Recombinant interleukin 4/BSF-1 promotes growth and differentiation of intrathymic T cell precursors from fetal mice in vitro.

Recombinant mouse interleukin 4/BSF-1 (rIL4/BSF-1) together with phorbol myristate acetate (PMA) promotes growth of one out of approximately four intrathymic T cell precursors from fetal mice (14-15 days gestation). This response is not inhibited by even high concentrations of monoclonal antibody against the receptor for interleukin 2. Fetal thymocytes activated by rIL4/BSF-1 plus PMA give rise to cytolytic T cells after 7-21 days of culture. All the proliferating cells are Thy1+, some of them express Lyt2 but none has detectable L3T4 T cell differentiation antigens nor T cell antigen receptor (F23.1) on the cell membrane as assessed by immunofluorescence staining and flow fluorocytometry analysis. It is concluded that rIL4/BSF-1 exerts both growth and differentiation activities on normal intrathymic T cell precursors. The results provide evidence for an alternative growth factor to interleukin 2 involved in proliferation of T cell precursors. These findings open new and direct ways of studying cellular and molecular events during the differentiation of normal intrathymic T cell precursors in vitro and extend the spectrum of target cells for IL4/BSF-1.     

Migration of putative progenitor T cells in response to thymus-derived chemotactic factors

Progenitor T cells reach the thymus through the circulation from hematopoietic organs and then migrate toward the site of differentiation in the thymus. The mechanism that regulates such intrathymic migration is not well understood. In order to clarify this mechanism, in vitro chemotactic activity for murine thymocytes was assayed in the extracts and culture supernatants of thymic tissue elements. A potent thymocyte chemotactic activity was found in the extract and culture supernatant from Ig-, Ia- thymic stromal cells. Peanut agglutinin-positive (PNA+1), Thy 1+, TL-, Lyt 1+2-, L3T4- thymocytes, Ig-, Thy 1- bone marrow cells, and mononuclear cells of spleen and peripheral blood, but neither B cells nor lymph node cells, were chemotactically attracted by the factor(s). The chemotactic activity was found in none of the following materials tested: the extract and culture supernatant of thymocytes, culture supernatant of lymph node stromal cells, normal mouse serum, and zymosan-activated serum. The chemotactic activity was found in three molecular fractions by gel chromatography. The activity in all three fractions was destroyed by trypsin digestion or by heating at 56 degrees C for 30 min. These results suggest that Ig-, Ia- thymic stromal cells but not thymocytes secrete a chemotactic factor(s) for progenitor T cells with three molecular species. The factor is considered to play an important role in the migration of intrathymic progenitor T cells into the site of differentiation.     

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.     

Differentiation of hematopoietic stem cells in irradiated mouse thymic lobes. Kinetics and phenotype of progeny

To define cell populations which participate in the very early stages of T cell development in the mouse thymus, we enriched hematopoietic stem cells from mouse bone marrow and injected them into thymic lobes of irradiated Ly-5 congenic recipients. The progeny of the stem cells were identified and their phenotypes were determined by two-color flow cytometry for the expression of various cell surface differentiation Ag during the course of their subsequent intrathymic development. The majority of the differentiation which occurred in the first 10 days after intrathymic cell transfer was myeloid in nature; hence, this study demonstrates that the irradiated thymus is not strictly selective for T cell development. Further, the maximum rate of T cell development was observed after intrathymic injection of 200 stem cells. Donor-derived cells which did not express Ag characteristic of the myeloid lineage could be detected and their phenotypes could be determined by flow cytometry as early as 7 days after intrathymic injection. At this time, the cells were still very similar phenotypically to the bone marrow hematopoietic stem cells. Exceptions to this were the expression of stem cell Ag 2 and a decrease in the level of MHC class I Ag expression. After 9 days, the donor-derived cells expressed high levels of the Thy-1 Ag and proceeded to change in cell surface phenotype as differentiation continued. These cell phenotypes are described for the time frame ending 18 days after injection, when most donor-derived cells were phenotypically small CD4+ CD8+ (double-positive) thymocytes.     

