Deletion of high-avidity T cells by thymic epithelium.

Tolerance induction by thymic epithelium induces a state of so-called "split tolerance," characterized in vivo by tolerance and in vitro by reactivity to a given thymically expressed antigen. Using a model major histocompatibility complex class I antigen, H-2Kb (Kb), three mechanisms of thymic epithelium-induced tolerance were tested: induction of tolerance of tissue-specific antigens exclusively, selective inactivation of T helper cell-independent cytotoxic T lymphocytes, and deletion of high-avidity T cells. To this end, thymic anlagen from Kb-transgenic embryonic day 10 mouse embryos, taken before colonization by cells of hemopoietic origin, were grafted to nude mice. Tolerance by thymic epithelium was not tissue-specific, since Kb-bearing skin and spleen grafts were maintained indefinitely. Only strong priming in vivo could partially overcome the tolerant state and induce rejection of some skin grafts overexpressing transgenic Kb. Furthermore, the hypothesis that thymic epithelium selectively inactivates those T cells that reject skin grafts in a T helper-independent fashion could not be supported. Thus, when T-cell help was provided by a second skin graft bearing an additional major histocompatibility complex class II disparity, tolerance to the Kb skin graft was not broken. Finally, direct evidence could be obtained for the avidity model of thymic epithelium-induced negative selection, using Kb-specific T-cell receptor (TCR) transgenic mice. Thymic epithelium-grafted TCR transgenic mice showed a selective deletion of those CD8+ T cells with the highest density of the clonotypic TCR. These cells presumably represent the T cells with the highest avidity for Kb. We conclude that split tolerance induced by thymic epithelium was mediated by the deletion of those CD8+ T lymphocytes that have the highest avidity for antigen.     

Regulation of thymic epithelium by keratinocyte growth factor

Here we demonstrate that keratinocyte growth factor (KGF) and FGFR2IIIb signaling can affect development and function of thymic epithelium (TE) and that alphabeta-lineage thymocytes contribute to intrathymic levels of KGF. Thymocyte expression of KGF is developmentally regulated, being undetectable in CD3-4-8- thymocytes and expressed at highest levels by mature CD4 or CD8 thymocytes. Exposure of thymocyte-depleted fetal thymic lobes to KGF resulted in reduced thymic epithelial expression of class II major histocompatibility complex (MHC), invariant chain (Ii), and cathepsin L (CatL) molecules involved in thymocyte-positive selection and also stimulated expression of the cytokines interleukin 6 (IL-6) and thymic stromal-derived lymphopoietin (TSLP), while having little effect on IL-7 or stem cell factor expression. Within intact fetal thymic organ culture (FTOC), exogenous KGF impairs the generation of CD4 thymocytes. Two lines of evidence point to responsiveness of the medullary TE compartment to KGF and FGFR2IIIb signaling. First, the medullary compartment is expanded in intact FTOC exposed to KGF in vitro. Second, in the RAG-deficient thymus, where the thymocytes do not express detectable levels of KGF message, the hypoplastic medullary TE compartment can be expanded by administration of recombinant KGF in vivo. This expansion is accompanied by restoration of the normal profile of medullary TE-associated chemokine expression in the RAG2(-/-) thymus. Collectively, these findings point to a role for KGF and FGFR signaling in the development and function of thymic epithelium.     

Lymphocytes selected in allogeneic thymic epithelium mediate dominant tolerance toward tissue grafts of the thymic epithelium haplotype.

Athymic mice grafted at birth with allogeneic thymic epithelium (TE) from day 10 embryos before hematopoietic cell colonization reconstitute normal numbers of T cells and exhibit full life-long tolerance to skin grafts of the TE haplotype. Intravenous transfers of splenic cells, from these animals to adult syngeneic athymic recipients, reconstitute T-cell compartments and the ability to reject third-party skin grafts. The transfer of specific tolerance to skin grafts of the TE donor strain, however, is not observed in all reconstituted recipients, and the fraction of nontolerant recipients increases with decreasing numbers of cells transferred. Furthermore, transfers of high numbers of total or CD4+ T cells from TE chimeras to T-cell receptor-anti-H-Y antigen transgenic immunocompetent syngeneic hosts specifically hinder the rejection of skin grafts of the TE haplotype that normally occurs in such recipients. These observations demonstrate (i) that mice tolerized by allogeneic TE and bearing healthy skin grafts harbor peripheral immunocompetent T cells capable of rejecting this very same graft; and (ii) that TE selects for regulatory T cells that can inhibit effector activities of graft-reactive cells.     

