Thymic stromal organization is regulated by the specificity of T cell receptor/major histocompatibility complex interactions

The thymic architecture is normally compartmentalized into a central medulla surrounded by a peripheral cortical region. We investigated how compartmentalization of the thymic stroma is regulated using T cell receptor (TCR)-transgenic mouse models. Our studies show that the signals generated by TCR/peptide/major histocompatibility complex interactions regulate thymic stromal cell compartmentalization. In TCR-transgenic mice, normal stromal cell compartmentalization occurs when the transgenic TCR is expressed on a background that does not result in skewing toward either positive or negative selection. In models representing strong positive selection, the thymic stromal elements do not fully organize into a central medulla. Instead, small medullary foci are dispersed throughout the thymus with some regions residing directly under the capsule. The highest degree of disorganization in medullary epithelial regions is observed in TCR-transgenic mice that exhibit negative selection. Although the medullary foci lack central organization, the expression in these regions of CD80, CD86 and CD40, as well as the clustering of dendritic cells, is similar to that observed in medullae of wild-type mice. Thus, the organization of the medulla appears to occur in two stages: (1) small medullary epithelial regions that are dispersed in fetal thymi expand and associate with antigen-presenting cells, and (2) the expanded medullary foci organize into a central medullary compartment. Our data suggest a model in which this second stage of stromal cell organization is increasingly inhibited as the normal balance of TCR-mediated signals is skewed by higher-avidity interactions between thymocytes and antigen-presenting cells.     

EphrinB1-EphB signaling regulates thymocyte-epithelium interactions involved in functional T cell development

The Eph and ephrin families are involved in numerous developmental processes. Recently, an increasing body of evidence has related these families with some aspects of T cell development. In the present study, we show that the addition of either EphB2-Fc or ephrinB1-Fc fusion proteins to fetal thymus organ cultures established from 17-day-old fetal mice decreases the numbers of both double-positive (CD4(+)CD8(+)) and single-positive (both CD4(+)CD8(-) and CD4(-)CD8(+)) thymocytes, in correlation with increased apoptosis. By using reaggregate thymus organ cultures formed by fetal thymic epithelial cells (TEC) and CD4(+)CD8(+) thymocytes, we have also demonstrated that ephrinB1-Fc proteins are able to disorganize the three-dimensional epithelial network that in vivo supports the T cell maturation, and to alter the thymocyte interactions. In addition, in an in vitro model, Eph/ephrinB-Fc treatment also decreases the formation of cell conjugates by CD4(+)CD8(+) thymocytes and TEC as well as the TCR-dependent signaling between both cell types. Finally, immobilized EphB2-Fc and ephrinB1-Fc modulate the anti-CD3 antibody-induced apoptosis of CD4(+)CD8(+) thymocytes in a process dependent on concentration. These results therefore support a role for Eph/ephrinB in the processes of development and selection of thymocytes as well as in the establishment of the three-dimensional organization of TEC.     

Thymic T cell export is not influenced by the peripheral T cell pool

The peripheral T cell pool is maintained both by export of naive T cells from the thymus and by post-thymic expansion of activated/memory T cells. However, it is not known whether the thymus can alter its output following peripheral T cell depletion. Using intrathymic injection of fluorescein isothiocyanate to detect recent thymic emigrants (RTE), we directly tested whether the thymus is able to alter the number of RTE or the CD4:CD8 ratio of RTE emigrating to the periphery in response to in vivo depletion of total peripheral T cells or CD4 T cells, respectively. Depletion of peripheral T cells was achieved with anti-Thy-1 or anti-CD4, at doses that did not affect thymocyte numbers. Depletion of greater than 70% of peripheral T cells by treatment with anti-Thy-1 in vivo did not alter the number or cell cycle status of RTE trafficking to lymph nodes or spleen during the peripheral reconstitution phase (6, 9, 12 days). Similarly, depletion of the majority of CD4 T cells, which significantly reduced the peripheral CD4:CD8 T cell ratio, did not alter the total number or the proportion of CD4⁺ CD8^? RTE in peripheral lymphoid organs. These data clearly indicate that thymic output is not influenced by downstream alterations in peripheral T cell pool size or CD4:CD8 ratio. Rather we contend that thymic T cell export is internally regulated by as yet undefined mechanisms.     

