Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-gamma in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-gamma and IL-13. Analysis of the thymic subsets, CD4(-)CD8(-) (DN), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-gamma and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-gamma(+) cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-gamma and IL-13 expression is dependent on Stat4 and NF-kappaB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-gamma. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-gamma-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.     

Lymphohematopoietic progenitors do not have a synchronized defect with age-related thymic involution

It has been speculated that aging lymphohematopoietic progenitor cells (LPC) including hematopoietic stem cells (HSC) and early T-cell progenitors (ETP) have intrinsic defects that trigger age-related thymic involution. However, using a different approach, we suggest that that is not the case. We provided a young thymic microenvironment to aged mice by transplanting a fetal thymus into the kidney capsule of aged animals, and demonstrated that old mouse-derived LPCs could re-establish normal thymic lymphopoiesis and all thymocyte subpopulations, including ETPs, double negative subsets, double positive, and CD4(+) and CD8(+) single positive T cells. LPCs derived from aged mice could turn over young RAG(-/-) thymic architecture by interactions, as well as elevate percentage of peripheral CD4(+)IL-2(+) T cells in response to costimulator in aged mice. Conversely, intrathymic injection of ETPs sorted from young animals into old mice did not restore normal thymic lymphopoiesis, implying that a shortage and/or defect of ETPs in aged thymus do not account for age-related thymic involution. Together, our findings suggest that the underlying cause of age-related thymic involution results primarily from changes in the thymic microenvironment, causing extrinsic, rather than intrinsic, defects in T-lymphocyte progenitors.     

Expression of interleukin-1 receptors in the later period of foetal thymic organ culture and during suspension culture of thymocytes from aged mice

Interleukin-1 has been reported to be involved in thymocyte development by exerting a variety of effects on immature CD4-CD8- double-negative (DN) thymocytes. In contrast to the well-documented involvement of IL-1 in thymocyte development, expression of IL-1 receptors (IL-1R) on thymocytes has not been well demonstrated. In the present study, expression of IL-1R on the developing thymocytes was investigated. Although normal thymocytes barely express IL-1R, expression of IL-1R (type I) substantially increased at days 12-15 of foetal thymic organ culture (FTOC), with an increase of the DN subset. The CD4/CD8 profile of the IL-1R (type I)+ cells showed that these cells were mostly restricted to the DN and CD4+CD8+ subsets. Interestingly, in vitro culture of the thymocytes from an aged mouse, but not those from young adult or newborn mice, revealed similar results to those of FTOC. In addition, half of the IL-1R+ cells that increased in the later period of FTOC were gammadelta thymocytes. These results demonstrate IL-1R expression on thymocytes during ex vivo culture and suggest that IL-1R is expressed in a certain environment during normal thymocyte differentiation.     

Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy

The roles that thymus cytokines might play in regulating thymic atrophy are not known. Reversing thymic atrophy is important for immune reconstitution in adults. We have studied cytokine mRNA steady-state levels in 45 normal human (aged 3 days to 78 years) and 34 myasthenia gravis thymuses (aged 4 to 75 years) during aging, and correlated cytokine mRNA levels with thymic signal joint (sj) TCR delta excision circle (TREC) levels, a molecular marker for active thymopoiesis. LIF, oncostatin M (OSM), IL-6, M-CSF, and stem cell factor (SCF) mRNA were elevated in normal and myasthenia gravis-aged thymuses, and correlated with decreased levels of thymopoiesis, as determined by either decreased keratin-positive thymic epithelial space or decreased thymic sjTRECs. IL-7 is a key cytokine required during the early stages of thymocyte development. Interestingly, IL-7 mRNA expression did not fall with aging in either normal or myasthenia gravis thymuses. In vivo administration of LIF, OSM, IL-6, or SCF, but not M-CSF, i.p. to mice over 3 days induced thymic atrophy with loss of CD4+, CD8+ cortical thymocytes. Taken together, these data suggest a role for thymic cytokines in the process of thymic atrophy.     