Identification of nuclear triiodothyronine receptors in the thymic epithelium.

Thymic epithelial cell physiology is known to be under neuroendocrine control. In particular, thyroid hormones modulate thymic hormone secretion by thymic epithelial cells in vivo and in vitro, thus suggesting the existence of specific receptors for those hormones in this component of the thymic microenvironment. Yet, thyroid hormone-binding sites have previously been detected only in crude thymus fractions and lymphocytes. We, thus, decided to search for T3 receptors in the thymic epithelium, by using an antinuclear T3 receptor monoclonal antibody. In situ immunohistochemical analysis of thymic frozen sections showed nuclear labeling of both lymphoid and nonlymphoid cells in the cortex and medulla. Moreover, in vitro studies using thymic epithelial cell lines and the so-called thymic nurse cells revealed a positive reaction in the chromatin, with nucleoli remaining negative. Immunoblot data clearly showed a single protein band of 57K reactive with the antinuclear T3 receptor antibody in murine thymus extracts as well as in the thymic epithelial cell lines. Lastly, in vitro treatment of these cells with T3 resulted in a transient, yet profound, down-modulation of the receptor. In conclusion, our findings provide molecular evidence that the action of thyroid hormones on thymic epithelium occurs via the typical 57K nuclear T3 receptors.     

Enhancement by tetragastrin of experimental induction of gastric epithelium in the duodenum.

The effects of tetragastrin and truncal vagotomy on the incidence of gastric type epithelium in the duodenum by intraduodenal instillation of 5% NaOH solution were investigated in Wistar rats. Prolonged administration of 1 mg tetragastrin/kg body weight in depot form starting one week after NaOH treatment resulted in a significant increase in gastric acid secretion and the incidence and number of villi with gastric epithelium in the duodenum in experimental week 10. Villi with gastric epithelium were found in five (23%) of 22 rats in control group, whereas abnormal villi were found in 13 (59%) of 22 rats in the tetragastrin treated group (p less than 0.05). The average number of villi with gastric epithelium rose from 0.6 (0.4) per 100 villi in control rats to 2.4 (0.6) per 100 villi in tetragastrin treated rats (p less than 0.01). On histological examination, gastric type epithelium was most often found on stunted or flattened villi, and was always within the boundaries of the area of Brunner's glands. These mucosal changes reverted toward normal with time. In week 35, the incidence of gastric epithelium was significantly less than at week 10 (p less than 0.05). In contrast, no villi with gastric epithelium were found in vagotomised rats in week 10 (p less than 0.05). Vagotomy also caused a significant decrease in gastric acid secretion. These results show that exposure of the duodenal mucosa to high levels of gastric acid enhance the induction of gastric eithelium in the duodenum.     

Thymic rejuvenation and the induction of tolerance by adult thymic grafts

The thymus, the site of origin of T cell immunity, shapes the repertoire of T cell reactivity through positive selection of developing T cells and prevents autoimmunity through negative selection of autoreactive T cells. Previous studies have demonstrated an important role for the thymus not only in central deletional tolerance, but also in the induction of peripheral tolerance by vascularized renal allografts in juvenile miniature swine recipients. The same protocol did not induce tolerance in thymectomized recipients nor in recipients beyond the age of thymic involution. We subsequently reported that vascularized thymic lobe grafts from juvenile donors were capable of inducing tolerance in thymectomized juvenile hosts. However, the important question remained whether aged, involuted thymus could also induce tolerance if transplanted into thymectomized hosts, which, if true, would imply that thymic involution is not an intrinsic property of thymic tissue but is rather determined by host factors extrinsic to the thymus. We report here that aged, involuted thymus transplanted as a vascularized graft into juvenile recipients leads to rejuvenation of both thymic structure and function, suggesting that factors extrinsic to the thymus are capable of restoring juvenile thymic function to aged recipients. We show furthermore that rejuvenated aged thymus has the ability to induce transplant tolerance across class I MHC barriers. These findings indicate that it may be possible to manipulate thymic function in adults to induce transplantation tolerance after the age of thymic involution.     