Thymic epithelial tumour progression in an SV40T transgenic mouse model

 There have been several reports that thymoma in human is a progressive disease, and that thymoma and thymic carcinoma form a continuum. We established a stable line of SV40T transgenic mice, which consistently produced thymic epithelial tumours progressing to thymic carcinoma within a predictable time span. Using this animal model and a morphological approach, thymic epithelial tumour progression was studied with reference to sequential changes at different time points in animals aged from 3 to 32 weeks. At all ages, SV40T was expressed in the nuclei of thymic epithelial cells; in these transgenic mice we observed the entire spectrum from cortical type thymoma to thymic carcinoma. Thymic size tended to increase with ageing in SV40T TG mice. While younger mice had predominantly cortical (organoid) or cortical thymoma, older mice had well-differentiated thymic carcinoma (WDTC) or poorly differentiated thymic carcinoma. When SV40T TG mice (248 line) reached a certain age, carcinoma of the thymus was present in all of them. Cortical-type thymoma became malignant within a predictable time span, suggesting a cortical thymoma–carcinoma sequence. When the mice were 9 weeks of age, the thymuses formed gross masses compatible with cortical thymoma. At 14 weeks of age, WDTC appeared against the background of cortical thymoma. Poorly differentiated thymic carcinoma was found after 15 weeks and affected all animals over 23 weeks of age. Most thymic carcinomas coexisted in varying proportions with cortical-type thymoma. Medullary thymomas did not develop in the mice, and no transition from medullary-type thymomas to thymic carcinomas was observed. In this SV40T transgenic mouse model, thymic carcinoma is clearly preceded by cortical-type thymoma. These transgenic mice may provide an interesting model for the progression from cortical thymoma to WDTC and/or high-grade carcinoma. Received: 6 August 1996 / Accepted: 23 June 1997     

Effect of reduced EPHB4 expression in thymic epithelial cells on thymocyte development and peripheral T cell function

The Eph kinase (EPH) and ephrin (EFN) families are involved in a broad range of developmental processes. Increasing evidence is demonstrating the important roles of EPHBs and EphrinBs in the immune system. In this study on epithelial cell-specific Ephb4 knockout (KO) mice, we investigated T-cell development and function after EPHB4 deletion. KO mice presented normal thymic weight and cellularity. Their thymocyte subpopulation percentages were in the normal range. KO mice had normal T-cell numbers and percentages in the spleen, and T cells were activated and proliferated normally upon TCR ligation. Furthermore, naïve spleen CD4 cells from KO and wild type mice were capable of differentiating, in a comparable manner, into Th1, Th17 and Treg cells. In vivo, KO mice mounted effective delayed type hypersensitivity responses, indicating that thymocytes develop normally in the absence of TEC EPHB4, and T cells derived from EPHB4-deleted thymic epithelian cells (TEC) have normal function. Our data suggest that heavy redundancy and promiscuous interaction between EPHs and EFNs compensate for the missing EPHB4 in TECs, and TEC EPHB4's role in T cell development might only be revealed if multiple EPHs are ablated simultaneously. We cannot exclude the possibility that (1) some immunological parameters not examined in this study are affected by the deletion; (2) the deletion is not complete due to the leaky Cre-LoxP system, and the remaining EPHB4 in TEC is sufficient for thymocyte development; or (3) EPHB4 expression in TEC is not required for T cell development and function.     

Thymic production of human FOXP3(+) regulatory T cells is stable but does not correlate with peripheral FOXP3 expression

In humans functionally mature FOXP3⁺ regulatory T (Treg) cells can be found already in the fetus, but the kinetics of their maturation is still unknown. Here, we show that from birth to until 10 years of age the thymic production of FOXP3⁺ Treg cells is very stable and correlates with T-lymphopoiesis in general. The level of FOXP3 expression in the blood was also very stable, even when children and adults were compared, but there was no correlation between thymic and peripheral FOXP3 levels. Analysis of the cell cycle-associated marker Ki67 showed that a substantial fraction of peripheral FOXP3⁺ cells is dividing. This characteristic was obtained in the periphery, since it was not observed in thymic CD4⁺ FOXP3⁺ cells. These data suggest that the thymic output of human Treg cells is intrinsically stable, while in the periphery the increased rate of proliferation severs the connection between production and homeostatic maintenance of the FOXP3⁺ Treg cell population.     

