Evidence for elevated prothymocyte activity in the bone marrow of New Zealand Black (NZB) mice. Elevated prothymocyte activity in NZB mice.

New Zealand Black (NZB) mice spontaneously develop an autoimmune syndrome similar to that of systemic lupus erythematosus. Numerous abnormalities in T lymphocyte development have been reported in NZB mice, and the autoimmune syndrome can be adoptively transferred to naive recipients using bone marrow cells. In the present study, we have used an adoptive transfer system to study quantitatively the relative prothymocyte activity of marrow in either young (4-6 weeks of age) or older (8-10 months of age) NZB mice. Our results demonstrate that NZB marrow has approximately 7-fold more prothymocyte activity than that of marrow in SEA mice, a histocompatible, non-autoimmune prone normal strain of mice. This was ascertained by a competitive repopulation assay, in which mixtures of NZB and SEA prothymocytes were compared directly for their ability to repopulate the thymus of adoptive recipients. This increase in prothymocyte activity in the primary recipient of NZB marrow was associated with an increased competitive advantage of NZB marrow prothymocytes over that of SEA marrow prothymocytes in repopulating the hemopoietic (bone marrow) compartment of the primary host. These findings suggest that elevated prothymocyte activity in NZB mice, along with our previously presented evidence for abnormalities of thymocytopoiesis in NZB mice, may be important in their predisposition for autoimmunity.     

Functional demonstration of intrathymic binding sites and microvascular gates for prothymocytes in irradiated mice

Quantitative intrathymic (i.t.) and i.v. adoptive transfer assays for prothymocytes show strict log dose saturation kinetics, consistent with a finite number of i.t. binding sites (microenvironmental niches). This inference is supported here by demonstration of competitive antagonism obeying one-on-one receptor occupancy kinetics during the establishment of thymic chimerism in irradiated adult mice. The results of primary and secondary transfer experiments suggested that hematogenous precursors (i) enter specific i.t. niches between 4 and 24 h after injection, (ii) compete reversibly with subsequently introduced precursors, (iii) establish insurmountable competition within 5-7 days, (iv) mature through the initial stages of thymocytopoiesis preceding proliferative expansion, and (v) vacate the niches between 7 and 14 days after entry. The results also suggested that, as in non-irradiated mice, prothymocyte importation in irradiated mice is a gated phenomenon. Gate closure was indicated by the inability of i.v.-, but not i.t.-, injected bone marrow (BM) cells to induce thymic chimerism when administered 7--14 days after a primary injection and gate opening by the ability of i.v.-injected BM cells to induce thymic chimerism in competition with circulating host prothymocytes. Gate closing was log dose-responsive and could be induced in individual thymic lobes by unilateral i.t. injection, whereas gate opening, which occurs bilaterally, was not initiated until most of the niches for prothymocytes had been vacated. We therefore posit the existence of a series of associated microvascular gates and microenvironmental niches that act in concert to regulate prothymocyte importation and early thymocyte differentiation.     

Interleukin-2 and coculture with thymic epithelial cells synergistically induce prothymocyte differentiation and proliferation

T cell precursors or prothymocytes present in the spleen and bone marrow of mice lack the differentiation antigens found on mature thymocytes. Incubation of prothymocytes with the hormone products of thymic epithelial cells can trigger induction of antigens like Thy 1.2. We have recently found that interleukin-2 will also induce Thy 1.2. We have found that a 4 day incubation of prothymocytes (derived from the spleens of nu/nu mice) with natural and recombinant interleukin-2 in the presence of human thymic epithelial cells results in their synergistic effect on prothymocyte differentiation as measured by Thy 1.2 induction using a fluorescence activated cell sorter (FACS). This coculture condition also caused a synergistic enhancement of proliferation of the prothymocytes and the appearance of a population of large lymphoblasts. Analysis of the presence of Thy 1.2 surface antigen indicated that induction of this surface antigen occurs on both medium sized and large lymphoblast populations. Under these conditions induction of Ly 2 surface antigen was not detected and prothymocytes at the end of the coculture did not manifest IL-1 or mitogen responsiveness. Incubation of prothymocytes in the presence of thymic epithelial cells alone resulted in a lesser degree of Thy 1.2 induction and proliferation and in the consistent induction of low levels of interleukin-2 (0.5-1 unit/ml). Thymosin fraction V did not have a synergistic effect with IL-2 on the cell proliferation. The enhanced response of prothymocytes to IL-2 in the presence of thymic epithelial cells presumably results from the enhanced induction of IL-2 receptors and/or responsiveness. The observation offers further support for a role of interleukin-2 in the regulation of T cell ontogeny.     