同种异体胰岛移植免疫耐受的诱导

同种异体胰岛移植为1型糖尿病患者带来了治愈疾病的希望,临床应用的成功显示了胰岛移植良好的发展前景。但移植后患者需终身服用免疫抑制剂,使胰岛移植仅适合用药物难以控制的糖尿病或需进行肾移植的患者。显然,诱导免疫耐受无疑将大大扩展胰岛移植的指征,更多的1型糖尿病患者将从中得益,避免糖尿病远期并发症的发生并提高生活质量。本文综述了诱导免疫耐受、延长同种异体移植胰岛存活的最新实验进展,包括外周和中央型耐受的诱导。对一些细胞表面分子,如共刺激分子、黏附素及细胞因子受体等进行干预,均可延长移植于非自身免疫疾病背景小鼠的同种异体胰岛存活时间。去除同种异体反应性T细胞、诱导供体特异的调节性T细胞是建立外周耐受的重要途径,甚至对有抵抗耐受倾向的自身免疫性非肥胖糖尿病小鼠也有效。通过骨髓移植,结合无须清除原始粒细胞的方案,能获得造血细胞嵌合,使胰岛成功移植于自身免疫性糖尿病受体中,并尽可能减少移植物抗宿主疾病的发生。     

Tolerance induction by lentiviral gene therapy with a nonmyeloablative regimen

Antibodies (Abs) directed at the Gal alpha1,3Gal beta1,4GlcNAc-R (alphaGal) carbohydrate epitope initiate xenograft rejection. Previously, we have shown that bone marrow transplantation (BMT) with lentivirus-mediated gene transfer of porcine alpha1,3 galactosyltransferase (GalT) is able to induce tolerance to alphaGal-expressing heart grafts following a lethal dose of irradiation. Here we show the first demonstration of permanent survival of alphaGal+ hearts following transplantation with autologous, lentivirus-transduced BM using a nonmyeloablative regimen. Autologous BM from GalT knockout (GalT-/-) mice was transduced with a lentiviral vector expressing porcine GalT and transplanted into sublethally irradiated (3 Gy) GalT-/- mice. Chimerism in the peripheral blood cells (PBCs) remained low but was higher in the BM, especially within the stromal cell population. Mice reconstituted with GalT did not produce anti-alphaGal Abs over time. We immunized these mice with alphaGal-expressing cells and assessed humoral immune responses. Anti-alphaGal xenoantibodies were not produced in mice reconstituted with GalT, but normal Ab responses to other xenoantigens were detected. Mice reconstituted with GalT accepted alphaGal+ heart grafts over 100 days. Transduction with lentiviral vectors results in chimerism at levels sufficient to induce long-term tolerance under nonmyeloablative conditions.     

Tolerance induction by bone marrow transplantation in a multiple sclerosis model

Experimental autoimmune encephalomyelitis (EAE) in rats is a highly valuable model of multiple sclerosis (MS) because it mimics major hallmarks of the human disease. EAE induced with myelin-oligodendrocyte-glycoprotein (MOG) in DA rats is relapsing/remitting, and lesions in the central nervous system show inflammation, demyelination, and axonal and neuronal loss. Recently, bone marrow transplantation (BMT) was introduced as a novel strategy to treat MS, but its efficiency and the underlying mechanism are debatable. In MOG-induced EAE we found that BMT at the peak of EAE but not in the chronic phase leads to disease attenuation. In both settings, rats receiving bone marrow (BM) transplants were protected from subsequently induced relapses. These findings could be confirmed by histopathology in which rats receiving BM transplants did not have lesions compared with controls not receiving transplants. Importantly, the protective effect was achieved by allogeneic, syngeneic, and BM grafts from diseased rats. BMT resulted in increased numbers of CD4(+)CD25(bright) regulatory T cells, increased Foxp3 expression, a shift in T-cell epitope recognition, and a strong reduction of autoantibodies even after rechallenge with MOG. Thus, our results indicate potential mechanisms of how BMT may contribute to the improvement of MS and provide a rationale for its application in patients suffering from various autoimmune diseases.     

Androgen receptors in thymic epithelium modulate thymus size and thymocyte development

Castration of normal male rodents results in significant enlargement of the thymus, and androgen replacement reverses these changes. Androgen-resistant testicular feminization (Tfm) mice also show significant thymus enlargement, which suggests that these changes are mediated by the androgen receptor (AR). The cellular targets of androgen action in the thymus are not known, but may include the lymphoid cells (thymocytes) as well as nonlymphoid epithelial cells, both of which have been believed to express AR. In the present study immunohistochemical analysis and hormone binding assays were used to demonstrate the presence of AR in thymic epithelial cells. The physiological significance of this epithelial cell AR expression was defined by further studies performed in vivo using chimeric mice, produced by bone marrow transplantation, in which AR expression was limited to either lymphoid or epithelial components of the thymus. Chimeric C57 mice engrafted with Tfm bone marrow cells (AR(+) epithelium and AR(-) thymocytes) had thymuses of normal size and showed the normal involutional response to androgens, whereas chimeric Tfm mice engrafted with C57 bone marrow cells (AR(-) epithelium and AR(+) thymocytes) showed thymus enlargement and androgen insensitivity. Furthermore, phenotypic analyses of lymphocytes in mice with AR(-) thymic epithelium showed abrogation of the normal responses to androgens. These data suggest that AR expressed by thymic epithelium are important modulators of thymocyte development.     