A requirement for IL-2/IL-2 receptor signaling in intrathymic negative selection

The nature of the signals that influence thymocyte selection and determine the fate of CD4(+)8(+) (double positive) thymocytes remains unclear. Cytokines produced locally in the thymus may modulate signals delivered by TCR-MHC/peptide interactions and thereby influence the fate of double-positive thymocytes. Because the IL-2/IL-2R signaling pathway has been implicated in thymocyte and peripheral T cell survival, we investigated the possibility that IL-2/IL-2R interactions contribute to the deletion of self-reactive, Ag-specific thymocytes. By using nontransgenic and transgenic IL-2-sufficient and -deficient animal model systems, we have shown that during TCR-mediated thymocyte apoptosis, IL-2 protein is expressed in situ in the thymus, and apoptotic thymocytes up-regulate expression of IL-2RS: IL-2R(+) double-positive and CD4 single-positive thymocytes undergoing activation-induced cell death bind and internalize IL-2. IL-2-deficient thymocytes are resistant to TCR/CD3-mediated apoptotic death, which is overcome by providing exogenous IL-2 to IL-2(-/-) mice. Furthermore, disruption or blockade of IL-2/IL-2R interactions in vivo during Ag-mediated selection rescues some MHC class II-restricted thymocytes from apoptosis. Collectively, these findings provide evidence for the direct involvement of the IL-2/IL-2R signaling pathway in the deletion of Ag-specific thymocyte populations and suggest that CD4 T cell hyperplasia and autoimmunity in IL-2(-/-) mice is a consequence of ineffective deletion of self-reactive T cells.     

Differences in the pattern of proliferative response with age in thymocytes undergoing spontaneous and induced proliferation

The proliferative capacity of thymocytes from C3H/HeJ mice decrease as the animals attain maturity. The proliferative response of thymocytes from 24- to 28-week-old mice to stimulation with concanavalin A (Con A) is only 20% of that observed at 4 weeks of age. The decreased proliferative capacity of thymocytes in response to Con A stimulation observed between 4 and 24 weeks of age closely correlates to the drop in thymic weight and cellularity observed during this period. In contrast, the spontaneous proliferative capacity of thymocytes, as well as proliferation of thymocytes in response to stimulation with phorbol myristate acetate (PMA) and ionomycin, drops only slightly during this period, as proliferation under these condition in thymocytes from 24- to 28-week-old mice is approximately 65-70% of that observed in 4-week-old animals. We have previously shown that cytoplasmic extracts from proliferating lymphoid cells contain a factor, termed the activator of DNA replication (ADR), which is capable of inducing DNA synthesis in isolated, quiescent nuclei. We show in this study that the decreased proliferative capacity of thymocytes during whole organism maturation and thymic involution is associated with decreased endogenous levels of ADR, while nuclear sensitivity of thymocyte to ADR was retained during these process. The diminution of ADR activity during thymic involution was quantitatively greater than the loss in proliferative capacity.     

An essential role for the IL-7 receptor during intrathymic expansion of the positively selected neonatal T cell repertoire

Intrathymic T cell development is a multistage process involving discrete phases of proliferation as well as differentiation. From studies on IL-7 or IL-7Ralpha-deficient mice, it is clear that the IL-7 receptor (IL-7R) plays a critical role during the initial stages of intrathymic CD4-8- precursor development. In contrast, the role of IL-7R in later stages of thymocyte development are unclear. Here, we have used various approaches to investigate directly the role of the IL-7R in thymocyte positive selection and the recently described phase of postselection proliferation. First, we show that positive selection involves selective up-regulation of IL-7Ralpha- and IL-7Rgamma-chains, with the majority of CD4+ and CD8+ cells being IL-7R+. Second, MHC class II+ thymic epithelium-which drives postselection proliferation-expresses IL-7 mRNA. Finally, analysis of positive selection and postselection proliferation in thymocytes from IL-7Ralpha-/- neonates shows that positive selection occurs normally, whereas postselection expansion is drastically reduced. Thus, our data provide the first evidence that, as well as playing a role during early phases of thymic development, IL-7R mediates intrathymic expansion of positively selected thymocytes, which may aid in establishment of the neonatal peripheral T cell pool.     