Chemokines define distinct microenvironments in the developing thymus

During thymus development, prothymocytes home to the thymus where they migrate as maturing thymocytes from the cortex to the medulla. Chemotaxis assays show that developing T cells of newborn mice respond to certain chemokines depending on their differentiation state. In situ expression analyses indicate that the same chemokines are expressed in distinct microenvironments within the thymic stroma. Expression of chemokines is regulated temporally during embryogenesis; in the alymphoid early thymic anlage, only TECK, SDF-1 and SLC but not ELC, MDC or TARC are expressed. Fetal blood prothymocytes destined to colonize the thymus respond to the embryonic chemokines TECK and SDF-1 in chemotaxis assays with high efficacy. The in vivo significance of this finding is demonstrated by studies in the nude mouse where the thymic anlage lacks TECK and SDF-1 expression and prothymocytes home to the parathyroid anlage rather than to the thymic anlage. Developing thymocytes respond to chemokines expressed in distinct microenvironments within the thymic stroma in a way that correlates well with the previously observed migration pattern from cortex to medulla. The complexity of these chemokine-defined microenvironments increases as the thymic anlage develops to a mature thymus.     

Thymic involution in viable motheaten (me(v)) mice is associated with a loss of intrathymic precursor activity.

Mice homozygous for the viable motheaten (me(v)) allele manifest abnormalities in thymocytopoiesis, are severely immunodeficient, and develop autoimmune disorders early in life. Premature thymic involution occurs in me(v)/me(v) mice, and their bone marrow prothymocytes are unable to repopulate the thymus of adoptive recipients following intravenous (i.v.) transfer. However, analysis of thymocytopoiesis following intrathymic (i.t.) adoptive transfer of bone marrow from me(v)/me(v) mice demonstrates the presence of normal numbers of prothymocytes. To investigate intrathymic development in me(v)/me(v) mice, we determined intrathymic precursor cell number and activity. Dual labeling analyses showed that an involuted me(v)/me(v) thymus is relatively enriched (fivefold) in CD4-CD8- thymocytes (intrathymic precursor phenotype) compared with wild-type (+/+) thymus. However, thymocytes from me(v)/me(v) mice were deficient in precursor activity when adoptively transferred i.t. into irradiated recipients. Thymocytes recovered from the involuted thymus of aged or steroid-treated normal mice also displayed reduced precursor activity. However, the phenotypic profile of thymocyte subsets from steroid-treated mice was enriched in single positive cells (mature phenotype) and was distinctly different from the subset distribution of thymocytes in me(v)/me(v) and aged mice. These results suggest that intrathymic precursor activity in me(v)/me(v) mice is decreased, and may be reflective of decreased prothymocyte seeding to the thymus in vivo. In addition, the results suggest that the thymic involution in me(v)/me(v) mice is not due solely to effects of corticosteroids.     

