human hematopoietic stem cells. After immune reconstitution, human DC, monocyte and T cell populations were detectable in the mice, and in vitro TLR responsiveness of DCs and monocytes reflected those observed in human PBMCs. Humanized mice were infected with HIV-1 and then treated with a TLR7/9 antagonist. The T cell activation markers were examined at each stage by flow cytometry. Results: BLT mice-derived human pDC responses to TLR7 stimulation were significantly blocked by in vitro treatment with the TLR7/9 antagonist (P<0.05). HIV-1 infection of BLT mice led to increased expression of immune activation marker CD38 on human T cells in vivo, and treatment of infected mice with the TLR7/9 antagonist led to a significant reduction in CD38 expression. Conclusion: Treatment of HIV-1-infected humanized BLT mice with TLR7/9 antagonist resulted in significant reduction of HIV-1-associated immune activation, indicating an important role of the TLR7/9 pathway in mediating immune activation in HIV-1.

In Vivo Suppression of HIV by Antigen Specific T Cells Derived from Engineered Hematopoietic Stem Cells

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. These results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.

Isolation strategies of regulatory T cells for clinical trials: Phenotype, function, stability, and expansion capacity

Recent clinical results demonstrate the highly effective potency of regulatory T cells (Tregs) to control graft-versus-host disease (GvHD). In this presented study, we directly compared different Treg subpopulations in order to define the most promising Treg target cell population for cellular intervention studies with respect to their phenotype, functional properties, stability, and expansion capacity. Different Treg cell populations have been isolated from healthy donors and characterized by fluorescence activated cell sorting (FACS) analysis for their phenotypic marker and purity, functional properties by suppression assay, stability by Treg-specific demethylated region (TSDR) of the Foxp3 promoter and their invitro expansion capacity. The direct comparison of the respective Treg target cell populations identified CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs as the most promising Treg population for fresh cell infusions in clinical trials with respect to cell yield, phenotype, function, and stability. The CD4(+)CD25(hi) Tregs qualified as the best candidate for invitro expansion combining a highly stable phenotype with strong suppressive potential and attractive cell yield after repetitive stimulation. The suppressive capacity of freshly isolated CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs is comparable to freshly isolated CD4(+)CD25(hi), but inferior to CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs. Invitro expansion of CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs resulted in cell populations with less suppressive potency compared with CD4(+)CD25(hi) expanded Tregs correlating well with a higher TSDR demethylation level. In conclusion, future clinical trials should favor CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs for direct Treg cell transfer, whereas CD4(+)CD25(hi) Tregs qualify as best candidate for invitro expansion.

DirectedTrans-Differentiation of Thymus Cells into Parathyroid-Like Cells Without Genetic Manipulation

Replacement of a diseased organ with an autologously derived tissue is an ideal therapy for some medical problems. However, it is difficult to recreate many adult human tissues in vitro due to the functionally necessary architecture of most organs and the lack of understanding of methods to direct the development of the organ of interest. The parathyroid gland is ideal for in vitro organ development because this gland is relatively simple, is transplantable, and is commonly affected by a surgical complication rather than an autoimmune disease. We have investigated thymus as a source of autologous endoderm and parathyroid-like precursor cells. Human thymus cells were treated with a differentiation protocol we developed with human embryonic stem cells (The Bingham Protocol) that utilizes timed exposures to Activin A and soluble Sonic hedgehog (Shh). We incrementally changed the protocol to optimize the differentiation of the thymus cells into parathyroid-like cells. The final protocol used 50 ng/mL Activin A and 100 ng/mL Shh over 13 weeks. The differentiated cells expressed the parathyroid markers parathyroid hormone (PTH), calcium sensing receptor, chemokine receptor type-4 (CXCR4), and chorian-specific transcription factor (GCM2) as measured by reverse transcription-polymerase chain reaction and PTH enzyme-linked immunosorbent assay. Cultured thymus cells without Activin A or Shh exposure did not secrete PTH nor express similar markers. The differentiated cells released PTH, which was suppressed in response to increased calcium concentration. The chemically differentiated cells did not form tumors in immune-compromised mice. Our protocol recreated cells with markers of parathyroid tissue that responded as parathyroid cells to physiologic stimuli. This approach is a further step toward a strategy to restore parathyroid function using autologous cells that were directed to differentiate by nongenetic in vitro manipulation.

Analysis of the CD1 Antigen Presenting System in Humanized SCID Mice

CD1 molecules are glycoproteins that present lipids and glycolipids for recognition by T cells. CD1-dependent immune activation has been implicated in a wide range of immune responses, however, our understanding of the role of this pathway in human disease remains limited because of species differences between humans and other mammals: whereas humans express five different CD1 gene products (CD1a, CD1b, CD1c, CD1d, and CD1e), muroid rodents express only one CD1 isoform (CD1d). Here we report that immune deficient mice engrafted with human fetal thymus, liver, and CD34+ hematopoietic stem cells develop a functional human CD1 compartment. CD1a, b, c, and d isoforms were highly expressed by human thymocytes, and CD1a+ cells with a dendritic morphology were present in the thymic medulla. CD1+ cells were also detected in spleen, liver, and lungs. APCs from spleen and liver were capable of presenting bacterial glycolipids to human CD1-restricted T cells. ELISpot analyses of splenocytes demonstrated the presence of CD1-reactive IFN-γ producing cells. CD1d tetramer staining directly identified human iNKT cells in spleen and liver samples from engrafted mice, and injection of the glycolipid antigen α-GalCer resulted in rapid elevation of human IFN-γ and IL-4 levels in the blood indicating that the human iNKT cells are biologically active in vivo. Together, these results demonstrate that the human CD1 system is present and functionally competent in this humanized mouse model. Thus, this system provides a new opportunity to study the role of CD1-related immune activation in infections to human-specific pathogens.

