Ligand-specific selection of MHC class II-restricted thymocytes in fetal thymic organ culture

Positive and negative selection of thymocytes is determined by the specificity of the TCR and signaling through its associated molecules. We have studied selection of thymocytes bearing a MHC class II-restricted TCR using fetal thymic organ culture. This system allows the addition of peptides to the already diverse panoply of endogenous peptide ligands and is useful for analyzing ligand-specific negative selection of CD4 single positive (CD4SP) thymocytes. The data reveal that the ability of a given ligand to mediate negative selection is related to its dissociation rate from the TCR. We find that negative selection is very sensitive, and only the weakest ligand that we can identify fails to induce negative selection. None of the numerous peptides tested were able to induce an increase in CD4SP thymocytes. In addition, the ligands that induce negative selection of CD4SP thymocytes also cause an increase in numbers of CD8SP thymocytes bearing high levels of the class II-restricted TCR. Although these cells have a cell surface phenotype consistent with positive selection, they most likely represent cells in the process of negative selection. Further analysis reveals that these cells are not induced by these ligands in intact adult animals and that their induction is probably only revealed in the organ culture system.     

Preferential proliferation and differentiation of double-positive thymocytes into CD8(+) single-positive thymocytes in a novel cell culture medium

The identification of factors that regulate the proliferation and differentiation of double-positive (DP) into CD4(+) and CD8(+) single-positive (SP) thymocytes has proven difficult due to the inability of DP thymocytes to proliferate, expand, and differentiate into SP thymocytes in available cell culture media. Here we report on the ability of DP thymocytes to differentiate in a novel conditioned medium, termed XLCM, derived from the supernatant of mitogen activated human cord blood mononuclear cells. During a 5-day culture in XLCM in the absence of thymic stromal cells, DP thymocytes from normal mice and MHC double knockout mice (lack SP thymocytes) proliferate, expand, and differentiate into several (alphabetaTCR(+), NK1.1(+)alphabetaTCR(+), and gammadeltaTCR(+)) subsets of CD4(+) and predominantly CD8(+) SP thymocytes. These studies suggest that the use of XLCM may aid in the characterization of factors that regulate the differentiation of DP thymocytes into CD8(+) SP thymocytes.     

Relation between arachidonic acid metabolism and development of thymocytes in fetal thymic organ cultures

Because products of arachidonic acid metabolism, particularly the PG, have been implicated as modulators of growth and differentiation of adult thymocytes, we investigated relations between metabolism of arachidonic acid and growth, as well as differentiation, of thymocytes during fetal thymic organ culture. Fetal thymic cells synthesized immunoreactive PGE2 during organ culture and were found to be capable of metabolizing exogenous arachidonic acid to products that cochromatographed with authentic 6-keto-PGF1 alpha, PGE2, PGF2 alpha. Synthesis of these products and growth and expression of Thy-1 and Lyt-1 Ag were inhibited by culture of fetal thymic lobes with indomethacin, a cyclooxygenase inhibitor, as well as meclofenamate and eicosatetraynoic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Only indomethacin inhibited expression of Lyt-2. Culture with eicosatetraynoic acid also inhibited the capacity of thymic lobes to synthesize 15-hydroxyeicosatetraenoic acid-like products. The inhibitory effects of indomethacin on growth and expression of Thy-1 were partially reversed by simultaneous addition of arachidonic acid. Thus, fetal thymic cells appear to require an intact cyclooxygenase, and possibly lipoxygenase, pathway of arachidonic acid metabolism for growth and differentiation. These data also provide evidence that Lyt-1 and Lyt-2 may be regulated by different requirements with respect to arachidonic acid metabolism.     

Development of fetal thymocytes in organ culture: effect of corticosteroids.

Corticosteroids affect the development of fetal foregut-derived organs in which epithelial-mesenchymal interactions are associated with the developmental process. The thymus is one such organ and is profoundly sensitive to corticosteroids when mature. In this study corticosterone (CS) effects on fetal thymocyte development were investigated using a fetal thymus organ culture system which allows the growth, differentiation, and function of developing thymocytes to be monitored in vitro. CS inhibited, but did not block growth of fetal thymocytes, although the appearance of mature thymocytes was inhibited, similar to previously reported effects of interleukin 2 (IL2). CS enhanced the proportion of Mac1+, Ia+ and FcR+ cells and maintained high levels of IL2 receptor (IL2R) positive immature cells. Functional cytotoxic cells were detected in CS-treated organ cultures which expressed a Thy 1-, CD8- phenotype, atypical for thymus derived killer cells. While this cytotoxicity may be stimulated by CS, it could simply be due to a relative depletion of the main pool of thymocytes. These cytotoxic cells may have a role in directing apoptotic mechanisms occurring during thymocyte development.     

