A chemotactic factor for rat thymocytes may regulate T-lymphocyte migration toward the thymic microenvironment

Using a modified Boyden chamber assay, extracts or culture supernatants of rat thymic stromal cells, or thymocytes were examined by chemotactic activity to rat leukocytes. Rat thymocytes responded chemotactically to the aqueous extract as well as to culture supernatants of thymic stromal cells. However, neither the extract and culture medium from concanavalin A-stimulated thymocytes nor any component of rat serum has shown such an activity. The thymic extract was fractionated into three molecular species with chemotactic activity for thymocytes. The thymocyte chemotactic factor(s) (TCFs) in the extract was distinct from known lymphocytic chemotactic factors, such as interleukin-1 (IL-1), IL-2, C5a, and the culture supernatant of stimulated thymocytes. In vitro, TCFs could attract, in addition to thymocytes, bone marrow cells, fetal liver cells, and nylon-wool nonadherent lymphocytes from peripheral blood and spleen. Lymph node cells, neutrophils, macrophages, and B cells from peripheral blood could not respond to TCFs. Thymocytes also responded to the extract of splenic stromal cells. Unlike the thymic extract, however, the splenic extract was chemotactically active for lymphocytes from lymph nodes but not for bone marrow cells. These results indicate that thymic stromal cells secrete a chemotactic factor(s) for a relatively immature type of T-lineage cells, which may by a thymus-homing progenitor T cell, while spleen may contain an attractant for a relatively mature type of T-lineage cells.     

Characterization of thymic progenitors in adult mouse bone marrow

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1(-)) (Thy1.1(-) progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1(-) progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1(-) population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.     

Generation of macrophages from early T progenitors in vitro

Early T progenitors in the thymus have been reported to have the capacity to develop into B cells, thymic dendritic cells, and NK cells. Here we describe conditions that induce early T progenitors to develop into macrophages. Initially, we observed that early T progenitors could be induced to develop into macrophages by cytokines produced from a thymic stromal cell line, TFGD, and later we found that the cytokine mixture of M-CSF plus IL-6 plus IL-7 also induced macrophage differentiation from pro-T cells. M-CSF by itself was unable to induce macrophage differentiation from early T progenitors. To correlate this observation with the developmental potential of early T progenitors, mouse embryonic thymocytes were sorted into four populations, pro-T1 to pro-T4, based on the expression of CD44 and CD25, and then cultured with TFGD culture supernatant. We found that pro-T1 and pro-T2 cells, but not pro-T3 and pro-T4 cells, generate macrophages. Limiting dilution analysis of the differentiation capability of sorted pro-T2 cells also confirmed that pro-T2 cells could generate macrophages. These results suggest that T cells and thymic macrophages could originate from a common intrathymic precursor.     

Fas is expressed early in human thymocyte development but does not transmit an apoptotic signal.

We investigated the expression and function of Fas on human thymocytes prepared from fetal and pediatric tissue specimens and from SCID-hu Thy/Liv grafts. Unlike mouse thymocytes, human thymocytes exhibited a pattern of Fas expression skewed to immature cells, in that the highest expression was seen on double negative thymocytes and on intrathymic T progenitor cells. Fas expression was intermediate on double positive human thymocytes, and low or negative on mature single positive CD4 and CD8 medullary thymocytes. In spite of this relatively abundant surface expression, cross-linking of Fas with agonist mAb was incapable of triggering an apoptotic signal in human thymocytes. Apoptotic signaling was not enhanced by treatment with cycloheximide, nor by restoring a cosignaling milieu by addition of thymic stromal cells. Mouse thymocytes were induced to apoptosis by cross-linked recombinant soluble human Fas ligand both in vitro and in vivo, though human thymocytes were also resistant to this mode of receptor ligation. Membrane-bound Fas ligand also induced apoptotic death in murine thymocytes but not in human thymocytes. Human thymocytes were as sensitive as Jurkat cells, however, to apoptosis induced by TNF-alpha, suggesting that these cells have a signaling defect before activation of the earliest caspases. These data demonstrate a durable and specific resistance of human thymocytes to apoptosis induced through Fas receptor engagement, and reveal significant species-specific differences in the biology of thymocyte-programmed cell death.     