Regulation of T cell production in T cell receptor transgenic mice

The thymus produces many more cells than it releases into the periphery. According to generally accepted models of T cell development most of this loss occurs in the thymic cortex, among CD4⁺8⁺ thymocytes. An interesting situation arises in the case of T cell receptor (TcR) transgenic mice in which all cells can potentially be positively selected, leading to a theoretical increase of about 30-fold in the survival rate of CD4⁺8⁺ cells and in their transition to mature CD4⁺8^?or CD4^?8⁺ thymocytes. This in turn should lead to a 30-fold increase in the size of the thymic medulla, in the emigration rate and in the size of the peripheral Tcell pool. Increases in medullary or peripheral pool sizes of this magnitude are not seen in TcR transgenic mice. The question was therefore asked whether some form of homeostatic process regulated the size of the mature T cell pool and at what level it might operate. In this report we demonstrate that the increased rate of double-positive to single-positive transition in the TcR transgenic mice is directly reflected in an increased emigration rate, and that the medulla seems to be relatively efficient regardless of the number of cells passing through it. However, the potential increases in emigrant numbers in TcR transgenic mice are offset by the reduced size of the CD4⁺8⁺ thymocyte pool. It would appear then that regulation of T cell production, if it occurs, probably does so through regulation of the size of the CD4⁺8⁺ thymocyte pool. Mechanisms for regulation of this kind are not yet known.     

Cassette vectors directing expression of T cell receptor genes in transgenic mice

We describe a pair of cassette vectors that can be used to express rearranged T cell receptor genes in transgenic mice. Short DNA fragments containing rearranged V and Vβ segments are readily amplified from T cells and introduced between artificial cloning sites. Transgene-derived mRNAs are transcribed under the control of the natural TCR and -β promoter/enhancer elements. Using this vector, we have obtained transgenic mouse lines which display transgene-encoded TCR and β chains on a majority of T cells.     

SP2/0细胞膜抗原负载DC诱导体外特异性CTL效应

目的 研究SP2／0细胞膜抗原负载树突状细胞（DC）诱导的特异性细胞毒性T淋巴细胞（CTL）对肿瘤细胞的杀伤活性。方法 密度梯度离心法分离SP2／0细胞膜，负载经rmGM-CSF和rmIL-4诱导扩增的小鼠骨髓来源的DC，活化T淋巴细胞获得肿瘤特异性CTL，MTT法检测对SP2／0细胞的杀伤效果。结果 骨髓单个核细胞在rmGM-CSF和rmIL-4作用下获得了高表达CDS0、CD86和MHC-Ⅱ分子的DC，致敏的CTL对SP2／0骨髓瘤细胞具有高杀伤率，显著高于对I5178Y淋巴瘤细胞的杀伤活性（P〈0．01）。结论 SP2／0细胞膜抗原负载DC能诱导明显的特异性抗肿瘤免疫应答。     

Ligand-dependent regulation of T cell development and activation

Mature CD4⁺ and CD8⁺T lymphocytes develop in the thymus from precursors with diverse clonally distributed receptors, possessing binding sites with negligible, intermediate, or high affinity for self-peptide: major histocompatibility complex (MHC) ligands. Positive-and negative-selection processes acting on this precursor pool yield a peripheral T cell population comprised of cells with receptors (TCR) capable of self-peptide: MHC ligand recognition, but largely depleted of those able to mediate overt self-responsiveness. The Lymphocyte Biology Section of the Laboratory of Immunology studies how self-ligand recognition guides T cell development in the thymus and influences the functionality of naive and activated T cells in the periphery. It also seeks to define the molecular basis for the discrimination between self-ligands and foreign antigens that controls T cell activation to effector function. Finally, it uses a combination of conventional cellular immunological methods, biochemical and biophysical studies, and advanced imaging techniques to visualize, quantitate, and model the various steps in the development of primary and memory T cell immune responses.     

Impaired survival of T cell receptor Vγ3+ cells in interleukin-4 transgenic mice

The mouse epidermis contains a network of Thy-1⁺ dendritic T cells. Most of these cells express a homogeneous T cell receptor (TCR) configuration (Vγ3/ Vδ1) with only negligible junctional diversity. Because fetal thymocytes are precursors of these dendritic epidermal T cells (DETC) and the addition of interleukin (IL)-4 to fetal thymic organ cultures causes an early arrest in thymopoiesis, we examined DETC development in transgenic (tg) mice expressing IL-4 under the control of major histocompatibility complex class I regulatory sequences. Immunohistologic examination of epidermal sheets and polymerase chain reaction analysis of total skin RNA from IL-4 tg mice failed to reveal TCR Vγ3⁺ DETC and Vγ3 mRNA, respectively. In contrast, the sizes of TCR γδ subpopulations in lymphoid organs were unchanged in these mice. Although the numbers and staining intensities of TCR Vγ3⁺ thymocytes in early fetal (days 14–17) IL-4 tg mice were similar to those of littermate controls, we observed a preferential death of these cells in thymic organ cultures from IL-4 tg mice. We observed further that epidermal sheets prepared from 9-day-old mice whose mothers had been treated with an IL-4-neutralizing antibody from day 12 to day 18 of pregnancy contained DETC numbers similar to those of controls. However, upon termination of the anti-IL-4 treatment, DETC ceased to expand. We conclude that IL-4 impairs the survival of TCR Vγ3⁺ cells.     