Recruitment of adult thymic progenitors is regulated by P-selectin and its ligand PSGL-1

The molecular mechanisms that direct the migration of early T lymphocyte progenitors to the thymus are unknown. We show here that P-selectin is expressed by thymic endothelium and that lymphoid progenitors in bone marrow and thymus bind P-selectin. Parabiosis, competitive thymus reconstitution and short-term homing assays indicated that P-selectin and its ligand PSGL-1 are functionally important components of the thymic homing process. Accordingly, thymi of mice lacking PSGL-1 contained fewer early thymic progenitors and had increased empty niches for prothymocytes compared with wild-type mice. Furthermore, the number of resident thymic progenitors controls thymic expression of P-selectin, suggesting that regulation of P-selectin expression by a thymic 'niche occupancy sensor' may be used to direct progenitor access.     

Defective T-cell differentiation in acquired immune deficiency syndrome (AIDS)

A decline in T-cell lymphocyte number is the central characteristic of acquired immune deficiency syndrome (AIDS). The reason for the loss of these cells is not well understood. We investigated the hypothesis that defects in T-cell differentiation contributed to T-cell loss using anin vitro colony assay that measures T-cell precursor (CFU-T) frequency. The results indicate a substantial generalized decrease in CFU-T in people with AIDS (P<0.01), most of whom have Kaposi's sarcoma, and an occasionally severe decrease in CFU-T in people with ARC. Some of the cells from low colony formers suppressed colony formation by control cells. In addition, plasma from people with AIDS was less supportive of colony growth than control plasma. Decreased Ia expression on adherent mononuclear cells did not correlate with colony formation. A defect in T-cell repopulation can help explain the loss of T cells associated with AIDS.     

Prothymocytes in mouse fetal liver

Prothymocytes in fetal liver were characterized in an in vivo thymus regeneration assay. The frequency of prothymocytes in fetal liver is about 10.7% of that determined in normal bone marrow cells whereas the frequency of colony forming unit-spleen (CFU-S) in fetal liver is 24.4% of that in normal bone marrow cells. The lower level of prothymocytes leads to a delay in the development of thymus derived cells after transplantation of fetal liver cells when compared to bone marrow transplantation. There were no significant changes in the frequencies of both CFU-S and prothymocytes between 12 and 16 days from gestation. Fetal liver prothymocytes and CFU-S have a low (1.065 g X cm-3) but similar buoyant density when compared to their density in bone marrow cells (1.070 g X cm-3). These results reflect the close relationship between prothymocytes and CFU-S and might explain the differences in the development of the cellular immune system after transplantation of bone marrow and fetal liver cells.     

Multilineage embryonic hematopoiesis requires hypoxic ARNT activity

Although most cells undergo growth arrest during hypoxia, endothelial cells and placental cytotrophoblasts proliferate in response to low O(2). We demonstrate that proliferation of embryonic multilineage hematopoietic progenitors is also regulated by a hypoxia-mediated signaling pathway. This pathway requires HIF-1 (HIF-1alpha/ARNT heterodimers) because Arnt(-/-) embryoid bodies fail to exhibit hypoxia-mediated progenitor proliferation. Furthermore, Arnt(-/-) embryos exhibit decreased numbers of yolk sac hematopoietic progenitors. This defect is cell extrinsic, is accompanied by a decrease in ARNT-dependent VEGF expression, and is rescued by exogenous VEGF. Therefore, "physiologic hypoxia" encountered by embryos is essential for the proliferation or survival of hematopoietic precursors during development.     

Persistence of stem cell activity within the murine thymus after transfer of a bone marrow fraction enriched in CFU-S

Mechanisms involved in prothymocyte migration, differentiation and self-commitment were investigated. We used a murine bone marrow fraction isolated on a discontinuous Ficoll gradient and enriched 10-20 times in CFU-S activity, and studied its fate after intrathymic transfer over a period of 200 days. In order to assess their hemopoietic activity, chimeric thymuses were intravenously transferred to secondary lethally irradiated hosts and both day 8 and day 12 spleen colonies were evaluated. The results show that transfer of a stem cell enriched fraction leads to long-term repopulation of the thymuses and that the input of progenitors is regulated by the size of the intrathymic precursor pool. Furthermore, stem cells can locate within the irradiated thymus and remain in a primitive stage for several months.     