In vivo administration of IL-1 induces thymic hypoplasia and increased levels of serum corticosterone

Administration of IL-1 alpha or IL-1 beta to normal mice induces a decrease in thymic cellularity, the magnitude of which depends on the number of injections and dose of IL-1. Twice daily injections of 200 ng of IL-1 alpha or -beta for 4 days results in a 90% decrease in thymic cellularity, which regenerated after cessation of treatment. Study of thymocyte subpopulations revealed that the number of CD4+/CD8+ thymocytes was dramatically decreased in IL-1-treated mice. Functional assessment of the CD4-/CD8- population from treated animals showed that these cells had adequate mitogenic responses in vitro and that the proportion of these cells in cycle was not different from control CD4-/CD8- cells. IL-1 treatment also prevented the regeneration of thymic cellularity after irradiation. The use of strains of mice differing genetically at the Ly 1 locus to construct radiation bone marrow chimeras demonstrated that bone marrow-derived thymocyte precursors were able to seed the thymus in the IL-1-treated animals. Again, however, the CD4+/CD8+ thymocyte population was significantly decreased. Thymic repopulation occurred upon cessation of IL-1 therapy. Finally, we determined that a single i.p. injection of IL-1 caused a three-fold increase in serum corticosterone levels, which peaked approximately 3 h after IL-1 administration. Thus, an IL-1-dependent increase in serum corticosterone levels may be responsible for the observed thymic hypoplasia.     

Downregulation of interleukin-7 and hepatocyte growth factor in the thymic microenvironment is associated with thymus involution in tumor-bearing mice

During mammary tumorigenesis, there is a profound thymic involution associated with severe depletion of the most abundant subset of thymocytes, CD4⁺CD8⁺ immature cells, and an early arrest in at least two steps of T cell differentiation. Thymic atrophy that is normally related with aging has been observed in other model systems, including graft-vs-host disease (GVHD) and tumor development. However, the mechanisms involved in this phenomenon remain to be elucidated. Vascular endothelial growth factor (VEGF) has been associated with thymic involution, when expressed at high levels systemically. In thymuses of D1-DMBA-3 tumor-bearing mice, this growth factor is diminished relative to the level of normal thymuses. Interestingly, the expression of hepatocyte growth factor (HGF), which has been associated with proliferation, cell survival, angiogenesis and B-cell differentiation, is profoundly down-regulated in thymuses of tumor bearers. In parallel, IL-7 and IL-15 mRNA, crucial cytokines involved in thymocytes development and cellular homeostasis, respectively, are also down-regulated in the thymuses of tumor hosts as compared to those of normal mice. Injection of HGF into mice implanted with mammary tumors resulted in normalization of thymic volume and levels of VEGF, IL-7 and IL-15. While, injections of IL-7 partially restored the thymic involution observed in the thymuses of tumor-bearing mice, injection of IL-15 did not have any significant effects. Our data suggest that the downregulation of HGF and IL-7 may play an important role in the thymic involution observed in tumor-bearing hosts.     

Selective stimulation of human thymocyte subpopulations by recombinant IL-4 and IL-3

Human thymocytes and thymocyte subsets were examined for their proliferative response to recombinant interleukin-4 (IL-4) and interleukin-3 (IL-3) in serum-free cultures. IL-4 induced marked proliferation of thymocytes after PHA and TPA stimulation, in contrast to the marginal response of T cells from adult peripheral blood. However, depletion of thymocytes bearing the CD3 antigen diminished the IL-4-induced proliferation of thymocytes, indicating that the response of thymocytes to IL-4 is mainly mediated by the CD3-positive cells. Phenotypic changes after culture with IL-4 showed an increase in the percentage of total thymocytes expressing mature T cell antigens (CD3, CD5, and TCR-1) and a decrease in CD1-positive cells. In addition there was an increase in the percentage of CD4+8- cells in both nylon wool-separated thymocytes and CD3-depleted cells with the disappearance of most of the CD4+8+ cells. However, an increase in the percentage of CD4-8- cells was also observed. The IL-4-responding cells do, however, express the mature T cell antigen, CD5, in high density. The effect of IL-3 on the proliferation of human thymocytes was very low and detected only when the thymocytes were cultured in serum-free medium. Depletion of CD3-positive cells did not diminish the IL-3-mediated proliferation of thymocytes, indicating that IL-3-responsive thymocytes are more immature than the subset of thymocytes which responds to IL-4. These results suggest that IL-4 and IL-3 play different roles in the development of human T cells.     