Human thymic epithelial cells produce interleukin 1

Although the thymus plays a critical role in generation of immunocompetent T lymphocytes, the precise role of the epithelial component of the thymus in the induction of T cell proliferation and maturation remains unknown. Since interleukin 1 (IL 1) is required for mature T cell activation, we have determined whether human thymic epithelial (TE) cells produce IL 1. By using a system for longterm culture of human TE cells, we found that human TE cells produced an IL 1-like factor (TE-IL 1) that augmented the proliferation of C3H/HeJ mouse thymocytes to phytohemagglutinin. IL 1 activity (20 to 200 U/ml) was detected in supernatants of TE cultures from all individuals (2 to 13 yr old) tested. IL 1 activity was also detected in supernatants of TE cultures from a 17-wk fetus but not from a 10-wk fetus. Production of TE-IL 1 was dependent on TE cell density and time in culture with optimal TE-IL 1 activity observed at 10(6) TE cells/ml after 48 to 72 hr of culture. With the use of high performance liquid chromatography, TE-IL 1 chromatographed as a molecule of 18,000 to 20,000 relative molecular mass, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, TE-IL 1 migrated at 15,000 to 17,000 Mr. With the use of isoelectrofocusing gels, charge heterogeneity of TE-IL 1 was demonstrated with two major isoelectric points of 5.7 to 5.8 and 6.9 to 7.0. Polyclonal antibody to human monocyte IL 1 markedly inhibited the TE-IL 1 activity. In indirect immunofluorescence assay of frozen human thymic sections, rabbit anti-IL 1 antibody reacted with epithelial cells in human thymic cortex and medulla. Furthermore, high performance liquid chromatography-purified TE-IL 1 augmented human thymocyte proliferation to suboptimal concentrations of phytohemagglutinin. Thus, thymic epithelial cells are capable of providing an intrathymic source of IL 1-like cytokine (TE-IL 1), which affects thymocyte proliferation. We propose that TE-IL 1 may play an important role in intrathymic proliferation and differentiation of human thymocytes.     

Development of CD4+ macrophages from intrathymic T cell progenitors is induced by thymic epithelial cells

It was recently demonstrated that there are CD4(+) macrophages, which exhibit strong phagocytic activity, in the thymus. They are suggested to play an important role for the elimination of apoptotic thymocytes. However, the origin and nature of CD4(+) macrophages in the thymus remain unexplored. In this study, we describe that the most immature intrathymic progenitors (CD25(-)/CD44(+)/FcR(+)) give rise to CD4(+) macrophages by oncostatin M-responsive thymic epithelial cells (ORTEC) in an IL-7-dependent manner. Neither conditioned medium of ORTEC nor a mixture of cytokines induced CD4(+) macrophages, and oncostatin M receptor was not expressed in thymocytes, suggesting that the development of CD4(+) macrophages from the immature thymocytes requires a direct interaction with ORTEC. These results collectively suggest that the development of CD4(+) macrophages from the intrathymic T cell progenitors is induced by thymic epithelial cells.     

Extrathymic hemopoietic progenitors committed to T cell differentiation in the adult mouse

The role of the thymus in T cell commitment of hemopoietic precursor is yet controversial. We previously identified a major T cell progenitor activity in precursor cells isolated from bone marrow-derived spleen colonies. In this study, we characterize the properties of these pre-T cells. We demonstrate that they have unique phenotype and can be generated in a total absence of any thymic influence. Indeed, even when studied at the single-cell level, extrathymic T cell-committed precursors express T cell-specific genes. Moreover, these cells are not committed to a particular T cell differentiation pathway because they can generate both extrathymic CD8alphaalpha+ intraepithelial lymphocytes and thymus-derived conventional thymocytes. We also compared these pre-T cells with fully T cell-committed thymic progenitors. When tested in vitro or by direct intrathymic transfer, these cells have a low clonogenic activity. However, after i.v. transfer, thymus repopulation is efficient and these precursors generate very high numbers of peripheral T cells. These results suggest the existence of extra steps of pre-T cell maturation that improve thymus reconstitution capacity and that can be delivered even after full T cell commitment. Consequently, our studies identify a source of extrathymic progenitors that will be helpful in defining the role of the thymus in the earliest steps of T cell differentiation.     

