Detection of rearranged T cell receptor beta-chain gene and induction of cytolytic function in interleukin 2-responsive day 14-15 murine fetal thymocytes

A subpopulation of interleukin 2 (IL 2) receptor-positive day 14-15 murine fetal thymocytes can be induced by recombinant IL 2 to proliferate over prolonged time periods in dissociated cell cultures. The proliferating day 14-15 fetal thymocytes exhibit no cytolytic effector function, nor do they rearrange T cell receptor beta chain genes. This contrasts with thymic organ cultures in which day 14-15 thymocytes do rearrange beta chain genes and give rise to immunocompetent cells. However, such events can also take place in dissociated cell cultures, provided the IL 2-responsive thymocytes are cultured on syngeneic feeder cells in the presence of IL 2 and the mitogen concanavalin A. Under such conditions rearrangement of the beta chain gene complex becomes detectable and cytolytic effector cells are generated. The frequency of inducible cytolytic precursor cells in day 14-15 thymocytes is 1/7000. These data either imply that immunocompetent cells are already present in the day 14-15 fetal thymus, or differentiation from precursors to immunocompetent cells must occur in dissociated cell cultures.     

Differentiation of thymocytes in fetal organ culture: lack of evidence for the functional role of the interleukin 2 receptor expressed by prothymocytes

Thymic rudiments of 14-day-old mouse embryos were put into organ culture in the presence of interleukin 2 (IL2) and PC.61, a monoclonal antibody that binds to the IL2 receptor (IL2R). After 5 and 7 days of culture, we found no influence of PC.61 on the growth of the thymus and the composition of the thymocyte subpopulations as studied with a panel of monoclonal antibodies. High-dose IL2 treatment of the organ culture resulted in a reduction of the number of thymocytes and a decrease in the single CD4+ and CD4+CD8+ thymocytes, whereas the number of CD8+Ly-1+ thymocytes and the number of CD4-CD8-Ly-1- IL2R+ thymocytes increased. The effect of high-dose IL2 treatment was ascribed to the induction of a nonspecific LAK activity. Our findings argue against a functional role of IL2R on prothymocytes during early T cell development.     

Detection of interleukin 2 receptors on murine lymphocytes using fluorescent interleukin 2.

Murine interleukin 2 receptors found on freshly isolated and on in vitro activated lymphocytes were identified using a fluorescent interleukin 2 (IL2F). Three percent of freshly isolated small thymocytes bound the IL2F; these cells appeared to be dual CD4 and CD8 positive cells. Ten percent of the larger thymocytes also bound the IL2F; phenotypically, these cells were more heterogenous in their CD4/CD8 composition than the small IL2F+ thymocytes. Freshly isolated splenocytes bound more IL2F than did the thymocytes. Twenty-four percent of the small splenocytes were IL2F+ and they were mostly B220+ cells. Half of the larger splenocytes were IL2 receptor positive and these cells consisted of B and T cells. Using mitogen stimulated splenocytes, three times as many LPS stimulated B220+ blasts bound the fluorescent IL2 than freshly isolated large B220+ cells; this level of IL2F binding was maintained for four days. Of the Con A blasts, more CD8+ cells (30%) bound IL2F than did CD4+ blasts (19%); these cells maintained this level of IL2F binding for only three days. The IL2F binding could be completely inhibited by excess unlabeled IL2 and could be inhibited by 92% using a monoclonal antibody directed against the IL2 binding region of the IL2 alpha receptor, indicating that IL2F can bind to both IL2 alpha and IL2 beta receptors.     

