Quantitative and functional expression of somatostatin receptor subtypes in human thymocytes

We recently demonstrated the expression of somatostatin (SS) and SS receptor (SSR) subtype 1 (sst1), sst2A, and sst3 in normal human thymic tissue and of sst1 and sst2A on isolated thymic epithelial cells (TEC). We also found an inhibitory effect of SS and octreotide on TEC proliferation. In the present study, we further investigated the presence and function of SSR in freshly purified human thymocytes at various stages of development. Thymocytes represent a heterogeneous population of lymphoid cells displaying different levels of maturation and characterized by specific cell surface markers. In this study, we first demonstrated specific high-affinity 125I-Tyr(11)-labeled SS-14 binding on thymocyte membrane homogenates. Subsequently, by RT-PCR, sst2A and sst3 mRNA expression was detected in the whole thymocyte population. After separation of thymocytes into subpopulations, we found by quantitative RT-PCR that sst2A and sst3 are differentially expressed in intermediate/mature and immature thymocytes. The expression of sst3 mRNA was higher in the intermediate/mature CD3+ fraction compared with the immature CD2+CD3- one, whereas sst2A mRNA was less abundant in the intermediate/mature CD3+ thymocytes. In 7-day-cultured thymocytes, SSR subtype mRNA expression was lost. SS-14 significantly inhibited [3H]thymidine incorporation in all thymocyte cultures, indicating the presence of functional receptors. Conversely, octreotide significantly inhibited [3H]thymidine incorporation only in the cultures of immature CD2+CD3- thymocytes. Subtype sst3 is expressed mainly on the intermediate/mature thymocyte fraction, and most of these cells generally die by apoptosis. Because SS-14, but not octreotide, induced a significant increase in the percentage of apoptotic thymocytes, it might be that sst3 is involved in this process. Moreover, sst3 has recently been demonstrated on peripheral human T lymphocytes, which derive directly from mature thymocytes, and SS analogs may induce apoptosis in these cells. Interestingly, CD14+ thymic cells, which are cells belonging to the monocyte-macrophage lineage, selectively expressed sst2A mRNA. Finally, SSR expression in human thymocytes seems to follow a developmental pathway. The heterogeneous expression of SSR within the human thymus on specific cell subsets and the endogenous production of SS as well as SS-like peptides emphasize their role in the bidirectional interactions between the main cell components of the thymus involved in intrathymic T cell maturation.     

mTORC1 in Thymic Epithelial Cells Is Critical for Thymopoiesis,T-Cell Generation,and Temporal Control of γδT17 Development and TCRγ/δ Recombination

Thymus is crucial for generation of a diverse repertoire of T cells essential for adaptive immunity. Although thymic epithelial cells (TECs) are crucial for thymopoiesis and T cell generation, how TEC development and function are controlled is poorly understood. We report here that mTOR complex 1 (mTORC1) in TECs plays critical roles in thymopoiesis and thymus function. Acute deletion of mTORC1 in adult mice caused severe thymic involution. TEC-specific deficiency of mTORC1 (mTORC1KO) impaired TEC maturation and function such as decreased expression of thymotropic chemokines, decreased medullary TEC to cortical TEC ratios, and altered thymic architecture, leading to severe thymic atrophy, reduced recruitment of early thymic progenitors, and impaired development of virtually all T-cell lineages. Strikingly, temporal control of IL-17-producing γδT (γδT17) cell differentiation and TCRVγ/δ recombination in fetal thymus is lost in mTORC1KO thymus, leading to elevated γδT17 differentiation and rearranging of fetal specific TCRVγ/δ in adulthood. Thus, mTORC1 is central for TEC development/function and establishment of thymic environment for proper T cell development, and modulating mTORC1 activity can be a strategy for preventing thymic involution/atrophy.     

Distinct functional properties of native somatostatin receptor subtype 5 compared with subtype 2 in the regulation of ACTH release by corticotroph tumor cells