Developmental regulation of the intrathymic T cell precursor population

The maturation potential of CD4-8- thymocytes purified from mice of different developmental ages was examined in vivo after intrathymic injection. As previously reported, 14-day fetal CD4-8- thymocytes produced fewer CD4+ than CD8+ progeny in peripheral lymphoid tissues, resulting in a CD4+:CD8+ ratio of less than or equal to 1.0. In contrast, adult CD4-8- thymocytes generated CD4+ or CD8+ peripheral progeny in the proportions found in the normal adult animal (CD4+:CD8+ = 2 to 3). Here we have shown that CD4-8- precursor cells from the 17-day fetal thymus also produced peripheral lymphocytes with low CD4+:CD8+ ratios. Precursors from full term fetuses produced slightly higher CD4+:CD8+ ratios (1.1-1.6) and precursors from animals three to 4 days post-birth achieved CD4+:CD8+ ratios intermediate between those produced by fetal and adult CD4-8- thymocytes. Parallel changes in the production of alpha beta TCR+ peripheral progeny were observed. Fetal CD4-8- thymocytes generated fewer alpha beta TCR+ progeny than did adult CD4-8- thymocytes. However, peripheral lymphocytes arising from either fetal or adult thymic precursors showed similar proportions of gamma delta TCR+ cells. The same pattern of progeny was observed when fetal CD4-8- thymocytes matured in an adult or in a fetal thymic stromal environment. In contrast to fetal thymic precursors, fetal liver T cell precursors resembled adult CD4-8- thymocytes by all parameters measured. These results suggest that fetal thymic precursors are intrinsically different from both adult CD4-8- thymocytes and fetal liver T cell precursors. Moreover, they lead to the hypothesis that the composition of the peripheral T cell compartment is developmentally regulated by the types of precursors found in the thymus. A model is proposed in which migration of adult-like precursors from the fetal liver to the thymus approximately at birth triggers a transition from the fetal to the adult stages of intrathymic T cell differentiation.     

小鼠胸腺上皮细胞系MTEC1产生30kDr MCP—1类趋化因子

A medullary type mouse thymic epithelial cell line MTEC1 can produce chemokines. Crude culture supernant of MTEC1 cells was concentrated by controlled-pore glass beads. One of these chemotactic proteins was isolated from MTEC1 supernatant and purified to homogeneity by heparin-Sepharose affinity chromatography, cation-exchange FPLC and Reverse Phase-HPLC. The chemotactic factor for both lymphocytes and monocytes was identified as a 30 kD protein by SDS-PAGE analysis under reducing conditions. After cleavage of the NH2-terminally blocked protein with formic acid, the amino acid sequence of the internal fragment was analyzed and found to be identical to the amino acid sequence of mouse MCP-1/JE. The protein was hence identified as a glycosylated MCP-1/JE-like chemokine secreted by thymic epithelial cells. The characterization of chemokines produced by thymic stromal cells will benefit the analysis of the mechanism of thymus homing and the finding of new chemokines.     

ChT1, an Ig superfamily molecule required for T cell differentiation

The thymus is colonized by circulating progenitor cells that differentiate into mature T cells under the influence of the thymic microenvironment. We report here the cloning and function of the avian thymocyte Ag ChT1, a member of the Ig superfamily with one V-like and one C2-like domain. ChT1-positive embryonic bone marrow cells coexpressing c-kit give rise to mature T cells upon intrathymic cell transfer. ChT1-specific Ab inhibits T cell differentiation in embryonic thymic organ cultures and in thymocyte precursor cocultures on stromal cells. Thus, we provide clear evidence that ChT1 is a novel Ag on early T cell progenitors that plays an important role in the early stages of T cell development.     

Thymic lymphocytes. III. Cooperative phenomenon in the proliferation of thymocytes under Con A stimulation

In the present paper, the response of thymocytes to Con A is analyzed in terms of a cooperative phenomenon between medullary thymocytes, cortical thymocytes, thymic accessory cells, and interleukin 2. Medullary thymocytes respond spontaneously to Con A and produce IL-2. The addition of exogenously produced IL-2 enhances their proliferation. Small numbers of cortical (PNA+) thymocytes do not respond to Con A, even in the presence of IL-2-containing supernatant. By increasing the number of PNA+ cells per well, sensitivity to Con A and IL-2 appears. This response may be linked either to the increase in a minor PNA+-responding population and/or to the enhanced contamination by medullary thymocytes and macrophages in non-responding PNA+ thymocyte population. In this hypothesis, either the contaminating cells respond by themselves and/or cooperate with PNA+ cells to induce their proliferation. Coculture of non-responding low numbers of PNA+ thymocytes with Con A- and IL-2-containing supernatant in the presence of PNA- cells containing thymic medullary thymocytes and macrophages always produces a higher response than that of each individual population. These results show that a cooperative phenomenon occurs in the cocultures of PNA+ and PNA- thymic cells. We can show using PNA+ and PNA- thymocytes with different Thy 1 alleles, that indeed both PNA+ and populations participate PNA-thymocytes with different Thy 1 alleles, that indeed both PNA+ and PNA- populations participate in the generation of proliferating cells. We can demonstrate, by lysis experiments with monoclonal antibodies and complement that at the end of coculture, most of the proliferating cells are Lyt 1+, and part are Lyt 2+ or L3T4+. We discuss the fact that the phenotype of the cells after activation does not allow us to deduce the phenotype of their precursors. Lysis of Ia+ cells prior to coculture, reduces the level of the proliferative response but does not modify the percentage of cooperation produced by the coculture. Cooperation with medullary mature thymocytes or the presence of active Ia- accessory cells possibly able to convert to Ia expression during coculture experiments may account for these results.     

