Contact-mediated maturational effects of thymic stromal cells on murine thymocytes in culture.

The effect of direct contact between thymic stromal cells and thymocytes on differentiation markers and functions of the latter was studied. The thymic stromal cells included two epithelial and one fibroblast cell lines, previously described. Murine thymocytes were incubated with confluent monolayers of these cells or their supernatants for 24 hr, using monolayers of non-thymic cells and their supernatants as controls. Then, the thymocytes were tested for changes in expression of several surface antigens [Thy-1, Lyt-1, Lyt-2, L3T4, IL-2-receptor (IL-2R)], spontaneous [3H]thymidine incorporation (STI), lectin-induced proliferative response (PR) and lymphokine (IL-2 and IL-3) production. All three thymic stromal cell lines reduced the expression of Thy-1, Lyt-1 and Lyt-2 significantly. The expression of L3T4 was also reduced in some of the experiments, while IL-2R was not expressed by the thymocytes, neither before nor after the co-culture. The thymic stromal cell lines also increased the spontaneous [3H]thymidine incorporation and lymphokine production by the thymocytes and inhibited their proliferative response to lectins. Under the same experimental conditions, the culture supernatants of the thymic stromal cells and the non-thymic cells did not have any effect on the thymocytes, either when collected and used separately or when used in a co-culture system which allowed thymocyte contact with the medium but not with the stromal cells (Transwell system). These results suggest a specific effect of thymic stromal cells, epithelial as well as fibroblasts, on thymocyte maturation. The effect is mediated by direct cell contact and not by secreted factors.     

初代培养的胸腺基质细胞对小鼠胸腺细胞的粘附和吞噬作用

目的 探讨胸腺基质细胞（TSC）对胸腺细胞的调节作用。方法 应用光镜、电镜和流式细胞术等，观察初代培养的TSC对胸腺细胞发育的调节作用。结果 光镜和电镜均可见，TSC可大量粘附和吞噬胸腺细胞，其中部分胸腺细胞发生凋亡。尽管与TSC共培养的胸腺细胞存活率高，凋亡率低，但其总数却显著减少。结论 TSC可通过粘附和吞噬作用，清除部分胸腺细胞。     

Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. IV. Cytokines secreted by human thymic epithelial cells in culture and their activities on murine thymocytes and bone marrow cells.

In previous reports we described our approach to the cultivation of murine and human thymic epithelial cells in primary cultures, using defined, serum-free growth factor-supplemented medium and extracellular matrix-coated culture plates. The cells in these cultures displayed high metabolic activity and their supernatant was highly active on thymocytes. In the study reported here we analysed cytokine activities in the supernatant of human thymic epithelial cell cultures (HTES), by using the respective cytokine-dependent cell lines and by neutralization with specific monoclonal antibodies. Three cytokine activities were detected--interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF) and macrophage (M)-CSF. Other cytokine activities tested for [IL-1, IL-2, IL-7, interferon (IFN) and tumour necrosis factor (TNF)] were negative. The effect of HTES on concanavalin A (Con A)-induced proliferation of murine thymocytes could be completely abolished by anti-IL-6 antibodies, but not by antibodies to CSF, whereas enhancement of bone marrow cell proliferation by HTES was partially inhibited by either anti-G-CSF or anti-M-CSF antibodies and completely inhibited by both antibodies, but not at all by anti-IL-6. We can thus distinguish between thymocyte-related cytokines (IL-6) and bone marrow (myeloid/monocyte) related ones (G-CSF, M-CSF) in HTES.     