In a series of human corticotroph adenomas, we recently found predominant mRNA expression of somatostatin (SS) receptor subtype 5 (sst5). After 72 h, the multiligand SS analog SOM230, which has a very high sst5 binding affinity, but not Octreotide (OCT), significantly inhibited basal ACTH release. To further explore the role of sst5 in the regulation of ACTH release, we conducted additional studies with mouse AtT-20 cells. SOM230 showed a 7-fold higher ligand binding affinity and a 19-fold higher potency in stimulating guanosine 5'-O-(3-thiotriphosphate) binding in AtT-20 cell membranes compared with OCT. SOM230 potently suppressed CRH-induced ACTH release, which was not affected by 48-h dexamethasone (DEX) pretreatment. However, DEX attenuated the inhibitory effects of OCT on ACTH release, whereas it increased the inhibitory potency of BIM-23268, an sst5-specific analog, on ACTH release. Quantitative PCR analysis showed that DEX lowered sst(2A+2B) mRNA expression significantly after 24 and 48 h, whereas sst5 mRNA levels were not significantly affected by DEX treatment. Moreover, Scatchard analyses showed that DEX suppressed maximum binding capacity (B(max)) by 72% when 125I-Tyr3-labeled OCT was used as radioligand, whereas B(max) declined only by 17% when AtT-20 cells were treated with [125I-Tyr11]SS-14. These data suggest that the sst5 protein, compared with sst2, is more resistant to glucocorticoids. Finally, after SS analog preincubation, compared with OCT both SOM230 and BIM-23268 showed a significantly higher inhibitory effect on CRH-induced ACTH release. In conclusion, our data support the concept that the sst5 receptor might be a target for new therapeutic agents to treat Cushing's disease.     

Failure of rearranged TCR transgenes to prevent age-associated thymic involution.

After puberty, the thymus undergoes a dramatic loss in volume, in weight and in the number of thymocytes, a phenomenon termed age-associated thymic involution. Recently, it was reported that age-associated thymic involution did not occur in mice expressing a rearranged transgenic (Tg) TCRalphabeta receptor. This finding implied that an age-associated defect in TCR rearrangement was the major, if not the only, cause for thymic involution. Here, we examined thymic involution in three other widely used MHC class I-restricted TCRalphabeta Tg mouse strains and compared it with that in non-Tg mice. In all three TCRalphabeta Tg strains, as in control mice, thymocyte numbers were reduced by approximately 90% between 2 and 24 mo of age. The presence or absence of the selecting MHC molecules did not alter this age-associated cell loss. Our results indicate that the expression of a rearranged TCR alone cannot, by itself, prevent thymic involution. Consequently, other presently unknown factors must also contribute to this phenomenon.     

The initial age-associated decline in early T-cell progenitors reflects fewer pre-thymic progenitors and altered signals in the bone marrow and thymus microenvironments

Age-related thymus involution results in decreased T-cell production, contributing to increased susceptibility to pathogens and reduced vaccine responsiveness. Elucidating mechanisms underlying thymus involution will inform strategies to restore thymopoiesis with age. The thymus is colonized by circulating bone marrow (BM)-derived thymus seeding progenitors (TSPs) that differentiate into early T-cell progenitors (ETPs). We find that ETP cellularity declines as early as 3 months (3MO) of age in mice. This initial ETP reduction could reflect changes in thymic stromal niches and/or pre-thymic progenitors. Using a multicongenic progenitor transfer approach, we demonstrate that the number of functional TSP/ETP niches does not diminish with age. Instead, the number of pre-thymic lymphoid progenitors in the BM and blood is substantially reduced by 3MO, although their intrinsic ability to seed and differentiate in the thymus is maintained. Additionally, Notch signaling in BM lymphoid progenitors and in ETPs diminishes by 3MO, suggesting reduced niche quality in the BM and thymus contribute to the early decline in ETPs. Together, these findings indicate that diminished BM lymphopoiesis and thymic stromal support contribute to an initial reduction in ETPs in young adulthood, setting the stage for progressive age-associated thymus involution.     

The human thymus: A new perspective on thymic function,aging, and hiv infection

Shortly after birth, the human thymus begins a life long process of involution, whereby the net size of the thymus is not altered but the organ is replaced by adipose tissue. As a result, it has long been believed that thymic involution is indicative of a nonfunctional organ. Recently, however, with the use of computed tomography analysis and innovative molecular approaches that measure T-cell receptor circles, indicative of recent thymic emigrants, doubt has been placed on that dogma. The thymus appears to be active in thymopoiesis throughout the adult life, albeit inversely correlated with age. Being faced with diseases that deplete T-cells such as the acquired immunodeficiency syndrome (AIDS), this recent finding has the potential to exploit novel approaches that enhance thymic output as a mechanism to reconstitute the immune system. In this review, we will revisit the role of T-cells in immunity, the relationship between thymic function and age, and closely examine the impact of HIV-mediated thymic dysregulation on thymopoiesis.     

DKK1 Mediated Inhibition of Wnt Signaling in Postnatal Mice Leads to Loss of TEC Progenitors and Thymic Degeneration

Background

Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus.

Methodology/Principal Findings

Here we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of ΔNP63⁺ Foxn1⁺ and Aire⁺ TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments.

Conclusions/Significance

Taken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants.     