NZB mice exhibit a primary T cell defect in fetal thymic organ culture

Defects in T cell development have been suggested to be a factor in the development of systemic autoimmunity in NZB mice. However, the suggestion of a primary T cell defect has often been by extrapolation, and few direct observations of T cell precursors in NZB mice have been performed. Moreover, the capacity of NZB bone marrow T cell precursors to colonize the thymus and the ability of the NZB thymic microenvironment to support T lymphopoiesis have not been analyzed. To address this important issue, we employed the fetal thymic organ culture system to examine NZB T cell development. Our data demonstrated that NZB bone marrow cells were less efficient at colonizing fetal thymic lobes than those of control BALB/c or C57BL/6 mice. In addition, NZB bone marrow cells did not differentiate into mature T cells as efficiently as bone marrow cells from BALB/c or C57BL/6 mice. Further analysis revealed that this defect resulted from an intrinsic deficiency in the NZB Lin-Sca-1+c-kit+ bone marrow stem cell pool to differentiate into T cells in fetal thymic organ culture. Taken together, the data document heretofore unappreciated deficiencies in T cell development that may contribute to the development of the autoimmune phenotype in NZB mice.     

Hypoxic vasodilation in porcine coronary artery is preferentially inhibited by organ culture

Hypoxia (95% N₂-5%CO₂) elicits an endothelium-independent relaxation(45-80%) in freshly dissected porcine coronary arteries. Pairedartery rings cultured at 37°C in sterile DMEM (pH ~7.4) for 24 h contracted normally to KCl or 1 µM U-46619. However, relaxation inresponse to hypoxia was sharply attenuated compared with control (fresharteries or those stored at 4°C for 24 h). Hypoxicvasorelaxation in organ cultured vessels was reduced at both high andlow stimulation, indicating that both Ca²⁺-independent andCa²⁺-dependent components are altered. In contrast,relaxation to G-kinase (sodium nitroprusside) or A-kinase (forskolinand isoproterenol) activation was not significantly affected by organculture. Additionally, there was no difference in relaxation afterwashout of the stimulus, indicating that the inhibition is specific toacute hypoxia-induced relaxation. Simultaneous force and intracellularcalcium concentration ([Ca²⁺]_i) measurementsindicate the reduction in [Ca²⁺]_i concomitantwith hypoxia at low stimulus levels in these tissue is abolished byculture. Our results indicate that organ culture at 37°C specificallyattenuates hypoxic relaxation in vascular smooth muscle by alteringdynamics of [Ca²⁺]_i handling and decreasing aCa²⁺-independent component of relaxation. Thus organculture can be a novel tool for investigating the mechanisms ofhypoxia-induced vasodilation.

     

Effects of cyclosporine A on thymocyte differentiation in fetal thymus organ culture

During the course of differentiation in the thymus, precursor T cells are negatively selected by a self-tolerance mechanism or positively selected to acquire restriction specificity to self major histocompatibility complexes. We investigated the process of T cell differentiation and those selections using a fetal thymus organ culture with or without cyclosporine A. The agent blocked the maturation step from CD4+8+ double positive cells to mature CD4-8+ or CD4+8- single positive cells. On the other hand, the agent did not inhibit the development of CD3+4-8- T cell receptor (TCR)alpha beta- cells, which were supposed to be T cells bearing gamma delta-TCR chains. These results suggest that the development of thymocytes bearing alpha beta- or gamma delta-TCR chains differ in requirement for thymocyte-stromal cell interaction.     