Possible activation of murine T lymphocyte through CD98 is independent of interleukin 2/interleukin 2 receptor system

CD98 is a widely expressed cell surface heterodimeric protein of 125 kDa. Its expression is upregulated during lymphocyte activation induced by mitogen, superantigen, conventional antigen, and a combination of phorbol myristate acetate (PMA) and ionomycin. However, the role of CD98 in the immune system is not so well understood. The role of CD98 in murine T lymphocyte proliferation was investigated, especially in correlation with the interleukin 2 (IL-2)/interleukin 2 receptor (IL-2R) system. Monoclonal antibody (mAb) directed against murine CD98 heavy chain (mCD98HC) suppressed the proliferation of lymphocytes stimulated with concanavalin A (Con A). Anti-mCD98HC mAb did not suppress the expression of IL-2Ralpha. Anti-IL-2Ralpha mAb, which suppressed DNA synthesis, did not inhibit the expression of CD98HC. Murine IL-2 (recombinant), which induced considerable DNA synthesis by lymphocytes stimulated with a sub-optimal dose of Con A, did not induce CD98HC expression in lymphocytes. In addition, anti-mCD98HC mAb did not inhibit the production of IL-2 by lymphocyte stimulated with Con A. Taken together with these findings, it was speculated that the CD98 system is independent of the IL-2/IL-2R system in murine T lymphocyte activation.     

Maturation of fetal thymocytes in organ culture: secretion of interleukin 2, interferon and colony stimulating factors

This paper describes the differentiation of fetal thymocytes in organ cultures of embryonic thymuses. After one week in organ culture, fetal thymocytes from 14 d old mouse embryos could secrete IL-2, CSF and IFN upon stimulation with Con A. No constitutive secretion of lymphokines was observed. The only type of CSF produced was granulocyte-macrophage (GM) CSF. In contrast to thymocytes from adult mice, organ-cultured embryonic thymocytes did not secrete IFN-gamma, but IFN alpha/beta, in response to Con A. This is the first indication that secretion of IFN alpha/beta, but not IFN-gamma, can be induced in cells of the T cell lineage by a T cell specific mitogen. These results show that the embryonic thymus provides a sufficient environment for the development of some of the secretory functions of T cells and suggest that differentiating T cells acquire the ability to secrete IFN alpha/beta before IFN-gamma.     

Murine thymic nurse cell clone supports the growth of fetal thymocytes in the presence of interleukin 2.

To investigate the role of thymic nurse cells (TNC) in activation and differentiation of fetal CD4-CD8- (double-negative) thymocytes, we have co-cultured murine fetal thymocytes (14-15 days of gestation) with an established murine TNC clone. We show here that TNC induced the growth of the fetal double-negative thymocytes in the presence of recombinant interleukin 2 (rIL2). Activated fetal thymocytes markedly formed lymphocyte-TNC complexes and proliferated extensively after 5 days in the co-culture. The activated fetal thymocytes in this co-culture condition remained double negative after 10 days in culture. None of them gave rise to phenotypically and functionally competent lymphocytes during this period. TNC alone and the supernatant of TNC had no effect on activation. The presence of both TNC and rIL2 was necessary for the growth of fetal thymocytes in our system. The proliferation of fetal thymocytes was inhibited by a monoclonal antibody against mouse IL2 receptors (IL2R). The fetal thymocytes could be maintained further in this co-culture condition. The prolonged cultivation of fetal thymocytes resulted in the establishment of the fetal thymocyte line and its several clones. CD4 single-positive cells of activated fetal thymocytes first appeared 14 days after the onset of culture and their number increased, whereas CD8+ cells or CD4CD8 double-positive cells were not observed. These results indicate that fetal CD4-CD8- thymocytes underwent phenotypic change after long periods of culture. All established clones of fetal thymocytes are CD4 single positive showing lymphocyte-TNC interactions but do not express CD3 complex. Northern blot analysis detected mRNA for the gamma T cell receptor, but no messages for the delta, alpha or beta T cell receptor. Chemical cross-linking of 125I-labelled IL2 revealed that the 90-kDa band (presumably considered to be the IL2R beta chain) was clearly present in IL2-responsive fetal clones, whereas freshly isolated day 14-15 fetal thymocytes lacked the band. Taken together, TNC might be involved in the differentiation and/or expansion of murine fetal thymocytes by inducing IL2R beta chain, which forms the functional IL2R together with IL2R alpha chain and CD4, one of the T cell accessory molecules, on the cell surface through direct cell-cell interaction.     