The effects of age, thymectomy, and HIV Infection on alpha and beta TCR excision circles in naive T cells

Due to homeostasis total naive T cell numbers remain fairly constant over life despite a gradual involution of the thymus. The contribution of the thymus to maintaining naive T cell pools is typically measured with TCR excision circles (TRECs) that are formed in thymocytes. The mechanisms underlying thymic involution are poorly understood. Some data suggest that thymocytes undergo fewer divisions in old (small) than young (large) thymi, and other data suggest that the number of TRECs per thymocyte is independent of age. If thymic involution were associated with a decreased number of divisions of the thymocytes, this would markedly complicate the interpretation of TREC data. To study this we develop a mathematical model in which the division rate of thymocytes decreases with increasing age. We describe the dilution of TRECs formed during the arrangement of both chains of the TCR by division of thymocytes, recent thymic emigrants, and mature naive T cells. The model behavior is complicated as TREC contents in naive T cells can increase with age due to decreased dilution in the thymus. Because our model is consistent with current data on the effects of age and thymectomy on TRECs in peripheral T cells, we conclude that aging may well affect thymocyte division, which markedly complicates the interpretation of TREC data. It is possible, but more difficult, to let the model be consistent with the rapid changes in alpha and beta TRECs observed shortly after HIV infection.     

Altered T cell differentiation and Notch signaling induced by the ectopic expression of keratin K10 in the epithelial cells of the thymus

Transgenic mice expressing hK10 under the keratin K5 promoter display several alterations in the epidermis including decreased cell proliferation, and reduced susceptibility to tumor development. Given that K5 promoter is also active in the epithelial cells of the thymus, we explored the possible alterations of the thymus because of K10 transgene expression. We found severe thymic alterations, which affect not only the thymic epithelial cells (TEC), but also thymocytes. We observed altered architecture and premature thymus involution in the transgenic mice associated with increased apoptosis and reduced proliferation of the thymocytes. Interestingly, prior to the development of this detrimental phenotype, thymocytes of the transgenic mice also displayed altered differentiation, which is aggravated later on. Molecular characterization of this phenotype indicated that Akt activity is reduced in TEC, but not in thymocytes. In addition, we also observed altered expression of Notch family members and some of their ligands both in TEC and T cells. This produces reduced Notch activity in TEC but increased Notch activity in thymocytes, which is detectable prior to the disruption of the thymic architecture. In addition, we also detect altered Notch expression in the epidermis of bK5hK10 transgenic mice. Collectively the present data indicate that keratin K10 may induce severe alterations not only in a cell autonomous manner, but also in neighboring cells by the modulation of signals involved in cell-cell interactions.     

Mechanism of thymic involution (analysis of intrinsic and extrinsic aspects)

The mechanisms of thymic involution with age are analyzed in terms of both intrinsic and extrinsic aspects. The intrinsic factors include: decrease in the number of thymocyte-precursors (pro-T cells) in the bone marrow, in emigration of pro-T cells into the thymus, in proliferation of thymic lymphocytes and decrease in extrathymic factors promoting proliferation of thymic lymphocytes. The extrinsic factors capable to exert modulating effects on the thymic function are humoral factors of the hypothalamic-pituitary origin. It seems much easier to manipulate the extrinsic factors by controlling neuro-endocrine stimulation, and to restore the thymic involution. This is quite encouraging, because it permits restoring the impaired immune functions of the aged people.     

Laminin-211 controls thymocyte--thymic epithelial cell interactions

Thymocyte differentiation occurs within the thymic microenvironment, consisting of distinct cell types and extracellular matrix (ECM) elements. One of these ECM proteins is laminin. Previous experiments showed that laminin mediates interactions between thymocytes and thymic epithelial cells (TEC) in mice. Since, laminin comprises a family of related isoforms, we searched for laminin isoform expression in the human thymus. We found constitutive gene expression of various laminin chains in TEC preparations, comprising laminin-111 and laminin-211 isoforms. Immunocytochemistry revealed a selective laminin-211 distribution in the thymic lobules. In vitro functional assays revealed that laminin-211 enhances TEC/thymocyte adhesion and thymocyte release from thymic nurse cells, as well as the reconstitution of these complexes. Conversely, these interactions are blocked by monoclonal antibodies specific for laminin-211 and the laminin receptor VLA-6. Our results reinforce the notion that distinct laminin isoforms in the human thymus are relevant for lymphoepithelial interactions.     

Influence of genital hormones on guinea pig thymus gland cytology]

The changes of the thymic cytologia under the action of genital hormones were studied in giunea pigs. The conclusion of the experiments was: the oestrogens are the only hormones to induce a fast and complete involution of the thymic tissue with appearance of Foa-Kurloff cells'. The cell seems to have all the necessary characters to be the morphologic substratum of an eventual cytocrine secretion of the thymus.     