Expansion of large granular lymphocytes in IL-2-driven 14-day-old fetal thymocytes in organ culture

These experiments were designed to evaluate the role of cytokines in early T cell development within the thymus. By using a thymic organ culture model, we have studied the influence of high dose of IL-2 (10 to 1000 IU/ml) on the cell populations that are generated during 12 days starting from a thymic rudiment of 14-day-old mouse embryo. The IL-2 treatment resulted in the expansion of Thy-1+/-, CD4-, CD8-, CD3-, Fc gamma RII+, CD5 (Lyt-1)-, HSA-, Pgp- 1+, Mel-14- population. These cells had the morphology of large granular lymphocytes and displayed broad cytotoxic activity. In addition, IL-2-treated organ cultures had a dramatic decrease in CD4+CD8+ thymocytes, a marked reduction in TCR-alpha beta+ thymocytes--even more pronounced in the TCR-V beta 6+ and TCR-V beta 8+ thymocytes--and no significant changes in the number of TCR-gamma delta+ as compared to control organ cultures.     

The migration of bone marrow cells to thymic culture supernatants is inhibited by stress

The effect of immobilization stress on precursor cell migration from bone marrow to the thymus was studied in C57BL/6 mice. The in vitro migration assays, using Nuclepore chambers, showed that precursor cell migration to thymus supernatants was strongly inhibited in stressed animals. This inhibition of migration seemed to be cell-associated what can explain the thymic involution observed in mice under stress conditions. The migration of precursor cells from bone marrow may be one of the mechanisms by which the thymus gland is involuted by stress.     

The effect of soluble LAG-3 (CD223) treatment in fetal thymic organ culture

Lymphocyte activation gene-3 (LAG-3; CD223) is structurally similar to CD4 and binds to MHC class II with a 100-fold higher affinity than that of CD4. Soluble LAG-3 (sLAG-3Ig) might be useful for immunotherapy by inducing MHC class II-mediated cell activation. A new form of sLAG-3Ig was constructed containing a critical binding site (D1 and D2 region) to MHC class II, combined with a Fc portion of an immunoglobulin gamma1. After treatment of sLAG-3Ig in fetal thymic organ culture from DO11.10 transgenic mouse, CD4(+) T cell precursors were increased in the positive selection but not affected in the negative selection. Further analysis by treating sLAG-3Ig on thymic epithelial cells revealed that CD40 and MHC class II were up-regulated. These results may demonstrate that the treatment of sLAG-3Ig increases the precursor frequency of CD4(+) T cells by activation of thymic epithelial cells.     

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.     

Treatment of fetal thymic organ culture with IL-1 leads to accelerated differentiation of subsets of CD4-CD8- cells.

Using fetal thymic organ culture (FTOC), we describe the effects of IL-1 on T cell differentiation, particularly within the CD4-CD8- subset. While treatment of FTOC with IL-1 led to a modest reduction in total thymocyte yield, it induced an increase in the percentage of CD4-CD8- cells that express IL-2R early in culture and a decrease in the number of their precursors (CD44+IL-2R- cells). The increase in the percentage of cells expressing IL-2R was not accompanied by an increase in the number of these cells. At later time points these IL-2R+ cells (and their precursors) were reduced relative to controls. The total number of CD4-CD8-CD3- precursor cells in IL-1-treated cultures was reduced to approximately half that in controls at Day 12 of culture. However, only minor inhibition of total cell number was observed, which, taken together with the greater frequency of IL-2R+ precursors, suggests that this depletion of the pool of precursors may have been due to the induction of premature differentiation rather than to its inhibition.     

Induction of murine erythroleukemia cell differentiation by proteinases is inhibited by aromatic poly-amidines

The effects of the tetra benzamidine serine-proteinase inhibitor 1,3-di-(p-amidinophenoxy) -2,2- bis- (p-amidinophenoxymethyl)propane (TAPP-H) and related compounds, including halo-derivatives, were determined on the erythroid differentiation of murine erythroleukemic cells induced by trypsin and kallikrein. These aromatic poly-amidines and their halo derivatives were found to be strong inhibitors of both trypsin and kallikrein mediated induction of commitment of MEL cells to erythroid differentiation, hemoglobin synthesis and accumulation, globin mRNA production. No inhibitory effects were detected by treating proteinase-induced MEL cells with benzamidine. Only slight inhibitory activity was found after treatment of trypsin-induced MEL cells with other antiproteinase compounds widely used in the control of proteinase-dependent functions, including leupeptin, antipain and Bowman-Birk proteinase inhibitor. MEL cells induced to erythroid differentiation by proteinases could be proposed as an experimental system to test the biological activity of proteinase inhibitors.     