Ontogeny of the response of mouse thymocytes to interleukin 1 and interleukin 2.

In the present report we demonstrate that the in vitro proliferative response of the newborn thymocytes to interleukin (IL) 1 and IL 2, which is remarkably stronger than the adult thymocyte response, is associated with a considerable increase of CD4-CD8- cells expressing a gamma/delta T cell receptor (TcR). By polymerase chain reaction analysis we show that the V gamma gene segment usage in the adult and newborn responding cells reflects the developmentally regulated expression of the V gamma gene segments, suggesting that the increase in TcR gamma/delta+ cells results from the polyclonal expansion of pre-existing clones. Surprisingly, although the fetal thymocyte populations contain higher numbers of TcR gamma/delta+ cells than the adult and newborn ones, the highest proliferative response to IL 1 and IL 2 is obtained with the newborn thymocytes. Non mutually exclusive hypotheses are discussed to explain these results.     

Binding of human recombinant interleukin 2 to murine erythrocytes is erythropoietin receptor mediated

Murine red blood cells (RBC) incubated with recombinant human interleukin 2 (rIL2) bound 10%–20% of the added cytokine with 41% ± 5% of the population positive for bound cytokine as determined by fluorescence activated cell scanning (FACS) analysis. Since a high degree of homology exists between the erythropoietin receptor (EPOR) and the IL2R chain (based both on amino acid sequence and hydrophobicity alignment), we hypothesised that the rIL2 was binding to residual EPOR on murine RBC. Upon bioassay, it was found that reticulocytes (RET), which express a higher percentage of EPOR than normal RBC, bound 400% more rIL2 as compared to normal RBC. FACS analysis using fluorescently labelled rIL2 revealed a log higher fluorescence intensity on RET compared to RBC. Therefore, the population of immature or young RBC in circulation which are expressing residual EPOR are binding rIL2 due to cross-reactivity between the EPOR and IL2R.Mention of a tradename, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Con A response and interleukin 2 receptor expression of thymocytes rosetting with phagocytic cells of the thymic reticulum

Thymocytes bind to the phagocytic cells of the thymic reticulum (P-TR) and spontaneously forming rosettes were isolated and characterized. Rosetting thymocyte subsets included cells expressing different surface phenotype: most of them (91%) were of the double positive L3T4+ Lyt-2+ phenotype; a minor subpopulation (2.4%) expressed the helper L3T4+ Lyt-2- phenotype and finally a small subpopulation (6.3%) was negative for both L3T4 and Lyt-2 markers. Although the rosetting thymocytes were not enriched in mature single positive cells as compared to the non rosetting population, they showed a higher responsiveness to ConA plus recombinant IL-2. Moreover the rosetting thymocytes were induced to express IL-2 receptors just after stimulation with ConA whereas co-stimulation with ConA plus IL-2 was essential for the generation of IL-2 receptors on the non rosetting and total thymocytes. The possibility that the rosetting thymocytes acquired this activity during the rosetting process is discussed.     

Expression and characterization of the interleukin 2 receptor in Theileria parva-infected bovine lymphocytes

We have previously shown that interleukin 2 receptors (IL2R) are constitutively expressed on the surface of bovine lymphocytes infected with the parasite Theileria parva (Dobbelaere, D.A.E. et al., Proc. Natl. Acad. Sci. USA 1988. 85: 4730). In the present work we characterized these further and showed that IL2R (Tac antigen) gene expression depended on the continuous presence of the parasite in the host cell cytoplasm. By Northern blot analysis we showed that elimination of the parasite, using a specific theilericidal drug, led to the arrest of Tac antigen mRNA expression. We also investigated receptor internalization and reappearance after receptor-mediated endocytosis. Binding of human recombinant interleukin 2 (hrIL2) to the bovine IL2R caused rapid internalization of the surface IL2/IL2R complex. Approximately 50% of 125I-labeled hrIL2 was internalized within 10 min. The reappearance of surface IL2R after ligand-mediated endocytosis was also studied. Fifty percent of the maximum level of free IL2R reappeared within 1-1.5 h, but approximately 12 h were needed to restore normal levels of free IL2R expression after blocking with excess unlabeled hrIL2.     