Impaired T-cell differentiation in the thymus at the early stages of acute pathogenic chimeric simian-human immunodeficiency virus (SHIV) infection in contrast to less pathogenic SHIV infection

One of the mechanisms by which HIV infection induces the depletion of CD4+ T cells has been suggested to be impairment of T-cell development in the thymus, although there is no direct evidence that this occurs. To examine this possibility, we compared T-cell maturation in the intrathymic progenitors between macaques infected with an acute pathogenic chimeric simian-human immunodeficiency virus (SHIV), which causes profound and irreversible CD4+ T-cell depletion, and macaques infected with a less pathogenic SHIV, which causes only a transient CD4+ T-cell decline. Within 27 days post-inoculation (dpi), the two virus infections caused similar increases in plasma viral loads and similar decreases in CD4+ T-cell counts. However, in the thymus, the acute pathogenic SHIV resulted in increased thymic involution, atrophy and the depletion of immature T cells including CD4(+)CD8(+) double-positive (DP) cells, whereas the less pathogenic SHIV did not have these effects. Ex vivo differentiation of CD3(-)CD4(-)CD8(-) triple-negative (TN) intrathymic progenitors to DP cells was assessed by a monkey-mouse xenogenic fetal thymus organ culture system. Differentiation was impaired in the TN intrathymic progenitors of the acute pathogenic SHIV-infected monkeys, while differentiation was not impaired in the TN intrathymic progenitors of the less pathogenic SHIV-infected monkeys. These differences suggest that dysfunction of thymic maturation makes an important contribution to the irreversible depletion of circulating CD4+ T cells in vivo.     

Sonic hedgehog regulates early human thymocyte differentiation by counteracting the IL-7-induced development of CD34+ precursor cells

The Hedgehog (Hh) family of signaling molecules normally functions in the development of numerous tissues by regulating cellular differentiation and proliferation. Recent results have demonstrated that the different components of the Hh signaling pathway are expressed in the human thymus. In this study, we investigate the potential role of Sonic hedgehog (Shh) in human intrathymic T cell maturation. Results show that the expression of the two components of the Hh receptor, Patched and Smoothened, is mostly restricted to CD34+ precursor cells that are committing to the T cell lineage. Shh significantly increased the viability of CD34+ T cell precursors modulating bcl-2 and bax protein expression, and also inhibited their proliferation. The treatment of chimeric human-mouse fetal thymus organ cultures with Shh resulted in an arrested thymocyte differentiation and an accumulation of CD34+ progenitor cells. This effect was mainly attributed to the ability of Shh to counteract the IL-7-induced proliferation and differentiation of CD34+ cells. Shh down-regulated in the precursor cell population the expression of IL-7R as well as stromal-derived factor-1 chemokine receptor, CXCR4, and inhibited IL-7-dependent STAT5 phosphorylation. Therefore, Shh may function as a maintenance factor for intrathymic CD34+ precursor cells.     

Parthenolide-induced apoptosis in multiple myeloma cells involves reactive oxygen species generation and cell sensitivity depends on catalase activity

The sesquiterpene lactone, parthenolide (PTL), possesses strong anticancer activity against various cancer cells. We report that PTL strongly induced apoptosis in 4 multiple myeloma (MM) cell lines and primary MM cells (CD38⁺ high), but barely induced death in normal lymphocytes (CD38^−/+low). PTL-mediated apoptosis correlated well with ROS generation and was almost completely inhibited by L-N-acetylcysteine (L-NAC), indicating the crucial role of oxidative stress in the mechanism. Among 4 MM cell lines, there is considerable difference in susceptibility to PTL. KMM-1 and MM1S cells sensitive to PTL possess less catalase activity than the less sensitive KMS-5 and NCI-H929 cells as well as normal lymphocytes. A catalase inhibitor 3-amino-1,2,4-triazole enhanced their PTL-mediated ROS generation and cell death. The siRNA-mediated knockdown of catalase in KMS-5 cells decreased its activity and sensitized them to PTL. Our findings indicate that PTL induced apoptosis in MM cells depends on increased ROS and intracellular catalase activity is a crucial determinant of their sensitivity to PTL.     