Expression of a CD3epsilon transgene in CD3epsilon(null) mice does not restore CD3gamma and delta expression but efficiently rescues T cell development from a subpopulation of prothymocytes

The TCR-associated CD3 complex consists of four subunits, i.e. CD3gamma, delta, epsilon and zeta, which are expressed very early in T cell development prior to the expression of the TCR and the pre-TCR alpha chain. It is unclear whether the expression of each CD3 protein is independent of, or is influenced by, other CD3 subunits. To study whether CD3epsilon regulates expression of CD3gamma and delta genes, we generated a strain of CD3epsilon-deficient mice termed CD3epsilon(deltaP/deltaP) (epsilon(deltaP)), in which the promoter of CD3E was disrupted, and subsequently reconstituted these mice with a CD3epsilon transgene. In the epsilon(deltaP) mice, T cell development is arrested at the double-negative stage and targeting the CD3epsilon gene caused severe inhibition of CD3gamma and delta gene expression. Introduction of the CD3epsilon transgene did not restore CD3gamma and delta expression. However, a very small fraction of prothymocytes that expressed CD3gamma and delta was rescued upon reconstitution of the CD3epsilon transgene. Remarkably, this rescue led to a very efficient differentiation and maturation of thymocytes, resulting in a significant T cell population in the periphery. These results demonstrate that CD3epsilon does not regulate expression of CD3gamma and delta genes, and underscore the capacity of each prothymocyte to give rise to a large number of mature peripheral T cells.      

Step-wise divergence of primitive and definitive haematopoietic and endothelial cell lineages during embryonic stem cell differentiation

BACKGROUND: The developmental processes leading from the mesoderm to primitive and definitive haematopoietic and endothelial lineages, although of great importance, are still poorly defined. Recent studies have suggested a model in which common precursors give rise to endothelial progenitors and haematopoietic progenitors, the latter subsequently generating both primitive and definitive haematopoietic lineages. However, this model is contradicted by findings that suggest the emergence of haematopoietic cells from the endothelial lineage. RESULTS: We found sequential steps in the differentiation of FLK1+ mesoderm into haematopoietic and endothelial lineages in an in vitro differentiation system of embryonic stem (ES) cells: (i) the GATA-1+ subset of FLK1+ mesodermal cells loses the capacity to give rise to endothelial cells and is restricted to primitive erythroid, macrophage and definitive erythroid progenitors; (ii) the remaining GATA-1- cells give rise to VE-cadherin+ endothelial cells; and subsequently (iii) multiple definitive haematopoietic progenitors and endothelial cells branch off from a subset of VE-cadherin+ cells. CONCLUSIONS: These observations strongly suggest that the divergence of primitive and multilineage definitive haematopoietic/endothelial lineages occurs first, and then multilineage definitive haematopoietic progenitors arise from VE-cadherin+ endothelial cells in the development of haematopoietic and endothelial cells.     

The entry of the prothymocyte into the thymus after lethal irradiation and bone marrow transplantation. II. Time of entry

The time of entry of prothymocytes into the thymus after lethal irradiation and bone marrow transplantation (BMT) was determined by exposing the thymus only or the whole body with the thymus shielded to a second irradiation after different intervals. The repopulation of the thymus by donor type cells was determined by a thymus repopulation assay using donor specific markers. Reirradiation of the thymus kills the prothymocytes that have entered the thymus during the interval. It was found that reirradiation of the thymus from 48 hours after BMT onwards increasingly delayed thymus regeneration. This shows that donor prothymocytes do not enter the thymus until about 2 days after BMT and that they continue to do so during at least three subsequent days. In the second reirradiation protocol thymus regeneration occurred earlier in the shielded thymus than in thymuses of whole body irradiated mice. Earlier thymus regeneration was not seen in mice that were reirradiated at 24 hours after BMT, but occurred only when irradiation took place at 48 hours and later. These data are consistent with those obtained in the first protocol. The results are in contradiction with results of direct homing experiments, which showed entrance of donor cells within 3 hours after BMT. Our functional assay demonstrated that the early appearing cells cannot be prothymocytes. In retransplantation experiments it was shown that the bone marrow (BM) may indeed be the initial homing site of prothymocytes.     