Soluble interleukin 2 receptor release, interleukin 2 production, and interleukin 2 receptor expression in activated T-lymphocytes in vitro.

Following activation in vitro, peripheral blood mononuclear cells (PBMC) express cell-associated interleukin-2 receptors (IL2R). The present study was undertaken to define the proportion of T lymphocyte subsets that express the IL2R (CD25 antigen) upon different mitogenic stimulation. Double immunofluorescence staining with different fluorochromes, fluorescein isothiocyanate and phycoethyrin, was applied for identification of IL2R positive cells and individual lymphocyte subset. The exact percentage of individual activated lymphocyte subset bearing IL2R was enumerated by photographic counting. There was paucity of IL2R in freshly isolated, unstimulated peripheral blood, PBMC cultured without mitogen, and cultured B lymphocytes. Following pokeweed mitogen stimulation in vitro, 19% of CD4 (T-helper/inducer) lymphocytes and 14% of CD8 (T-suppressor/cytotoxic) lymphocytes expressed IL2R. Similarly, 25% of CD4 lymphocytes and 19% of CD8 lymphocytes expressed IL2R following phytohemagglutinin stimulation in vitro. Contrary to the reported data of Tac-positive cells in human lymphoid tissues, our study revealed that, upon lectin mitogen stimulation, approximately 55% of IL2R positive PBMC were CD4 lymphocytes, and 45% of them were CD8 lymphocytes. These observations imply the plausible notion that interleukin-2 mediated immune activation of T lymphocytes in PBMC is different from that in local lymphoid organs. It was also demonstrated that the release of soluble IL2R (sIL2R) and IL2 production in supernatant from cultured PBMC varied with different lectin stimulation. A significant correlation was demonstrated between the cellular and soluble IL2R but the production of IL2 from activated mononuclear cells bore no good correlation with either the cellular IL2R expression or the release of sIL2R.     

Toll样受体2在人胎盘和胎膜组织中的表达

目的 检测正常胎盘和胎膜组织中Toll样受体2(TLR2)的表达.方法 收集5例足月剖宫分娩胎盘和胎膜样本,运用RT-PCR法检测胎盘和胎膜组织中TLR2 mRNA的表达,运用免疫组织化学和Westen blot印迹方法检测TLR2蛋白质在胎盘和胎膜组织中的表达.结果 RT-PCR显示在mRNA水平,胎盘和胎膜组织中均有TLR2表达;Westen blot印迹显示TLR2抗体在胎盘和胎膜组织中相对分子质量为50000左右的位置有一条明显的条带;免疫组化研究显示TLR2在胎盘的合体滋养细胞、胎膜的羊膜上皮细胞及平滑绒毛膜滋养细胞中有表达.结论 TLR2在人胎盘和胎膜组织中的表达,提示其在妊娠期先天性免疫中可能发挥重要作用.     

Toll样受体2在人胎盘和胎膜组织中的表达

目的 检测正常胎盘和胎膜组织中Toll样受体2(TLR2)的表达.方法 收集5例足月剖宫分娩胎盘和胎膜样本,运用RT-PCR法检测胎盘和胎膜组织中TLR2 mRNA的表达,运用免疫组织化学和Westen blot印迹方法检测TLR2蛋白质在胎盘和胎膜组织中的表达.结果 RT-PCR显示在mRNA水平,胎盘和胎膜组织中均有TLR2表达;Westen blot印迹显示TLR2抗体在胎盘和胎膜组织中相对分子质量为50000左右的位置有一条明显的条带;免疫组化研究显示TLR2在胎盘的合体滋养细胞、胎膜的羊膜上皮细胞及平滑绒毛膜滋养细胞中有表达.结论 TLR2在人胎盘和胎膜组织中的表达,提示其在妊娠期先天性免疫中可能发挥重要作用.     