胸腺细胞对胸腺基质细胞生长及功能的调节

目的研究胸腺细胞对不同亚群胸腺基质细胞生长及功能的调节作用。方法胸腺基质细胞增殖以３Ｈ－ＴｄＲ掺入法测定，ＩＬ－７活性以促ＩＬ－７依赖株法检测，其ｍＲＮＡ表达以ＲＴ－ＰＣＲ法检测。结果胸腺细胞与ＭＴＳＣ４细胞分别以８０∶１，４０∶１或２０∶１的比例共育时，能明显抑制ＭＴＳＣ４细胞的增殖，而培养上清中ＩＬ－７活性无明显改变；当两种细胞的比例为１０∶１或５∶１时，则对ＭＴＳＣ４细胞的增殖无任何影响，而培养上清中的ＩＬ－７活性明显升高。ＲＴ－ＰＣＲ证实，与胸腺细胞共育的ＭＴＥＣ１和ＭＴＳＣ４细胞，ＩＬ－７ｍＲＮＡ水平明显提高。结论胸腺细胞能促进ＭＴＥＣ１和ＭＴＳＣ４细胞分泌ＩＬ－７，对不同亚群基质细胞的生长和功能有不同调节作用     

Biochemical and immunological differentiation of human thymocytes induced by thymic hormones.

Changes in levels of purine degradative enzymes have been shown to occur during T-cell maturation in both rats and humans with a fall in adenosine deaminase (ADA) and a rise in purine nucleoside phosphorylase (PNP) and 5'-nucleotidase (5'NT) activities. We have investigated the effects of four thymic factors: thymosin fraction 5 (TMS-F5); thymosin alpha 1 (TMS-alpha 1); thymopoietin pentapeptide (TP-5); and thymic conditioned medium (CM) on TdT activity, purine enzyme levels and the phenotypic markers OKT3 (a marker for mature T cells) and NA1/34 (which reacts with immature cortical thymocytes) in human thymocytes and in the lymphoid leukaemic cell lines RPMI-8402 and JM1 (derived from Thy-ALL). All four thymic factors caused one or more maturation change in human thymocytes, e.g. TMS-F5 caused a significant increase in OKT3 expression, TMS-alpha 1 a fall in TdT and ADA activities and a rise in OKT3-positive cells, TP-5 an increase in PNP and CM a rise in 5'NT activity. TMS-F5 also caused a marked elevation of 5'NT in both the T lymphoblastic lines (P less than 0.001). On the other hand the non-physiological phorbol ester, 12-O-tetradecanoyl phorbol acetate (TPA), a tumour promotor with potency of inducing differentiation in some leukaemic cell lines, induced changes in both normal thymocytes and in the leukaemic line JM1 were inconsistent with maturation, e.g. a fall in the percentage of OKT3 cells. These observations suggest that maturation of normal thymocytes might proceed stepwise, each step requiring at least one of the thymic hormones. Although thymosin also induces differentiation changes in a malignant lymphoid line, the pattern of these differs from that induced in their normal counterparts.     

Proliferation and differentiation of immature thymocytes induced by a thymic stromal cell clone

The monolayer of our established thymic stromal cell clone (MRL104.8a) exhibited the capacity to maintain immature double-negative thymocytes. Such capacity was also expressed by a factor produced by the MRL104.8a monolayer. This factor designated as thymic stroma-derived T cell growth factor (TSTGF) was found to be distinct from IL-2 or IL-4 but similar to IL-7 of the previously described cytokines from the functional and molecular aspects. The MRL104.8a monolayer also exerted its differentiation-promoting effect on double-negative thymocytes. Culture for one day of purified double-negative thymocytes on the monolayer resulted in the induction of an appreciable per cent of CD3-4-8+ cells. This differentiation could also be induced by a semipurified TSTGF sample but not by recombinant IL-7, suggesting that the MRL104.8a cells elaborate a factor(s) responsible for initiating the differentiation of double-negative cells in addition to the growth-promotion factor identical or closely related to IL-7. When the culture period of double-negative thymocytes was extended to 2 or 3 days, an appreciable number of double-positive (CD4+8+) and single-positive (CD4+8-) cells were generated on the MRL104.8a monolayer. Thus, these observations provide strong support for the proposition that a specialized thymic stromal component plays an essential role in the intrathymic T cell development in the context of T cell growth and differentiation.     