Regulation of insulin gene expression by cytokines and cell–cell interactions in mouse medullary thymic epithelial cells

Aims/hypothesis  

The expression of tissue-specific self-antigens in the thymus is essential for self-tolerance. Genetic susceptibility to type 1 diabetes correlates inversely with thymic insulin expression and, in mice, lowered levels of this expression result in T cell responses against insulin. This study was undertaken to examine whether thymic insulin expression is regulated by the same metabolic stimuli as in beta cells or by different inputs, possibly of an immune nature.     

On the role of thymic epithelium vs. bone marrow-derived cells in repertoire selection of T cells

T lymphocytes mature in the thymus to become functional T cells. Studies with chimeric mice and T cell receptor (TCR) transgenic (tg) mice have indicated that the major histocompatibility gene complex (MHC) of thymic radio-resistant (presumed to be epithelial) cells positively select the MHC-restricted T cell repertoire. Surprisingly, mice without a thymus reconstituted with an MHC-incompatible thymus generate effector T cells which are, in general, specific for the host and not for the thymic MHC. The present study reanalyzed this longstanding paradox in nude mice that were reconstituted with an MHC-incompatible thymus plus or minus immunologically defective bone marrow-derived cells or in nude mice expressing a transgenic T cell receptor. A pathway of thymus-dependent but thymic MHC-independent T cell maturation is revealed where expansion of the antiviral T cell repertoire depends on the MHC of bone marrow-derived cells. These results indicate an alternative, if not a general, pathway of T cell maturation and selection: the thymus may function essentially as an organ promoting T cell receptor expression; T cell specificity, however, reflects repertoire expansion plus cell survival and effector T cell induction driven by the MHC of bone marrow-derived cells. Therefore pure thymus defects can be efficiently reconstituted by allo- and xenogeneic thymic grafts.     

TRAF3 enforces the requirement for T cell cross-talk in thymic medullary epithelial development