Pertussis toxin can replace T cell receptor signals that induce positive selection of CD8 T cells

CD4⁺ helper T lymphocytes and CD8⁺ killer T lymphocytes are both generated in the thymus from common precursor cells expressing CD4 and CD8. The development of immature CD4⁺ CD8⁺ thymocytes into mature ‘single-positive’ T cells requires T cell antigen-receptor (TCR)-mediated positive selection signals. Although it is known that the recognition specificity of TCR expressed by CD4⁺ CD8⁺ thymocytes determines their fate to become either CD4⁺ or CD8⁺ T cells, the molecular signals that direct precursor thymocytes to become CD4⁺ and CD8⁺ T cells are unclear. By using ZAP-70^? mutant thymus organ cultures in which T cell development is arrested at the CD4⁺ CD8⁺ thymocyte stage, the present study shows that distinct biochemical treatments can selectively restore the generation of mature CD4⁺ and CD8⁺ T cells, bypassing TCR-induced positive selection signals. The combination of phorbol ester and ionomycin selectively restores the generation of CD4⁺CD8^? TCR^high cells consistent with previous results. On the other hand, we find that the generation of CD4^? CD8⁺ TCR^high cells is selectively induced by pertussis toxin. Interestingly, the signals generated by pertussis toxin, which increase Notch expression, can dominate the signals by phorbol ester and ionomycin, steering thymocyte development to CD8 lineage. These results indicate that distinct biochemical signals replace TCR signals that selectively induce positive selection of CD4⁺ and CD8⁺ T cells, and that biochemical treatment can manipulate the development and choice of CD4⁺ and CD8⁺ T cells.     

Direct cell/cell contact with stimulated T lymphocytes induces the expression of cell adhesion molecules and cytokines by human brain microvascular endothelial cells

Upon inflammation, stimulated, but not resting T lymphocytes cross the blood-brain barrier and migrate into the central nervous system. This study shows that direct contact between stimulated T lymphocytes and human brain microvascular endothelial cells (HB-MVEC) induces phenotypic and functional changes on the latter cells. Plasma membranes isolated from stimulated T lymphocytes (S-PM) up-regulated the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on isolated HB-MVEC. In addition, HB-MVEC activated by S-PM secreted interleukin (IL)-6 and IL-8. The levels of ICAM-1, E-selectin, IL-6, and IL-8 expressed in S-PM-activated HB-MVEC were similar to those observed with 1000 U/ml tumor necrosis factor (TNF). In contrast, VCAM-1 expression was 15% of that induced by TNF. Inhibitors of TNF diminished (≤ 45 %), but did not abolish the expression of cell adhesion molecules and IL-6 induced by S-PM, IL-8 production being insignificantly affected (≤ 10 %). This suggests that membrane-associated TNF was partially involved in HB-MVEC activation. The present study demonstrates that stimulated T lymphocytes are able to activate HB-MVEC upon direct cell contact. This novel mechanism of inducing the expression of cell adhesion molecules may prompt the initial adhesion of stimulated T lymphocytes to brain endothelium.     

FOXN1 recombinant protein enhances T‐cell regeneration after hematopoietic stem cell transplantation in mice

A prolonged period of T‐cell recovery is the major challenge in hematopoietic stem cell transplantation (HSCT). Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T‐cell generation. However, TECs undergo degeneration over time. FOXN1 plays a critical role in TEC development and is required to maintain adult TECs for thymopoiesis. To investigate the potential application of FOXN1, we have cloned and expressed recombinant FOXN1 protein (rFOXN1) that was fused with cell‐penetrating peptides. We show here that the rFOXN1 protein can translocate from the cell surface into the cytoplasm and nucleus. Administration of rFOXN1 into both congenic and allogeneic HSCT recipient mice increased the number of TECs, resulting in enhanced thymopoiesis that led to an increased number of functional T cells in the periphery. The increased number of TECs is due to the enhanced survival and proliferation of TECs. Our results suggest that rFOXN1 has the potential to be used in enhancing T‐cell regeneration in patients following HSCT.     