Expression of HLA antigens by human thymic epithelial cells

Human thymuses were examined by tissue section staining with antibodies specific for monomorphic and polymorphic HLA-A, B, C, and DR determinants. The principal cell type expressing high levels of HLA antigens has the distribution of epithelial cells. Immunoelectron microscopy confirmed their epithelial nature. As in the mouse, both medullary and cortical epithelial cells express high levels of class II (DR) antigens, a finding that is remarkable in that these antigens were originally thought to be restricted to lymphoid and accessory cells. Class I (A, B, and C) antigens are also present on thymic epithelial cells. They are easily detectable on medullary epithelial cells, but two distinct patterns of cortical straining were observed. One group of antibodies produced intense dendritic staining throughout the cortex: the other group produced only faint or no corticol dendritic staining at all. These different staining patterns do not correlate with known properties of the antibodies and thus appear to be due to intrinsci properties of the different A, B, and C antigens.     

Analysis of lymphoproliferative cytokines produced by thymic myoid cells.

Lymphoproliferative activities produced by cloned thymic myoid cell 871207B were analysed by immunological and biochemical methods. The lymphoproliferative activities were separated into two fractions by DEAE-Sepharose CL-6B chromatography: one is in the fraction passed through the column and the other in the fraction eluated from the column with a low concentration of NaCl. The eluated fraction induced the proliferation of interleukin-1 (IL-1)-dependent D10N4 M cells. This activity was abrogated by an anti-IL-1 alpha antibody, but not an anti-IL-1 beta antibody. Expression of IL-1 alpha mRNA was also detected in 871207B cells. The thymocyte proliferative activity found in the fraction passed through the DEAE-Sepharose column was further separated into three fractions by heparin-Sepharose column chromatography: (1) the fraction passed through the column, (2) the fraction weakly bound to the column, and (3) the fraction firmly bound to the heparin column. The fraction passed through the heparin column sustained the growth of IL-6-dependent MH60.BSF-2 cells. IL-6-specific mRNA was found in 871207B cells. The thymocyte proliferative activity of the fraction firmly bound to the heparin column was neutralized with an anti-IL-7 antibody. The biological activity of the fraction weakly bound to the column remained to be elucidated. These results suggest that thymic myoid cells produce IL-1 alpha, IL-6, IL-7 and unidentified lympho-stimulatory factors, all of which play significant roles in many steps of T-cell development in the thymus.     

白血病和正常血细胞的一些细胞因子及受体表达的比较研究

目的:研究白血病细胞和正常血细胞的细胞因子及相应受体(R)的表达及其意义。方法:采用逆转录-聚合酶链反应方法检测红白血病细胞系(HEL、K562)、髓系白血病细胞系(HL-60)、单核细胞白血病细胞系(U937)、巨核细胞白血病细胞系(DAMI、MEG-01)、T淋巴瘤细胞系(HUT78)及EB病毒感染的B细胞系(CA)和正常血细胞(CD34⁺造血干细胞、外周血单个核细胞、成熟粒细胞、巨核细胞及血小板)的多种细胞因子及受体的表达。结果:①CD34⁺造血干细胞同时表达IL-1(α、β)、IL-3、IL-6、GM-CSF、G-CSF及相应受体和干细胞因子受体(SCFR)、血小板生成素受体(MPL)基因,在成熟粒细胞仅表达IL-6、IL-6R、G-CSFR、GM-CSF,巨核细胞、血小板仅表达IL-3R、IL-6、IL-6R、MPL。而TGFβ₁、TNFα及相应受体在上述正常血细胞均有持续表达。②HEL、K562、HL-60、U937、DAMI、MEG-01、HUT78及CA可同时表达至少两种以上正调控因子及相应受体,而负调控因子TGFβ₁、TNFα及受体在上述各细胞系均有表达。结论:白血病细胞株细胞存在正性多自泌环节,白血病细胞的正、负自分泌因子失衡与白血病发病有重要关系。     

Five novel antigens illustrate shared phenotype between mouse thymic stromal cells, thymocytes, and peripheral lymphocytes.