Central tolerance is impaired in the middle‐aged thymic environment

One of the earliest hallmarks of immune aging is thymus involution, which not only reduces the number of newly generated and exported T cells, but also alters the composition and organization of the thymus microenvironment. Thymic T‐cell export continues into adulthood, yet the impact of thymus involution on the quality of newly generated T‐cell clones is not well established. Notably, the number and proportion of medullary thymic epithelial cells (mTECs) and expression of tissue‐restricted antigens (TRAs) decline with age, suggesting the involuting thymus may not promote efficient central tolerance. Here, we demonstrate that the middle‐aged thymic environment does not support rapid motility of medullary thymocytes, potentially diminishing their ability to scan antigen presenting cells (APCs) that display the diverse self‐antigens that induce central tolerance. Consistent with this possibility, thymic slice assays reveal that the middle‐aged thymic environment does not support efficient negative selection or regulatory T‐cell (Treg) induction of thymocytes responsive to either TRAs or ubiquitous self‐antigens. This decline in central tolerance is not universal, but instead impacts lower‐avidity self‐antigens that are either less abundant or bind to TCRs with moderate affinities. Additionally, the decline in thymic tolerance by middle age is accompanied by both a reduction in mTECs and hematopoietic APC subsets that cooperate to drive central tolerance. Thus, age‐associated changes in the thymic environment result in impaired central tolerance against moderate‐avidity self‐antigens, potentially resulting in export of increasingly autoreactive naive T cells, with a deficit of Treg counterparts by middle age.     

Somatostatin and dopamine receptor expression in lung carcinoma cells and effects of chimeric somatostatin-dopamine molecules on cell proliferation

To study somatostatin/dopamine (SS/D) synergy in a human cell system constitutively expressing SS and D receptors (SSR and DR, respectively), we characterized the expression of SSR and DR subtypes in the non-small-cell lung cancer line Calu-6, and then we evaluated the effect on cell proliferation of SS/D chimeric molecules (BIM-23A387 and BIM-23A370), which bind with high affinity both sst(2) and D(2)R, and compared the results with those obtained by using SS-14 and subtype-selective SS analogs (SSA) and D agonists (DA). Because Calu-6 cells produce insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) peptides, which play a role in the autocrine/paracrine control of cell growth, we also investigated the effects of chimeric compounds on secretion and expression of IGF system components. Relative high levels of sst(2) and the long isoform of the D(2)R were detected by real-time RT-PCR and Western blot in Calu-6, together with sst(5) and to a lesser extent sst(3) and D(4)R. BIM-23A387 and BIM-23A370 significantly inhibited growth of Calu-6, whereas IGF-IGFBP secretion or expression was unaffected, suggesting a direct inhibitory effect. The inhibition of cell growth, measured by both [(3)H]thymidine incorporation and cell count, was significantly lower when individual SSA and DA control peptides or subtype-specific SSA and DA were tested. BIM-23A370 was more potent than BIM-23A387 (P < 0.001). These findings show that SS/D chimeras can inhibit Calu-6 proliferation in an IGF-independent manner and suggest that this enhanced potency might be because of the induction of SSR/DR dimerization. The Calu-6 cell line, constitutively expressing SSR and DR, provides a suitable model to elucidate the mechanism of action of SSA and DA on regulation of cell growth and to characterize the interaction between SSR and DR.     

Expression of nerve growth factor is upregulated in the rat thymic epithelial cells during thymus regeneration following acute thymic involution

Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. Nerve growth factor (NGF) is increasingly recognized as a potent immunomodulator, promoting "cross-talk" between various types of immune system cells. The present study describes the expression of NGF during thymus regeneration following acute involution induced by cyclophosphamide in the rat. Immunohistochemical stain demonstrated not only the presence of NGF but also its upregulated expression mainly in the subcapsular, paraseptal, and perivascular epithelial cells, and medullary epithelial cells including Hassall's corpuscles in both the normal and regenerating thymus. Biochemical data obtained using Western blot and RT-PCR supported these results and showed that thymic extracts contain NGF protein and mRNA, at higher levels during thymus regeneration. Thus, our results suggest that NGF expressed in these thymic epithelial cells plays a role in the T lymphopoiesis associated with thymus regeneration during recovery from acute thymic involution.     

Age-related involution and terminal disorganization of the human thymus

The terminal involution pattern of the human thymus was studied based on autopsy cases (both sexes, age range 63-91 years). Large sections through the entire thymic fat body were examined with the help of both conventional histological and immunohistochemical techniques. The findings demonstrate that thymic atrophy in old humans (a) goes far beyond the degree of involution observed in small rodents; (b) results in a system of thin, branching, in part interrupted, non-keratinizing epithelial plates containing no typical Hassall bodies; (c) concerns all components of the thymus except fat tissue which progressively replaces original thymic structures; and (d) involves various types of disorganization of individual lobules with T and B lymphocytes often located outside rather than within epithelial remnants. Effects of low-level radiation on this final regression of the human thymus are unknown.