CD25+ immunoregulatory CD4 T cells mediate acquired central transplantation tolerance

Transplantation tolerance is induced reliably in experimental animals following intrathymic inoculation with the relevant donor strain Ags; however, the immunological mechanisms responsible for the induction and maintenance of the tolerant state remain unknown. We investigated these mechanisms using TCR transgenic mice (TS1) that carry T cells specific for an immunodominant, MHC class II-restricted peptide (S1) of the influenza PR8 hemagglutinin (HA) molecule. We demonstrated that TS1 mice reject skin grafts that have transgene-encoded HA molecules (HA104) as their sole antigenic disparity and that intrathymic but not i.v. inoculation of TS1 mice with S1 peptide induces tolerance to HA-expressing skin grafts. Intrathymic peptide inoculation was associated with a dose-dependent reduction in T cells bearing high levels of TCR specific for HA. However, this reduction was both incomplete and transient, with a full recovery of S1-specific thymocytes by 4 wk. Peptide inoculation into the thymus also resulted in the generation of immunoregulatory T cells (CD4+CD25+) that migrated to the peripheral lymphoid organs. Adoptive transfer experiments using FACS sorted CD4+CD25- and CD4+CD25+ T cells from tolerant mice revealed that the former but not the latter maintain the capacity to induce rejection of HA bearing skin allografts in syngeneic hosts. Our results suggest that both clonal frequency reduction in the thymus and immunoregulatory T cells exported from the thymus are critical to transplantation tolerance induced by intrathymic Ag inoculation.     

Growth of immature (double negative) rat thymocytes in the presence of IL-1

It is unclear which growth factors, if any, are involved in the growth and differentiation of immature T cells in the thymus. Because IL-1 has been previously implicated in thymocyte proliferation, we examined the effects of IL-1 on precursor thymocytes, the CD4-CD8- or double-negative (DN) cells. We show that IL-1 (together with Con A) is a growth factor for DN, TCR- alpha beta-cells in vitro. After 5 days of culture in IL-1 and Con A, a number of phenotypic changes were observed and two subsets of DN cells were distinguished. One subset expressed full length TCR-alpha and -beta mRNA and surface alpha beta TCR, the other expressed low levels of full length TCR-gamma together with high levels of full length TCR-delta mRNA. Thus, DN cells are induced by IL-1 and Con A to proliferate and express TCR without parallel acquisition of CD4 or CD8 markers. These data suggest that IL-1 drives early steps of intrathymic T cell differentiation.     

Thymic microenvironment induces HIV expression. Physiologic secretion of IL-6 by thymic epithelial cells up-regulates virus expression in chronically infected cells

The hallmark of infection with HIV-1 is progressive depletion and qualitative dysfunction of the CD4+ Th cell population in infected individuals. Clinical trials of antiretroviral agents have shown that, despite suppression of virus replication, regeneration of the T cell pool does not occur. One proposed explanation for the defective regenerative capacity of the CD4+ T cell pool is infection of early T lymphocyte progenitors or stem cells. An additional explanation could be failure of cells of the intrathymic microenvironment (thymic epithelial (TE) cells) to carry out critical nurturing functions for developing thymocytes, i.e., secretion of thymocyte-trophic cytokines and expression of adhesion molecules. This study examines the effect of HIV on cultured TE cells and determines the role of TE cells in the regulation of viral expression in chronically HIV-infected cells. We found no evidence of infection of TE cells after exposure to HIV-1. However, normal human serum induced secretion of IL-6 by TE cells; induction of TE IL-6 was partially blocked by anti-IFN-gamma antibodies. Moreover, supernatants from TE cells maintained in normal human serum up-regulated HIV replication in chronically HIV-1-infected cells. Because intrathymic T cell precursors can be infected with HIV and T cell precursors come into close contact with TE cells in the thymus, IL-6 secreted by TE cells during normal intrathymic development may induce HIV expression in infected thymocytes in vivo and promote the intrathymic spread of HIV.     

Activation of extrathymic T cells in the liver and reciprocal inactivation of intrathymic T cells by bacterial stimulation.