Immunoreactive neuropeptides in the cells of human thymus

The study was designed to explore the expression of different neuropeptides, viz. vasoactive intestinal peptide (VIP), calcitonin gene related peptide (CGRP), substance P (SP), bombesin and motilin in the cells of fetal and adult human thymus. Immunohistochemical staining revealed that cortical and medullary thymocytes were labeled by all antibodies, except those specific for motilin. Immunoreactive VIP and SP were observed in the solitary epithelial cells located in the subcapsular/subtrabecular cortex, at the corticomedullary junction and in the medulla. The cells within the subcapsular/subtrabecular monolayer, rare solitary cells in the deep cortex and epithelial cell network in the medulla, were labeled with antibodies to CGRP and bombesin. Hassall?s corpuscles were labeled with all antibodies except that specific for SP. The obtained data obtained testify to the expression of different neuropeptides in human thymic lymphoid and non-lymphoid cells and suggest a role for neuroendocrine hormone-mediated mechanisms in the regulation of thymic homeostasis in humans.

Plasmacytoid dendritic cells resident in human thymus drive natural Treg cell development

AbstractThe generation of natural regulatory T cells (nTregs) is crucial for the establishment of immunologic self-tolerance and the prevention of autoimmunity. Still, the origin of nTregs and the mechanisms governing their differentiation within the thymus are poorly understood, particularly in humans. It was recently shown that conventional dendritic cells (cDCs) in human thymus were capable of inducing nTreg differentiation. However, the function of plasmacytoid DCs (pDCs), the other major subset of thymic DCs, remains unknown. Here we report that pDCs resident in the human thymus, when activated with CD40 ligand (CD40L) plus interleukin-3, efficiently promoted the generation of CD4+CD25+Foxp3+ nTregs from autologous thymocytes. The progenitors of these nTregs were selectively found within CD4+CD8+ thymocytes that had accomplished positive selection, as judged by their CD69hiTCRhi phenotype. Supporting the involvement of the CD40-CD40L pathway in pDC-induced nTreg generation, we show that positively selected CD4+CD8+ progenitors specifically transcribed CD40L in vivo and up-regulated CD40L expression on T-cell receptor engagement, thereby promoting the activation of pDCs. Finally, evidence is provided that nTregs primed by pDCs displayed reciprocal interleukin-10/transforming growth factor-β cytokine expression profiles compared with nTregs primed by cDCs. This functional diversity further supports a nonredundant tolerogenic role for thymic pDCs in the human thymus.

Characterization and Histological Localization of Multipotent Mesenchymal Stromal Cells in the Human Postnatal Thymus

The aim of this work was to characterize multipotent mesenchymal stromal cells (MSCs) in the postnatal human thymus and to localize these MSCs in the organ. Adherent cells isolated from thymus samples were characterized by cell-surface antigen expression. This showed that adherent cells have a MSC profile as assessed by the expression of CD73 and CD105 markers and the lack of CD45 expression. These cells are able to differentiate in vitro into adipocytes, osteoblasts, and chondrocytes and to inhibit mixed lymphocyte reaction. This indicates that isolated cells have all of the characteristics of MSC. The fibroblast colony-forming unit (CFU-F) assay was used to determine their frequency in the postnatal thymus. This frequency was 60.9 +/- 14.8 CFU-F per 1 x 10(5) freshly isolated mononuclear cells. Moreover, taking advantage of CD34 and CD105 expression, immunohistological staining allowed us to localize MSC within interlobular trabeculae in close contact with the outer cortex. Polymerase chain reaction experiments indicated that thymic MSC expressed interleukin-7 and stromal cell-derived factor-1 messenger RNA. Overall, these results confirm previous findings of the presence in the adult human thymus of multipotent MSCs with a phenotype similar to adipose-derived adult stem cells. These results also show for the first time a histological localization of MSC in an organ. This suggests a possible role of thymic MSC in intrathymic differentiation.

Two Functional Subsets of FOXP3 + Regulatory T Cells in Human Thymus and Periphery

Previous studies suggest that thymus produces a homogenous population of natural regulatory T (Treg) cells that express a transcriptional factor FOXP3 and control autoimmunity through a cell-contact-dependent mechanism. We found two subsets of FOXP3+ natural Treg cells defined by the expression of the costimulatory molecule ICOS in the human thymus and periphery. Whereas the ICOS+FOXP3+ Treg cells used interleukin-10 to suppress dendritic cell function and transforming growth factor (TGF)-beta to suppress T cell function, the ICOS-FOXP3+ Treg cells used TGF-beta only. The survival and proliferation of the two subsets of Treg cells were differentially regulated by signaling through ICOS or CD28, respectively. We suggest that the selection of natural Treg cells in thymus is coupled with Treg cell differentiation into two subsets imprinted with different cytokine expression potentials and use both cell-contact-dependent and independent mechanisms for immunosuppression in periphery.