Toll样受体2在人胎盘和胎膜组织中的表达

目的 检测正常胎盘和胎膜组织中Toll样受体2(TLR2)的表达.方法 收集5例足月剖宫分娩胎盘和胎膜样本,运用RT-PCR法检测胎盘和胎膜组织中TLR2 mRNA的表达,运用免疫组织化学和Westen blot印迹方法检测TLR2蛋白质在胎盘和胎膜组织中的表达.结果 RT-PCR显示在mRNA水平,胎盘和胎膜组织中均有TLR2表达;Westen blot印迹显示TLR2抗体在胎盘和胎膜组织中相对分子质量为50000左右的位置有一条明显的条带;免疫组化研究显示TLR2在胎盘的合体滋养细胞、胎膜的羊膜上皮细胞及平滑绒毛膜滋养细胞中有表达.结论 TLR2在人胎盘和胎膜组织中的表达,提示其在妊娠期先天性免疫中可能发挥重要作用.     

Toll样受体2在人胎盘和胎膜组织中的表达

目的 检测正常胎盘和胎膜组织中Toll样受体2(TLR2)的表达.方法 收集5例足月剖宫分娩胎盘和胎膜样本,运用RT-PCR法检测胎盘和胎膜组织中TLR2 mRNA的表达,运用免疫组织化学和Westen blot印迹方法检测TLR2蛋白质在胎盘和胎膜组织中的表达.结果 RT-PCR显示在mRNA水平,胎盘和胎膜组织中均有TLR2表达;Westen blot印迹显示TLR2抗体在胎盘和胎膜组织中相对分子质量为50000左右的位置有一条明显的条带;免疫组化研究显示TLR2在胎盘的合体滋养细胞、胎膜的羊膜上皮细胞及平滑绒毛膜滋养细胞中有表达.结论 TLR2在人胎盘和胎膜组织中的表达,提示其在妊娠期先天性免疫中可能发挥重要作用.     

Toll样受体2在人胎盘和胎膜组织中的表达

目的 检测正常胎盘和胎膜组织中Toll样受体2(TLR2)的表达.方法 收集5例足月剖宫分娩胎盘和胎膜样本,运用RT-PCR法检测胎盘和胎膜组织中TLR2 mRNA的表达,运用免疫组织化学和Westen blot印迹方法检测TLR2蛋白质在胎盘和胎膜组织中的表达.结果 RT-PCR显示在mRNA水平,胎盘和胎膜组织中均有TLR2表达;Westen blot印迹显示TLR2抗体在胎盘和胎膜组织中相对分子质量为50000左右的位置有一条明显的条带;免疫组化研究显示TLR2在胎盘的合体滋养细胞、胎膜的羊膜上皮细胞及平滑绒毛膜滋养细胞中有表达.结论 TLR2在人胎盘和胎膜组织中的表达,提示其在妊娠期先天性免疫中可能发挥重要作用.     

Toll样受体2在人胎盘和胎膜组织中的表达

目的 检测正常胎盘和胎膜组织中Toll样受体2(TLR2)的表达.方法 收集5例足月剖宫分娩胎盘和胎膜样本,运用RT-PCR法检测胎盘和胎膜组织中TLR2 mRNA的表达,运用免疫组织化学和Westen blot印迹方法检测TLR2蛋白质在胎盘和胎膜组织中的表达.结果 RT-PCR显示在mRNA水平,胎盘和胎膜组织中均有TLR2表达;Westen blot印迹显示TLR2抗体在胎盘和胎膜组织中相对分子质量为50000左右的位置有一条明显的条带;免疫组化研究显示TLR2在胎盘的合体滋养细胞、胎膜的羊膜上皮细胞及平滑绒毛膜滋养细胞中有表达.结论 TLR2在人胎盘和胎膜组织中的表达,提示其在妊娠期先天性免疫中可能发挥重要作用.