Previously we characterized by immunohistology a group of rat anti-mouse thymic stromal mAbs (MTS 12, 32, 33, 35, and 37), which recognized novel plasma membrane determinants on both thymic stromal cells (TSCs) and thymocytes. The present study investigates in more detail this incidence of shared phenotype by an extensive flow cytometric analysis of MTS mAb reactivity on TSCs, thymocyte subsets, peripheral lymphocytes, and bone marrow cells. Examination of freshly isolated or cultured heterogeneous TSCs and TSC clones confirmed that the mAb identified plasma membrane molecules on distinct subsets of these cells. All but MTS 12 reacted with epithelial cells. Triple-labelling illustrated that MTS 32, 33, and 37 were also reactive with more than 90% of total thymocytes, but varied in their distribution on the four major CD4 and CD8 defined subsets. MTS 12, staining with thymic vascular endothelium by immunohistology, labelled more than 95% of each subset. MTS 35 reactivity in each subset correlated strongly with only the immature populations. Examination of peripheral lymphocytes by triple- and double-labelling unexpectedly showed that MTS 33, 35, and 37 did not recognize peripheral T cells but labelled all B cells. MTS 32 was negative for B cells, but positive for all CD8+ T cells, yet only a subset of CD4+ T cells. Further, MTS 33, 35, and 37 were present on a significant percentage of bone marrow cells. MTS 12 reacted with virtually all peripheral T and B cells, and about 50% bone marrow leukocytes. Collectively these results reveal the same novel epitopes on different thymic cell types and subsets thereof, highlighting specific similarities between cells of apparently diverse lineages. These findings may be of importance in the delineation of intercellular communications within the thymus and emphasize the integrated nature of the microenvironment in this organ.     

Activity of neutral endopeptidase and aminopeptidase N in mouse thymic stromal cells which bind double-positive thymocytes

The activity of two peptidases was determined in immortalized lines of thymic stromal cells. A line of total stromal cells (T-TG-St) was grown from transgenic mouse expressing temperature-sensitive SV40T antigen under the control of the regulatory elements of the mouse major histocompatibility complex class I gene. From these cells we isolated a subset (DP-TG-St) that binds thymocytes which are mainly CD4⁺8⁺. We also assayed a clone of fetal thymic epithelial cells (BA/10) that binds CD4⁺8⁺ thymocytes. Both lines of double-positive cell-binding stroma exhibited strong activity of two peptidases, neutral endopepti dase (NEP; EC 3.4.24.11) and aminopeptidase N (APN; EC 3.4.11.2). In contrast, the activity of both enzymes was very low in the total thymic stromal line. Use of specific inhibitors confirmed that these two enzymes were responsible for the activity observed but also suggested the presence of additional unidentified aminopeptidase(s) in the same stromal cells. The high activity of the two peptidases on stromal cells that bind thymocytes at the double-positive stage raises the possibility that they might contribute to the microenvironment of the developing thymocytes.     

Expression of JAGGED1 in T-lymphocytes results in thymic involution by inducing apoptosis of thymic stromal epithelial cells

Proper development of the thymus and differentiation of T-lymphocytes requires cell-cell interactions between the developing T-lymphocytes and the thymic epithelia. The Delta/Serrate/Lag-2 (DSL)/Notch signal-transduction pathway is known to govern cell fate decisions required for proper development through direct cell-cell interactions. The functional consequences of specific DSL/Notch interactions during the development of a complex organ, such as the thymus, have not been thoroughly elucidated, however. In order to examine the role of DSL proteins during thymus development and T-lymphocyte differentiation, we targeted expression of JAGGED1 in T-lymphocyte progenitors via the control of the proximal lck promoter. Here, we report that expression of JAGGED1 in T cells causes premature involution of the thymus by directing thymic epithelial cells to undergo an apoptotic program. Adoptive transfer of JAGGED1 transgenic bone marrow into non-transgenic mice revealed that JAGGED1 expression on T cells does not alter T-cell differentiation, but is directly responsible for involution of the thymus. We propose that the phenotype of the lck-JAGGED1 transgenic mice is a direct result of specific DSL/Notch interactions and improper cell-to-cell signaling.     