Induction of self-tolerance in developing T cells depends on medullary thymic epithelial cells (mTECs), whose development, in turn, requires signals from single-positive (SP) thymocytes. Thus, the absence of SP thymocytes in Tcra^−/− mice results in a profound deficiency in mTECs. Here, we have probed the mechanism that underlies this requirement for cross-talk with thymocytes in medullary development. Previous studies have implicated nonclassical NF-κB as a pathway important in the development of mTECs, because mice lacking RelB, NIK, or IKKα, critical components of this pathway, have an almost complete absence of mTECs, with resulting autoimmune pathology. We therefore assessed the effect of selective deletion in TEC of TNF receptor-associated factor 3 (TRAF3), an inhibitor of nonclassical NF-κB signaling. Deletion of TRAF3 in thymic epithelial cells allowed RelB-dependent development of normal numbers of AIRE-expressing mTECs in the complete absence of SP thymocytes. Thus, mTEC development can occur in the absence of cross-talk with SP thymocytes, and signals provided by SP T cells are needed to overcome TRAF3-imposed arrest in mTEC development mediated by inhibition of nonclassical NF-κB. We further observed that TRAF3 deletion is also capable of overcoming all requirements for LTβR and CD40, which are otherwise necessary for mTEC development, but is not sufficient to overcome the requirement for RANKL, indicating a role for RANKL that is distinct from the signals provided by SP thymocytes. We conclude that TRAF3 plays a central role in regulation of mTEC development by imposing requirements for SP T cells and costimulation-mediated cross-talk in generation of the medullary compartment.A major role of the thymus is the generation of a functional T-cell repertoire that is broadly responsive to foreign antigens but is self-tolerant. Through their role in exposing developing thymocytes to a spectrum of self-antigens, the stromal cells of the thymus are integral to this tolerization. Of particular importance in this process are the epithelial cells comprising the thymic medulla, the region of the thymus where thymocytes selected into the CD4 and CD8 single-positive (SP) lineages reside before emigrating to the periphery (reviewed in refs. 1–3). The importance of thymic medullary epithelial cells (mTECs) in the maintenance of self-tolerance is illustrated by the destructive autoreactivity that results from disruption of mTEC development (reviewed in ref. 4).Just as mTECs have a central role in shaping the developing T-cell repertoire, thymocytes, in turn, are vital to the development and maintenance of the mTEC compartment, a bidirectional interaction that has been termed cross-talk (5–7). A number of recent reports have characterized the CD4 SP thymocyte–stromal cell interactions that are critical for mTEC development (5–11). However, the mechanism that enforces the requirement for SP thymocytes in mTEC development has not been fully identified.We therefore addressed the signaling requirements that mediate the cross-talk required for mTEC development. Previous studies have implicated nonclassical NF-κB as a pathway important in the development of mTECs. It has been shown that mice lacking RelB, NIK, or IKKα, components of the nonclassical NF-κB pathway, have an almost complete absence of mTECs and exhibit resulting autoimmune pathology (12–16). Engagement of TNF receptor (TNFR) family members including CD40, LTβR, and RANK has been shown to activate nonclassical NF-κB signaling via a pathway regulated by several members of the TRAF family of adaptor/ubiquitin ligase proteins. TRAF3 has a unique role in inhibiting nonclassical NF-κB signaling in resting cells (17), and it has been demonstrated that deletion of TRAF3 in B cells results in constitutive activation of the alternative NF-κB pathway (18, 19). We therefore tested the possibility that TRAF3-mediated inhibition of alternative NF-κB is responsible for the failure of mTEC development in the absence of signals from SP thymocytes. We made the striking observation that deletion of TRAF3 in thymic epithelium is sufficient to allow RelB-dependent mTEC development in the complete absence of TCRαβ SP thymocytes. TRAF3 deletion is also capable of overcoming all requirements for LTβR and CD40 during mTEC development, but is not sufficient to overcome the requirement for RANKL, indicating an essential role for RANKL that is distinct from the signals provided by SP thymocytes. Together, these results demonstrate that mTECs can develop in the complete absence of SP thymocytes and that TRAF3 plays a critical role in imposing the requirement for cross-talk with SP thymocytes, thus linking the appearance of mature thymocytes to the development of the thymic medullary environment necessary for imposing self-tolerance.     

Pretreatment biopsy for histological diagnosis and induction therapy in thymic tumors

Background

This study was to investigate the value of pretreatment biopsy for histological diagnosis and induction therapies in the management of locally advanced thymic malignancies.

Methods

The clinical pathological data of patients with thymic tumors in the Chinese Alliance for Research in Thymomas (ChART) who underwent biopsy before treatment from 1994 to December 2012 were retrospectively reviewed. The application trend of preoperative histological diagnosis and its influence on treatment outcome were analyzed.

Results

Of 1,902 cases of thymic tumors, 336 (17.1%) had undergone biopsy for histological diagnosis before therapeutic decision was decided. In recent years, percentage of pretreatment histological diagnosis significantly increased in the later ten years than the former during the study period (P=0.008). There was also a significant increase in thoracoscopy/mediastinoscopy/E-BUS biopsy as compared to open biopsy (P=0.029). Survival in Patients with preoperative biopsy for histology had significantly higher stage lesions (P=0.000) and higher grade malignancy (P=0.000), thus a significantly lower complete resection rate (P=0.000) and therefore a significantly worse survival than those without preoperative biopsy (P=0.000). In the biopsied 336 patients, those who received upfront surgery had significantly better survival than those received surgery after induction therapy (P=0.000). In stage III and IVa diseases, the R0 resection rate after induction therapies increased significantly as compared to the surgery upfront cases (65.5% vs. 46.2%, P=0.025). Tumors downstaged after induction had similar outcomes as those having upfront surgery (92.3% vs. 84.2%, P=0.51). However, tumors not downstaged by induction had significantly worse prognosis than those downstaged (P=0.004), and fared even worse than those having definitive chemoradiation without surgery (37.2% vs. 62.4%, P=0.216).

Conclusions

It is crucial to get histological diagnosis for thymoma before surgery or adjuvant treatment and minimally invasive biopsy should be undertaken. Although in our study we could not find the benefit of induction chemotherapy before surgery in survival and recurrence rate, it could increase the R0 resection rate compared with direct surgery in late stage (III and IVa).