小鼠胸腺基质细胞系分泌的趋化因子的分析

利用Boyden小室法和FACS分析法,我们分析了五株小鼠胸腺基质细胞系(MTSC)培养上清液对中性粒细胞,单核巨噬细胞和淋巴细胞的化学趋化因子(Chemokines)活性,及定向迁移的淋巴细胞中B细胞、CD 4~ CD 8~-和CD4~-CD8~ T细胞的比例。结果显示,五株MTSC的培养上清液对上述靶细胞均有不同程度的趋化作用.MTSC细胞分泌趋化因子的情况可分为三类:1.MTEC 1和MTEC 2产生的Chemokine(s)对中性粒细胞和淋巴细胞的趋化作用相对较强;2.MTDC 4分泌的Chemokine(s)主要作用于单核巨噬细胞;3.MTEC 3和MTEC 5分泌的Chemokine(s)对多种类型的靶细胞,包括中性粒细胞、单核巨噬细胞和淋巴细胞,表现的趋化作用没有明显的强弱之分。MTSC-SN对B细胞的趋化活性普遍高于对T细胞的趋化活性,对CD 4~-CK 8~ T细胞的趋化活性高于对CD 4~ CD 8~-T细胞的趋化活性。MTSC-SN中趋化因子的分析,有利于新型chemokines的发现及其生物功能的阐明,并可进一步研究Chemok-ine(s)在T细胞发育中的作用。     

小鼠胸腺上皮细胞系MTEC1产生30kD的MCP-1类趋化因子

髓质型小鼠胸腺上皮细胞系MTEC1自发分泌趋化因子(Chemokines),细胞培养上清先经CPG珠(Controlled-pore glass beads)初步浓缩纯化,再依次经肝素-Sepharose柱亲和层析、阳离子交换FPLC及RP-HPLC分离纯化,得到了一个30kD的蛋白质分子,对淋巴细胞和单核巨噬细胞均有趋化活性。此蛋白分子N端封闭,因而先经甲酸水解得到蛋白片段,分析了其中24个氨基酸的序列,发现其与小鼠MCP-1/JE(Monocyte Chemoattractant Pro-tein-1)完全相同。以上结果说明,MTEC1产生的是mMCP-1/JE类趋化因子。     

Neonatal infection with mouse thymic virus. Differential effects on T cells mediating the graft-versus-host reaction.

Neonatal infection with mouse thymic virus (TA), a murine herpes virus, produced extensive but temporary necrosis of the thymus which was maximal at 10 to 14 days of age. Studies of precursor and amplifier cells mediating graft-vs-host (GVH) reactivity of thymocytes, spleen cells (SC), and lymph node cells (LNC) of normal and TA-infected mice were made at 4 and 8 weeks of age. Infection with TA resulting in a profound reduction (70 to 80%) in the direct GVH reactivity of thymocytes at both ages; by comparison, the capacity of thymocytes to produce synergy when combined with normal LNC was normal at 8 weeks. Direct GVH reactivity of SC was depressed 90% 4 weeks after infection with TA but returned to near normal at 8 weeks. Direct GVH reactivity of LNC from TA-infected mice was normal at 4 and 8 weeks of age, but amplifier T cell activity in LNC was markedly depressed at 8 wekks. These results demonstrate that TA has highly selective effects upon subpopulations of T cells in thymus and lymph node.     