胸腺基质细胞对热应激小鼠胸腺细胞亚群变化及HSP70表达的影响

目的探讨胸腺基质细胞(TSC)对热应激小鼠胸腺细胞的调节作用。方法应用光镜、电镜、流式细胞术等观察初代培养的TSC对热应激胸腺细胞发育的调节作用。结果 TSC可大量粘附和吞噬胸腺细胞,使热应激胸腺细胞数量明显减少,但尚存的胸腺细胞中活细胞比例却明显增加,凋亡率明显减少。TSC可促进热应激胸腺细胞HSP70表达增加,使热应激胸腺细胞的DP及SP细胞,尤其是DP细胞数量明显减少。结论 TSC可清除大量的热应激胸腺细胞,TSC促进热应激胸腺细胞HSP表达增高可能具有双重意义:既可保护胸腺细胞,又可促进TSC识别和吞噬已受损的或凋亡的胸腺细胞。     

胸腺基质细胞的抗原提呈作用

目的 研究胸腺基质细胞的抗原提呈能力。方法 应用ＯＶＡ－特异的、受Ｉ－Ａ＾ｄ分子识别限制的辅助Ｔ细胞杂交瘤（３ＤＯ．１８．３）识别提呈的ＯＶＡ的ＣＮＢｒ水解片段而被活化后产生ＩＬ－２,测定ＩＬ－２活性来分析胸腺基质细胞的抗原提呈作用。结果ＩＦＮ－γ能促进ＭＴＥＣＩ和ＭＴＳＣ４表达Ｉ－Ａ＾ｄ分子,并促进ＭＴＳＣ４表达Ｂ７－１分子。经ＩＦＮ－γ作用后,ＭＴＥＣ１和ＭＴＳＣ４均有抗原提呈能力,ＭＴＳＣ     

Maintenance and polarization of human TH2 central memory T cells by thymic stromal lymphopoietin-activated dendritic cells

The identity of TH2 memory cells and the mechanism regulating their maintenance during allergic inflammation remain elusive. We report that circulated human CD4+ T cells expressing the prostaglandin D2 receptor (CRTH2) are TH2 central memory T cells, characterized by their phenotype, TH2 cytokine production, gene-expression profile, and the ability to respond to allergens. Only dendritic cells (DCs) activated by thymic stromal lymphopoietin (TSLP) can induce a robust expansion of CRTH2+CD4+ TH2 memory cells, while maintaining their central memory phenotype and TH2 commitments. CRTH2+CD4+ TH2 memory cells activated by TSLP-DCs undergo further TH2 polarization and express cystatin A, Charcot-Leydon crystal protein, and prostaglandin D2 synthase, implying their broader roles in allergic inflammation. Infiltrated CRTH2+CD4+ TH2 effector memory T cells in skin lesion of atopic dermatitis were associated with activated DCs, suggesting that TSLP-DCs play important roles not only in TH2 priming, but also in the maintenance and further polarization of TH2 central memory cells in allergic diseases.     

两种小鼠胸腺基质细胞对胸腺细胞凋亡的不同作用

采用两种体外建系的小鼠胸腺基质细胞（ＴＳＣ）系，即上皮样ＴＳＣ（ＭＴＥＣ１）和树突状ＴＳＣ（ＭＴＳＣ４），观察其对胸腺细胞凋亡的影响。小鼠胸腺细胞在体外培养过程中，可自发地出现细胞凋亡的特征，表现为ＤＮＡ呈梯度断裂片段，细胞经ＦＡＣＳ分析出现亚二倍体ＤＮＡ波峰，以及Ｆｅｕｌｇｅｎ′ｓ染色镜检所见的ＤＮＡ凝聚和断裂。胸腺细胞在与ＴＳＣ共育后，在ＭＴＥＣ１组可见其凋亡过程受到抑制和存活率的增加；在ＭＴＳＣ４组，仅在共育１２至１８小时时，见到胸腺细胞凋亡加强，而其存活率不受影响。结果提示在胸腺细胞发育过程中，其阴性选择作用的主要机制之一的ＰＣＤ过程受不同来源的胸腺基质细胞的调节。     

Effect of a human serum thymic factor on hydrocortisone-treated thymocytes.