Growth-promoting activity of IL-1 alpha, IL-6, and tumor necrosis factor-alpha in combination with IL-2, IL-4, or IL-7 on murine thymocytes. Differential effects on CD4/CD8 subsets and on CD3+/CD3- double-negative thymocytes

Many cytokines (including IL-1, IL-2, IL-4, IL-6, and TNF-alpha) have been shown to induce thymocyte proliferation in the presence of PHA. In this report, we demonstrate that certain cytokine combinations induce thymocyte proliferation in the absence of artificial comitogens. IL-1 alpha, IL-6, and TNF-alpha enhanced the proliferation of whole unseparated thymocytes in the presence of IL-2, whereas none of them induced thymocyte proliferation alone. In contrast, of these three enhancing cytokines, only IL-6 enhanced IL-4-induced proliferation. We also separated thymocytes into four groups based on their expression of CD4 and CD8, and investigated their responses to various cytokines. The results indicate that each cytokine combination affects different thymocyte subsets; thus, IL-1 alpha enhanced the proliferation of CD4-CD8- double negative (DN) thymocytes more efficiently than IL-6 in the presence of IL-2, whereas IL-6 enhanced the responses of CD4+CD8- and CD4-CD8+ single positive (SP) thymocytes to IL-2 or IL-4 better than IL-1 alpha. TNF-alpha enhanced the proliferation of both DN and both SP subsets in the presence of IL-2 and/or IL-7. None of these combinations induced the proliferation of CD4+CD8+ double positive thymocytes. Finally, DN were separated into CD3+ and CD3- populations and their responsiveness was investigated, because recent reports strongly suggest that CD3+ DN thymocytes are a mature subset of different lineage rather than precursors of SP thymocytes. CD3+ DN proliferated in response to IL-7, TNF-alpha + IL-2, and IL-1 + IL-2. CD3- DN did not respond to IL-7 or to IL-1 + IL-2, but did respond to TNF-alpha + IL-2. Finally, we detected TNF-alpha production by a cloned line of thymic macrophages, as well as by DN adult thymocytes. These results suggest that cytokines alone are capable of potent growth stimuli for thymocytes, and indicate that different combinations of these molecules act selectively on thymocytes at different developmental stages.     

Effects of IL-7 on early human thymocyte progenitor cells in vitro and in SCID-hu Thy/Liv mice

IL-7 is a critical component of thymopoiesis in animals and has recently been shown to play an important role in T cell homeostasis. Although there is increasing interest in the use of IL-7 for the treatment of lymphopenia caused by the HIV type 1, evidence that IL-7 may accelerate HIV replication has raised concerns regarding its use in this setting. We sought to identify the effects of IL-7 on human thymocyte survival and to determine the impact of IL-7 administration on in vivo HIV infection of the human thymus. Using in vitro analysis, we show that IL-7 provides potent anti-apoptotic and proliferative signals to early thymocyte progenitors. Analysis of CD34(+) subpopulations demonstrates that surface IL-7 receptor is expressed on most CD34(high)CD5(+)CD1a(-) thymocytes and that this subpopulation appears to be one of the earliest maturation stages responsive to the effects of IL-7. Thus, IL-7 provides survival signals to human thymocytes before surface expression of CD1a. CD4(+)CD8(+) thymocytes are relatively unresponsive to IL-7, although IL-7 protects these cells from dexamethasone-induced apoptosis. IL-7 has a predominantly proliferative effect on mature CD4(+)CD3(+)CD8(-) and CD8(+)CD3(+)CD4(-) thymocytes. In contrast to the in vitro findings, we observe that in vivo administration of IL-7 to SCID-hu Thy/Liv mice does not appear to enhance thymocyte survival nor does it appear to accelerate HIV infection. Given the growing interest in the use of IL-7 for the treatment of human immunodeficiency, these findings support additional investigation into its in vivo effects on thymopoiesis and HIV infection.     