Naive T cells can mediate delayed-type hypersensitivity response in T cell receptor transgenic mice

We produced transgenic mice expressing Tcell receptor-αβ chain genes, derived from the chicken ovalbumin (OVA)-specific I-A^d-restricted CD4⁺CD8^? T helper cell clone 7–3–7. In transgenic mice with H-2^d genetic background (Tg-d mice), delayed-type hypersensitivity (DTH) was induced in the hind footpad by one inoculation with OVA without any previous sensitization, suggesting that naive T cells have the potential to be involved in DTH response. Spleen cells from nonimmunized Tg-d mice showed a strong T cell proliferative response to in vitro stimulation with OVA. Furthermore, these spleen cells produce cytokines including interleukin(IL)-2, IL-3, interferon-γ, granulocyte/macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-1α and MIP-1β, which may play an important role in the attraction of mononuclear cells to an antigen-challenging site.     

T cell development in a major histocompatibility complex class II-deficient patient

In this report we show that the major histocompatibility complex (MHC) class II-negative thymus of a bare lymphocyte syndrome (BLS) patient contains a reduced CD4⁺ CD8^? T cell population when compared to thymocytes derived from a MHC class II-expressing thymus. Of these CD4⁺ CD8^? BLS thymocytes, approximately only one third co-expressed the CD3 antigen, moreover at a lower expression level when compared to control thymocytes. This suggests a partial maturation of the CD4⁺ CD8^? T cells in the absence of MHC class II expression. Among the BLS thymocytes, CD4⁺ CD8⁺ thymocytes could easily be detected. Noteworthy, the number of CD4^? CD8⁺ thymocytes was significantly increased. CD4⁺ CD8^? T cells could also be found among the BLS peripheral blood mononuclear cells, albeit at reduced numbers. Despite the absence of peripheral MHC class II expression, the majority of these CD4⁺ CD8^? T cells co-expressed the CD45RO marker. In the BLS patient, thymocytes as well as peripheral CD4⁺ CD8^? T cells were not restricted in the use of the available T cell receptor (TcR) V gene family pool. However, the lack of detectable levels of thymic and peripheral MHC class II antigen expression in the BLS patient had altered the CD4^?skewing patterns of TcR V gene families which were present in normal individuals. In conclusion, the lack of MHC class II expression in the BLS patient does not completely inhibit the CD4+ CD8^? T cell development.     

Extensive proliferation of T cell lineage-restricted progenitors in the thymus: an essential process for clonal expression of diverse T cell receptor beta chains

For clonal diversification of TCR, a large number of T cell progenitors are required in which highly diverse TCRbeta chains are accommodated individually. In the present study, we examined the proliferative potential of thymic progenitors that have been defined to be T cell lineage restricted. We show that the earliest fetal thymus (FT) cells from Rag2(-/-) mice, when cultured individually in a thymic organ culture system, produced 150-1,800 CD25(+) cells. Since differentiation and proliferation of Rag2(-/-) thymocytes are arrested at the stage of TCRbeta chain gene rearrangement, the observed proliferation was considered to represent the proliferative potential of progenitors prior to the TCRbeta rearrangement. A comparable level of proliferation was revealed to occur by analyzing the Dbeta-Jbeta rearrangement profiles of T cells generated from single progenitors in the earliest population of FT from normal mice. The proliferative potential of progenitors declined along with the progression of developmental stages. Such an extensive proliferation of progenitors after the restriction to the T cell lineage may be an essential process ensuring the clonal diversification of TCRbeta chains.     

The ability of murine dendritic cell subsets to direct T helper cell differentiation is dependent on microbial signals

Dendritic cells (DC) initiate T cell responses and direct the class of T cell immunity through the production of Th-polarizing cytokines. In the mouse, immunization with CD8alpha(+) DC has led to Th1 priming whereas immunization with CD8alpha(-) DC has been associated with Th2 induction. Here, we use a direct T cell priming assay in vitro to re-examine the Th-directing potential of total DC or purified CD4(+) DC, CD8alpha(+) DC or CD4(-) CD8alpha(-) (double-negative; DN) DC subsets from mouse spleen. We show that the default Th effector phenotype induced by priming with DC depends on the protocol used for T cell purification, the T cell:antigen-presenting cell ratio and the antigen dose but is only marginally affected by DC subtype. All DC subsets can direct increased Th1 development in response to microbial stimuli known to elicit IL-12 production. Similarly, all subsets can suppress Th1 development and allow Th2 cellsto expand upon exposure to IL-10-inducing microbial agents. The flexibility of DC in directing Th development in function of microbial signals argues against the notion of pre-determined "DC1" and "DC2" subsets and suggests that multiple DC subtypes can direct an appropriate Th response to different classes of infectious agents.