A human serum thymic factors (SF) stimulated cyclic adenosine-3' ,5'-monophosphate (cAMP) synthesis in normal mouse thymocytes. Such stimulation was no longer observed when thymocytes were depleted of hydrocortisone (HC)-sensitive cells. It was concluded that SF selectively stimulate cAMP in HC-sensitive cells. Furthermore, incubation of thymocytes with SF enlarged the population of HC-resistant thymocytes. These results suggest that SF might act on HC-sensitive thymocytes increasing their cellular cAMP level and inducing their transformation in HC-resistant cells.     

Expression of CR2/EBV receptors on human thymocytes detected by monoclonal antibodies

The biologic effects of the third component of complement, C3, are mediated via receptors which specifically bind the enzymatic degradation products resulting from the cleavage of C3. One of the products, C3d, has been associated with binding to the second complement receptor CR2 (CD21). This receptor, which is identical to the receptor for Epstein-Barr virus (EBV), has been primarily found on cells of the B lineage, but not on mature T cells or other cells of erythroid or myeloid lineages. In the present investigation, we report the presence of CR2 on human thymocytes. Indirect immunofluorescence analysis employing monoclonal anti-CR2 antibodies revealed a range of thymocyte reactivity from 15% to 63% in thirteen experiments using cells of different donors. Reactivity was always greater with the monoclonal anti-CR2 (CD21) antibody HB-5 than with two other antibodies which recognize distinct epitopes on the CR2 molecule. Two-color immunofluorescence analysis indicated that the brightest of the HB-5-stained thymocytes also reacted with the monoclonal anti-CD1 antibody T6 (immature thymocyte marker) while some of the duller HB-5-staining cells reacted with the monoclonal anti-CD3 antibody Leu-4 (mature thymocyte marker). Immunoprecipitation of CR2 on thymocytes with antibody HB-5 and polyacrylamide gel electrophoretic analysis revealed a protein of 145 kDa molecular mass which is consistent with the size of CR2 found on B lymphocytes. These findings raise several questions regarding the biologic role of CR2-EBV receptor on cells of the T lineage.     

Expression of ADAMTS-5/implantin in human decidual stromal cells: regulatory effects of cytokines

BACKGROUND: The restricted expression of ADAMTS-5 (A Disintegrin And Metalloproteinase with ThromboSpondin repeats-5) to the maternal-fetal interface in mice has led to this novel metalloproteinase being assigned the trivial name 'implantin'. METHODS: As a first step in determining whether ADAMTS-5 also contributes to the implantation process in humans, we have examined the spatiotemporal expression of this ADAMTS subtype in the endometrium during the menstrual cycle and pregnancy by immunohistochemical analysis. A quantitative competitive PCR (QC-PCR) strategy and western blotting were subsequently used to determine whether interleukin (IL)-1beta and transforming growth factor (TGF)-beta1, two cytokines involved in the formation of the maternal-fetal interface, were capable of regulating ADAMTS-5 messenger RNA (mRNA) and protein levels in primary cultures of stromal cells isolated from first trimester decidual tissues. RESULTS: ADAMTS-5 expression in the stroma of the human endometrium correlates with decidualization of this cellular compartment in vivo. IL-1beta was found to increase (P < 0.05) whereas TGF-beta1 decreased (P < 0.05) ADAMTS-5 mRNA and protein levels in decidual stromal cell cultures in a concentration- and time-dependent manner. These regulatory effects were attenuated by function-perturbing antibodies directed against either cytokine. CONCLUSIONS: ADAMTS-5 expression is restricted to decidualized stromal cells of the human endometrium in vivo and is subject to regulation by cytokines in vitro.