Further differentiation of early neural tube explants in organ culture as studied by electron microscopy

Summary Chick embryo spinal cord has been explanted at 2-day stages, when few or no cells have formed axons, and cultured organotypically with adjacent tissues or isolated from all other tissues.Relatively mature nerve cells and glial cells, neurites and synapses could be seen in electron micrographs of cultures maintained for three to four weeks. Histological organization and some aspects of cell differentiation differed in the two types of cultures. Ependymal cells and randomly arranged cells, possibly modified glia, were seen only in cultures of neural tube with adjacent tissue; neurons and macroglia seemed to be more numerous in cultures of isolated neural tube.The development of characteristic cells, with variations according to culture conditions, provides the opportunity for further study of factors controlling patterns of proliferation and differentiation in the central nervous system from very early to advanced stages.This work was supported by a grant from the United States Public Health Service (5 ROI NB 0637).The author wishes to thank Miss Geraldine McTiernan for her competent technical assistance.     

Permissive effect of glutamine on the differentiation of fetal mouse small intestine in organ culture

The proximal third of the small intestine of 15-day-old mouse embryo can be cultured for 72 h at 37 degrees C. When Trowell-T8 medium is used, the integrity of the explants is maintained, but villi do not form and absorptive cells are poorly differentiated. However, when Leibovitz L-15 or RPMI-1640 medium is used, one can observe the formation of medium-sized villi, and absorptive cells in the explants are more differentiated. Since the chemical composition of T8 medium is quite different from that of the other two media, we decided to test the importance of two major differences, i.e., three amino acids and five vitamins, in order to find out which element(s) is necessary to permit the formation of intestinal villi. Subsequent testing demonstrated that the three amino acids are responsible for the effect on differentiation, and that glutamine is the only critical difference between T8 and the two other media. The results show that the addition of L-glutamine to T8 medium permits the formation of villi, the initiation of absorptive cell differentiation, an increase in DNA synthesis, and finally, an increase in the number of epithelial cells. These findings indicate that undifferentiated fetal mouse small intestine is able to express its phenotype in organ culture, even without any extrinsic regulatory influences, provided that L-glutamine is present at a sufficient level in the culture medium. The use of inhibitors indicated that L-glutamine may be essential as an energetic substrate and/or a precursor for glucosamine.     

Phagocytosis of apoptotic and necrotic thymocytes is inhibited by PAF-receptor antagonists and affects LPS-induced COX-2 expression in murine macrophages

There is evidence that apoptotic cells and oxidized low density lipoprotein (oxLDL) particles have common ligands on their surface consisting of oxidized phospholipids which bind to scavenger receptors in macrophages leading to phagocytosis. Some effects of oxLDL binding to its receptor(s) were shown to be inhibited by Platelet Activating Factor (PAF)-receptor antagonists. Thus, we investigated the effect of PAF-receptor antagonists on the phagocytosis of apoptotic, necrotic and viable thymocytes by murine peritoneal macrophages. It was found that phagocytosis of altered cells is significantly increased compared to viable cells, a phenomenon reversed by pre-treatment of macrophages with PAF-receptor antagonists (WEB2170 and CV3988), PAF or oxLDL. Phagocytosis of altered cells induced negligible expression of cyclooxygenase-2 (COX-2) but strongly potentiated the LPS-induced expression of this enzyme. This phenomenon was restricted to altered cells and was reversed by pre-treatment of macrophages with PAF-receptor antagonists. These findings indicate that apoptotic and necrotic cells share common ligands with PAF and oxLDL and suggest the involvement of PAF-like receptors in the enhanced clearance of these cells.     

R5 human immunodeficiency virus type 1 infection of fetal thymic organ culture induces cytokine and CCR5 expression