We recently demonstrated that the liver might be a major site of extrathymic T cell differentiation, including both alpha beta and gamma delta T cells. This extrathymic pathway in the liver, which has a relatively minor role in normal young mice, is activated in mice under bacterial stimulation. In the present study, we investigated how the extrathymic and intrathymic T cell differentiations were mutually related in mice injected intravenously with 10(8) heat-killed Escherichia coli. Three days after stimulation, extrathymic T cells in the liver were observed to be prominently activated in terms of increases in the total number of cells yielded, spontaneous cell proliferation in in vitro culture, and intermediate alpha beta TCR cells. Intermediate alpha beta TCR cells were extrathymic T cells uniquely seen in the liver. However, at the same time intrathymic T cells were profoundly inactivated, showing decreases in the number of thymocytes (more than 90% atrophy), spontaneous cell proliferation, and dull TCR cells with double positive CD4+8+ phenotype. With time, these responses were reversed and normal states were regained. These results suggested that extrathymic and intrathymic T cells are always activated or inactivated in the opposite direction, and that the liver and the thymus are dynamic immune organs. It raises the possibility that the extrathymic T cell differentiation in the liver and the intrathymic T cell differentiation may be reciprocally regulated by certain factors.     

Thymus hormones do not induce proliferative ability or cytolytic function in PNA+ cortical thymocytes

A variety of thymus hormone preparations, as well as drugs known to perturb cell differentiation, were tested for their ability to induce nonfunctional cortical thymocytes to become functional precursor cells. Murine cortical thymocytes, defined as the high peanut agglutinin (PNA) binding or as the low H-2K, major [86%] thymocyte subpopulation, were isolated by fluorescence-activated cell sorting. Their function was assessed in a high cloning efficiency, growth factor saturated, concanavalin A-stimulated limit-dilution culture system, determining the number of precursors of extended clones (PTL-p), or determining with a lectin-mediated tumor-lysis readout the number of precursors of cytolytic clones (CTL-p). The hormone preparations tested were crude or partially purified culture supernatants from thymus "epithelial" monolayers (TES), soluble extracts of thymic nonlymphoid tissue (STF), semipure thymus humoral factor (THF), and the pure peptides thymopoietin 32-36 (TP5) and "facteur thymique sérique" (FTS). These preparations were either added directly to the limit dilution cultures, or were first preincubated with the cells, which were then subjected to limit-dilution culture. In no case did the hormone preparations cause any increase in the level of PTL-p or CTL-p in the PNA+ or low H-2K thymocyte population, even though a conversion of only a few percent to functional cells could have been detected. Two possible explanations are considered. One is that the main function of these materials is to control post-thymic peripheral T cells, rather than to induce intrathymic differentiation. Another is that the typical cortical thymocyte is beyond the stage at which thymocytes can be induced by hormones, a view that is strengthened by the failure of either 5-azacytidine or the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate to activate these cells. In this latter explanation the true intrathymic target of hormone action may be an earlier, and very minor, thymus subpopulation.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. IV. Significance of intrathymic chimerism of blood-born Ia+ cells in Mls tolerance.

The significance of thymus cell chimerism in the induction and maintenance of tolerance was investigated. Mls-1b BALB/c mice were neonatally tolerized by the intravenous administration of either bone marrow (BM) cells or peritoneal cavity (PerC) cells from Mls-1b/a (BALB/c x AKR) F1 mice. Tolerance was long-lasting in the BM cell group, but transient in the PerC cell group, probably because PerC cells lack hemopoietic stem cells required for a continuous supply of tolerance-inducing cells. The degree of anti-Mls-1a responsiveness of these BALB/c thymus cells was correlated with the degree of intrathymic distribution of the inoculated F1 cells. The effect of BM cell inoculation, resulting in a year-long deletion of Mls-1a-reactive V beta 6-bearing T cells is in marked contrast to that of PerC cell inoculation which causes only a transient loss of V beta 6+ mature thymocytes (for about 1 week after birth). This functional profile of the tolerant state correlates well with the degree and persistence of the intrathymic presence of F1 type Ia+ cells. The long-lasting presence of donor-derived cells throughout the thymus tissue in the BM cell group is also in marked contrast to the early disappearance of Ia+ cells (within 2-3 weeks) from the cortex and then from the medulla in the PerC cell group, although these Ia+ cells were once spread throughout the thymus tissue 4 days after the tolerance-inducing cell inoculation. Taken together with a failure to induce consistent unresponsiveness to Mls-1a determinants in Mls-1b thymocytes regenerating in Mls-1a-thymic epithelial environments, all the above data indicate that intrathymic chimerism caused by hemopoietic stem cell-derived MHC-class II-bearing cells is a requisite for the induction and maintenance of unresponsiveness by means of clonal deletion in experimentally as well as naturally induced tolerance to Mls determinants.     