CD45R as a primary signal transducer stimulating IL-2 and IL-2R mRNA synthesis by CD3-4-8- thymocytes

CD45R is a high molecular weight (p205/220) form of a series of transmembrane glycoproteins, collectively known as CD45 and present in some form on all lymphoid cells. We have proposed that CD45R+ thymocytes, a minority (15 to 30%) of total thymocytes, represent the generative thymic lineage whereas CD45 p180+ thymocytes are destined for intrathymic death. To test this hypothesis, we prepared human thymus fractions enriched for the expression of CD45R by exhaustive depletion of CD45 p180+ cells, as well as progenitor CD3-4-8- "multinegative" thymocytes which are predominantly CD45R+. Northern analysis of RNA extracted from CD45 p180- and multinegative thymus fractions demonstrated that these populations are enriched for cells able to synthesize mRNA encoding IL-2 and IL-2R after mitogenic stimulation, as compared to unfractionated thymus, consistent with the properties expected for generative thymocytes. Postulating that the CD45R glycoprotein might represent an important signal delivery molecule, we analyzed the ability of mAb specific for CD45 epitopes to synergize with suboptimal amounts of PHA and PMA in the stimulation of IL-2 mRNA production by multinegative thymocytes. We found that CD45R-specific mAb synergizes strongly with PHA/PMA to stimulate IL-2 and IL-2R mRNA expression. In contrast, mAb to CD45 common determinants were unable to synergize. Multinegative thymocytes depleted of all CD45 p180+ cells were compared to total multinegative cells and found to synthesize fourfold greater levels of IL-2 mRNA after stimulation with anti-CD45R mAb. This CD45 p180- multinegative subset is enriched for cells expressing a high density of CD45R, and for CD45- thymus cells, suggesting a possible enrichment for nonlymphoid cells which may play a role in the stimulation process. Our results suggest that the extended amino acid insert of CD45R plays a fundamental role in transmembrane signalling, and that CD45R may be a primary signal transducer for developing thymic progenitor cells.     

Deficiency of the Metalloproteinase-Disintegrin ADAM8 Is Associated with Thymic Hyper-Cellularity

Background

Thymopoiesis requires thymocyte-stroma interactions and proteases that promote cell migration by degrading extracellular matrix and releasing essential cytokines and chemokines. A role for several members of the A Disintegrin and Metalloprotease (ADAM) family in T cell development has been reported in the past.

Methodology/Principal Findings

Here, we present data indicating that the family member ADAM8 plays a role in thymic T cell development. We used qrtPCR on FACS sorted thymic subsets together with immunofluorescene to analyze thymic ADAM8 expression. We found that ADAM8 was expressed in murine thymic stromal cells and at lower levels in thymocytes where its expression increased as cell matured, suggesting involvement of ADAM8 in thymopoiesis. Further flow cytometry analysis revealed that ADAM8 deficient mice showed normal development and expansion of immature thymocyte subsets. There was however an intrathymic accumulation of single positive CD4 and CD8 T cells which was most noticeable in the late mature T cell subsets. Accumulation of single positive T cells coincided with changes in the thymic architecture manifest in a decreased cortex/medulla ratio and an increase in medullary epithelial cells as determined by histology and flow cytometry. The increase in single positive T cells was thymus-intrinsic, independent of progenitor homing to the thymus or thymic exit rate of mature T cells. Chemotaxis assays revealed that ADAM8 deficiency was associated with reduced migration of single positive thymocytes towards CCL21.

Conclusions/Significance

Our results show that ADAM8 is involved in T cell maturation in the medulla and suggest a role for this protease in fine-tuning maturation of thymocytes in the medulla. In contrast to ADAM10 and ADAM17 lack of ADAM8 appears to have a relatively minor impact on T cell development, which was unexpected given that maturation of thymocytes is dependent on proper localization and timing of migration.     

Thymus cell maturation: II. Differentiation of three “mature” subclasses in Vivo

Several thymus cell subclasses may be defined on the basis of their sedimentation velocity, their light-scattering properties (a measure of cell volume), or binding of a fluoresceinated anti-Thy 1.2 antiserum. Using a multiparameter fluorescence-activated cell sorter (FACS), cells with distinguishable light-scattering or fluorescence intensity (after staining with fluorescein anti-Thy 1.2) were separable for analysis of intrathymic maturation pathways. Outer thymic cortical large and medium lymphocytes were the only cells labeled within 1 hr after transcapsular diffusion of administered [³H]thymidine. These labeled cells were also entirely contained in the brightest fluorescence intensity (with fluorescein anti-Thy 1.2) subclass. Under conditions of [¹H] thymidine “chase” in vivo, label shifted proportionately and apparently in parallel to three “mature” subclasses: (1) small thymocytes with high surface concentrations of Thy 1.2, representing ~ 80% of all thymus cells; (2) slightly larger cells, with very low surface Thy 1.2, which are indistinguishable from cortisone-resistant thymocytes, and which make up less than 10% of all thymus cells; (3) dead or fragile cells.