IL-2, IL-6, and IFN-gamma have distinct effects on the IL-4 plus PMA-induced proliferation of thymocyte subpopulations

We have previously reported complex effects of cytokine-containing T cell supernatants on the interleukin (IL)4 plus phorbol 12-myristate 13-acetate (PMA)-induced proliferative response of murine thymocytes. Here we show that recombinant murine IL-2, IL-6, and IFN-gamma each differentially regulate the IL-4/PMA-driven growth of thymocyte subpopulations. Thymocytes fractionated into four subpopulations on the basis of CD4 and CD8 expression were stimulated to proliferate by IL-4/PMA. Interferon-gamma (IFN-gamma) caused almost complete inhibition of the CD4+/CD8- response but had no measurable effect on the growth of CD4-/CD8+ or CD4-/CD8- populations. This inhibitory effect was also observed on splenic CD4+/CD8- T cells. In contrast, IL-6 strongly enhanced the proliferative response of CD4+/CD8- thymocytes, but showed no effect on peripheral CD4+/CD8- T cells, suggesting that IL-6 may be an important regulator of growth in the thymus. IL-2 also enhanced the proliferation of both CD4-/CD8+ and CD4-/CD8- thymocytes to IL-4 and PMA. To test whether the IL-4/PMA stimulus provided all the signals required to initiate growth in each subpopulation, we titrated cell number and examined the relationship between cell dose and cell response. Growth of CD8+/CD4- cells was cell density independent, indicating that IL-4/PMA is sufficient stimulus to induce growth of these cells. In contrast, growth of CD4-/CD8- and CD4+/CD8- cells is cell density dependent, suggesting a requirement for another signal provided by the cells themselves. These observations suggest that more signals remain to be identified in this thymocyte growth system.     

Absence of IFN-gamma or IL-12 has different effects on experimental myasthenia gravis in C57BL/6 mice

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-gamma and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-gamma and IL-12 knockout mutants (IFN-gamma-/-, IL-12-/-) with Torpedo AChR (TAChR). WT and IFN-gamma-/- mice developed EMG with similar frequency, IL-12-/-mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-gamma-/- mice had significantly less IgG2c, and IL-12-/- mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-gamma-/- mice had complement; WT mice had both IgG2b and IgG2c, IFN-gamma-/- only IgG2b, and IL-12-/- neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-gamma-/- mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12-/- mice secreted less IFN-gamma, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12-/- mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-gamma-/- mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.     

A quantitative study of the mechanisms behind thymic atrophy in Gαi2-deficient mice during colitis development

Mice deficient for the G protein subunit Gαi2 spontaneously develop colitis, a chronic inflammatory disease associated with dysregulated T cell responses. We and others have previously demonstrated a thymic involution in these mice and an aberrant thymocyte dynamics. The Gαi2(-/-) mice have a dramatically reduced fraction of double positive thymocytes and an increased fraction of single positive (SP) thymocytes. In this study, we quantify a number of critical parameters in order to narrow down the underlying mechanisms that cause the dynamical changes of the thymocyte development in the Gαi2(-/-) mice. Our data suggest that the increased fraction of SP thymocytes results only from a decreased number of DP thymocytes, since the number of SP thymocytes in the Gαi2(-/-) mice is comparable to the control littermates. By measuring the frequency of T cell receptor excision circles (TRECs) in the thymocytes, we demonstrate that the number of cell divisions the Gαi2(-/-) SP thymocytes undergo is comparable to SP thymocytes from control littermates. In addition, our data show that the mature SP CD4(+) and CD8(+) thymocytes divide to the same extent before they egress from the thymus. By estimating the number of peripheral TREC(+) T lymphocytes and their death rate, we could calculate the daily egression of thymocytes. Gαi2(-/-) mice with no/mild and moderate colitis were found to have a slower export rate in comparison to the control littermates. The quantitative measurements in this study suggest a number of dynamical changes in the thymocyte development during the progression of colitis.     

Role of IL-12 in intrathymic negative selection.

Cytokines are central regulatory elements in peripheral lymphocyte differentiation, but their role in T cell ontogeny is poorly defined. In the present study, we evaluated the role of IL-12 in thymocyte selection more directly by determining its role in two models of in vivo negative selection. In initial studies we demonstrated that abundant intrathymic IL-12 synthesis occurs during OVA peptide-induced negative selection of thymocytes in neonatal OVA-TCR transgenic mice, and such synthesis is associated with increased IL-12R beta2-chain expression as well as STAT4 intracellular signaling. In further studies, we showed that this form of negative selection was occurring at the alphabetaTCRlowCD4lowCD8low stage and was prevented by the coadministration of anti-IL-12. In addition, the IL-12-dependent thymocyte depletion was occurring through an intrathymic apoptosis mechanism, also prevented by administration of anti-IL-12. Finally, we showed that IL-12 p40-/- mice displayed aberrant negative selection of double positive CD4+CD8+ thymocytes when injected with anti-CD3 mAb. These studies suggest that intact intrathymic IL-12 production is necessary for the negative selection of thymocytes occurring in relation to a high "self" Ag load, possible through its ability to induce the thymocyte maturation and cytokine production necessary for such selection.     