Specific destruction of host-reactive mature T cells of donor origin prevents graft-versus-host disease in cyclophosphamide-induced tolerant mice.

In cyclophosphamide (CP)-induced tolerance, a long lasting skin allograft tolerance was established in many H-2-identical strain combinations without graft vs host disease. Destruction of donor-reactive T cells of host origin, followed by intrathymic clonal deletion of these cells, has been revealed to be the chief mechanisms of this system. Here, we studied the fate of host-reactive populations in donor-derived T cells of C3H/He (C3H) (H-2k, Mls-1b, Mls-2a) mice rendered CP-induced tolerant to AKR/J (AKR) (H-2k, Mls-1a, Mls-2b), by assessing AKR-derived Thy-1.1+ T cells bearing TCR V beta 3 that are specifically reactive with Mls-2a-encoded Ag of the recipient C3H mice. In the AKR-derived Thy-1.1+ lymph node cells of the C3H mice that had been treated with AKR spleen cells plus CP, CD4(+)-V beta 3+ T cells were obviously decreased by day 10 after the CP treatment. At this stage, the Thy-1.1+ T cells were not detected in the C3H thymus, suggesting that the obvious decrease of CD4(+)-V beta 3+ T cells of AKR origin was not due to intrathymic clonal deletion in the recipient C3H mice. Therefore, the destruction of the host-reactive mature T cells of donor origin, as well as that of the donor-reactive mature T cells of host origin, occurred by the CP treatment at the induction phase. Furthermore, after the establishment of intrathymic mixed chimerism in the recipient C3H mice, V beta 3+ T cells were not detected among the Thy-1.1+ T cells of AKR origin in the mixed chimeric thymus, suggesting that the host-reactive immature T cells repopulated from the injected donor hematopoietic cells were clonally deleted in the recipient thymus. These two mechanisms appear to prevent graft vs host disease in CP-induced tolerance.     

The triglyceride lipases of the pancreas

Pancreatic triglyceride lipase (PTL) and its protein cofactor, colipase, are required for efficient dietary triglyceride digestion. In addition to PTL, pancreatic acinar cells synthesize two pancreatic lipase related proteins (PLRP1 and PLRP2), which have a high degree of sequence and structural homology with PTL. PLRP1 has no known activity. PTL and PLRP2 differ in substrate specificity, behavior in bile salts and dependence on colipase. Each protein has a globular amino-terminal (N-terminal) domain, which contains the catalytic site for PTL and PLRP2, and a beta-sandwich carboxyl-terminal (C-terminal) domain, which includes the predominant colipase-binding site for PTL. Inactive and active conformations of PTL have been described. They differ in the position of a surface loop, the lid domain, and of the beta5-loop. In the inactive conformation, the lid covers the active site and, upon activation by bile salt micelles and colipase or by lipid-water interfaces, the lid moves dramatically to open and configure the active site. After the lid movement, PTL and colipase create a large hydrophobic plateau that can interact with the lipid-water interface. A hydrophobic surface loop in the C-terminal domain, the beta5' loop, may also contribute to the interfacial-binding domain of the PTL-colipase complex.     

Role of chorionic gonadotropin in the differentiation of thymocytes

The effects of chorionic gonadotropin, a basic hormone of pregnancy, on the differentiation of the human thymocytes were studied. The hormone does not affect the phenotype as determined by expression of the membrane molecules CD3, CD4, CD8 and functional activity of intrathymic pre-T-lymphocytes, but stimulates production of autocrine growth factors by these cells. Cultivation of cortical thymocytes in the presence of chorionic gonadotropin induces their phenotypic maturation with predominant development of CD4+ CD8- cells. In addition, at a high physiological dose (100 MU/ml) the hormone induces functional maturation of the cortical thymocytes. Thus, the data obtained allow us to consider this hormone as a factor regulating antigen-independent differentiation of T-lymphocytes during pregnancy and determining development of the immune system in embryogenesis.