Late-stage CCR5 tropic human immunodeficiency virus type 1 (HIV-1) isolates (R5 HIV-1) can deplete nearly all CD4+ thymocytes from human thymus/liver grafts, despite the fact that fewer than 5% of these cells express CCR5. To resolve this paradox, we studied the replication and cytopathic effects (CPE) of late-stage R5 HIV-1 biological clones from two progressors and two long-term nonprogressors (LTNP) in fetal thymic organ culture (FTOC) with and without added cytokines. We found that R5 HIV-1 clones from progressors but not LTNP were cytopathic in untreated FTOC. Moreover, R5 HIV-1 clones from progressors replicated to higher levels than LTNP-derived R5 HIV-1 clones in this system. In contrast, when FTOC was maintained in the presence of interleukin 2 (IL-2), IL-4, and IL-7, both progressor and LTNP clones exhibited similar replication and CPE, which were equal to or greater than the levels achieved by progressor-derived R5 HIV-1 clones in untreated FTOC. This finding was likely due to IL-2-induced CCR5 expression on CD4+ thymocytes in FTOC. R5 HIV-1 clones showed greater pathogenesis for CCR5+ cells but also showed evidence of CPE on CCR5- cells. Furthermore, infection of FTOC by R5 HIV-1 induced IL-10 and transforming growth factor beta (TGF-beta) expression. Both IL-10 and TGF-beta in turn induced CCR5 expression in FTOC. Induction of CCR5 expression via cytokine induction by R5 HIV-1 infection of CCR5+ thymocytes likely permitted further viral replication in newly CCR5+ thymocytes. CCR5 expression, therefore, is a key determinant of pathogenesis of R5 HIV-1 in FTOC.     

Effect of IL-7 on the growth of fetal thymocytes in culture

The effects of IL-7 on the in vitro growth and differentiation of day 12 to 14 murine fetal thymocytes were examined in three culture systems. In single cell suspension cultures, IL-7 and IL-2 induced a DNA synthetic response in a short term (1 day) assay, but neither cytokine supported continued cell growth. In conventional fetal thymus organ cultures, the addition of exogenous IL-7 resulted in a twofold increase in cell number over that which normally develops in unsupplemented fetal thymus organ cultures during a 7-day period. The most striking effects of IL-7 were noted in lobe submersion cultures (LSC), a system in which thymocyte growth was totally dependent on the addition of exogenous cytokine. Cells proliferated for a period of approximately 2 wk in IL-7, and cell viability could be maintained even longer. A high percentage of cells recovered after 7 to 14 days from IL-7-supplemented LSC resembled the earliest detectable fetal thymocytes with regard to cell surface markers: they expressed Pgp-1, lacked CD4, CD8, and CD3 and many expressed the IL-2R. These results suggest that IL-7 promotes the growth of cells that occur early in the T cell lineage. Cell populations recovered from LSC supplemented with IL-7 and IL-2 exhibited differential expression of some surface markers, particularly CD3 and NK1.1. In addition, cells from LSC supplemented with IL-7 were found to proliferate upon subsequent exposure to IL-2, but cells from LSC containing exogenous IL-2 were no longer responsive to IL-7. These results imply that IL-7 and IL-2 may act at different stages of thymocyte differentiation. Together with previous observations of IL-7-specific mRNA expression in the thymus, this study provides evidence highly suggestive of a pivotal role for IL-7 in T cell development.     

Role of CTLA-4 in the activation of single- and double-positive thymocytes

CTLA-4, a homologue of CD28, is a negative regulator of T cell activation in the periphery and is transiently expressed on the cell surface after T cell activation. However, the role of CTLA-4 in T cell activation in the thymus is not clear. This investigation was initiated to determine the role of CTLA-4 in the activation of CD4(+)CD8(+) double-positive (DP) and CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes using fetal thymic organ cultures (FTOC) of MHC class II-restricted, OVA(323-339)-restricted TCR transgenic mice (DO11.10). We found that treatment of the FTOC with anti-CTLA-4-blocking Ab during activation with OVA(323-339) increased the proportion and number of DP thymocytes, but decreased the proportion and number of SP thymocytes compared with OVA(323-339)-stimulated FTOC without anti-CTLA-4 Ab treatment. In addition, anti-CTLA-4 Ab treatment inhibited OVA(323-339)-induced expression of the early activation marker, CD69, in DP thymocytes, but increased CD69 in SP thymocytes. Similarly, CTLA-4 blockage decreased phosphorylation of ERK in DP thymocytes by Ag-specific TCR engagement, but increased phosphorylation of ERK in SP thymocytes. CTLA-4 blockage inhibited deletion of DP thymocytes treated with a high dose of OVA(323-339), whereas CTLA-4 blockage did not inhibit deletion of DP thymocytes treated with a low dose of OVA(323-339). We conclude that CTLA-4 positively regulates the activation of DP thymocytes, resulting in their deletion, whereas blocking CTLA-4 suppresses the activation of DP thymocytes, leading to inhibition of DP thymocyte deletion. In contrast, CTLA-4 negatively regulates the activation of SP thymocytes.