Recombinant IL-7/HGFβ Hybrid Cytokine Enhances T Cell Recovery in Mice Following Allogeneic Bone Marrow Transplantation

T cell immunodeficiency is a major complication of bone marrow (BM) transplantation (BMT). Therefore, approaches to enhance T cell reconstitution after BMT are required. We have purified a hybrid cytokine, consisting of IL-7 and the β-chain of hepatocyte growth factor (HGFβ) (IL-7/HGFβ), from a unique long-term BM culture system. We have cloned and expressed the IL-7/HGFβ gene in which the IL-7 and HGFβ genes are connected by a flexible linker to generate rIL-7/HGFβ protein. Here, we show that rIL-7/HGFβ treatment enhances thymopoiesis after allogeneic BMT. Although rIL-7 treatment also enhances the number of thymocytes, rIL-7/HGFβ hybrid cytokine was more effective than was rIL-7 and the mechanisms by which rIL-7 and rIL-7/HGFβ increase the numbers of thymocytes are different. rIL-7 enhances the survival of double negative (DN), CD4 and CD8 single positive (SP) thymocytes. In contrast, rIL-7/HGFβ enhances the proliferation of the DN, SP thymocytes, as well as the survival of CD4 and CD8 double positive (DP) thymocytes. rIL-7/HGFβ treatment also increases the numbers of early thymocyte progenitors (ETPs) and thymic epithelial cells (TECs). The enhanced thymic reconstitution in the rIL-7/HGFβ-treated allogeneic BMT recipients results in increased number and functional activities of peripheral T cells. Graft-versus-host-disease (GVHD) is not induced in the rIL-7/HGFβ-treated BMT mice. Therefore, rIL-7/HGFβ may offer a new tool for the prevention and/or treatment of T cell immunodeficiency following BMT.     

Intrathymic radioresistant stem cells follow an IL-2/IL-2R pathway during thymic regeneration after sublethal irradiation

Sublethally irradiated mice undergo thymic regeneration which follows a phenotypic pattern of events similar to that observed during normal fetal development. Thymic regeneration after irradiation is the product of a limited pool of intrathymic radioresistant stem cells undergoing simultaneous differentiation. We show that in this model of T cell development, thymic regeneration follows a pathway in which the IL-2R is transiently expressed on CD4-/CD8- cells. IL-2R expression occurred during the exponential growth period of thymic regeneration, and IL-2R blocking prevented this explosive growth. Flow cytometry analysis revealed that the IL-2R blockade affected primarily the development of the immature CD3-/CD4-/CD8- (triple negative) cells and their ability to generate CD3+/CD4+/CD8+ or CD3+/CD4+/CD8- and CD3+/CD4-/CD8+ thymocytes. Thus, our findings demonstrate that blocking of the IL-2R resulted in an arrest in proliferation and differentiation by intrathymic radioresistant stem cells, indicating that the IL-2/IL-2R pathway is necessary for the expansion of immature triple negative T cells.     

Ontogeny and regulation of IL-7-expressing thymic epithelial cells

Epithelial cells in the thymus produce IL-7, an essential cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We identified IL-7-expressing thymic epithelial cells (TECs) throughout ontogeny and in the adult mouse thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the early primordium by embryonic day 11.5 and is expressed in a Foxn1-independent pathway. Marked changes occur in the localization and regulation of IL-7-expressing TECs during development. IL-7-expressing TECs are present throughout the early thymic rudiment. In contrast, a major population of IL-7-expressing TECs is localized to the medulla in the adult thymus. Using mouse strains in which thymocyte development is arrested at various stages, we show that fetal and postnatal thymi differ in the frequency and localization of IL-7-expressing TECs. Whereas IL-7 expression is initiated independently of hemopoietic-derived signals during thymic organogenesis, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Moreover, different thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium, suggesting that despite phenotypic similarities, the cortical TEC compartments of wild-type and RAG-1(-/-) mice are developmentally and functionally distinct.