T cell involvement in the decrease of antigen-responsive B cells in aged mice

The role of T cells in the reduced frequency of splenic B cells specific for several antigens in aged mice was studied by assessing B cell responsiveness in (a) aged nude mice and (b) irradiated young mice repopulated with splenic B cells or with Ig- bone marrow cells from young mice and T cells from aged vs. young mice. Using the fragment culture technique to assess B cells specific for 2,4-dinitrophenyl (DNP) and for (4-hydroxy-3,5-dinitrophenyl) acetyl, we found that the frequency of responsive splenic B cells in aged BALB/c nude mice was very similar to that of young nude mice. In addition, we found that in chimeric mice constructed with either bone marrow or splenic B cells from young mice and T cells from aged mice the frequency of DNP-specific splenic B cells was significantly lower than that in control chimeras constructed with T cells from young mice. These results indicate that T cells from aged mice can down regulate B cell responsiveness and that a mature, naive B cell may be its possible target. The results of both experimental approaches are consistent with a role for T cells in the regulation of responsive B cells in aging.     

The entry of the prothymocyte into the thymus after lethal irradiation and bone marrow transplantation. I. Seeding of bone marrow cells into the thymus

Bone marrow (BM) cells arrive in the thymus of lethally irradiated mice as early as three hours after bone marrow transplantation (BMT). They can be recognized by labeling of the injected cells with Hoechst 33342 (direct homing assay). In order to relate the immigrated BM cells to thymocyte precursor cells, direct homing and thymus repopulation experiments were compared. It was shown that homing of BM cells depends on the time between lethal irradiation and BMT, while it was previously shown that thymus repopulation does not. In addition, thymic immigrants were smaller than precursor cells committed to the T cell limeage (prothymocytes) and their progenitors. A cell population obtained from normal BM cells and enriched in stem cells (purified stem cells) was previously shown to repopulate the thymus similarly as BM cells from mice pretreated in vivo with 5-fluorouracil (FUBM). Both cell suspension showed a delayed thymus repopulation when compared to normal BM. This is indicative for a depletion of prothymocytes in these cell suspensions. In the direct homing assay, however, it was found that relatively many cells from FUBM seeded into the thymus, while purified stem cells did not. These results indicate that most if not all donor cells that are present in the thymus at three hours after BMT are not thymocyte precursor cells.     

KLF17对裸鼠移植瘤生长的影响及其在体调控靶基因的筛选和功能分析

目的:研究Krüppel样因子17(Krüppel-like factor 17,KLF17)在裸鼠移植瘤中的作用并分析KLF17在体调控的靶基因及其功能和参与的信号通路。方法:慢病毒转染技术稳定上调人肺腺癌A549细胞及下调人肺腺癌H322细胞中KLF17的表达。11只BLAB/c nu/nu裸鼠分为上调组5只和下调组6只,分别通过左、右侧躯体皮下注射KLF17上调和下调的相应细胞及其对照细胞,观察KLF17对裸鼠移植瘤生长的影响;Real-time PCR及免疫组织化学染色分析裸鼠移植瘤组织中KLF17 mRNA及蛋白的表达,转录组测序技术分析上调KLF17表达后裸鼠移植瘤中差异表达的基因,通过The Cancer Genome Atlas(TCGA)数据库分析KLF17调控的差异基因的功能,Gene Ontology和KEGG PATHWAY富集分析差异基因的功能和可能参与的信号通路。结果 :上调A549细胞中KLF17的表达后,其裸鼠移植瘤生长速率显著低于空载体对照组(P0.05),下调H322细胞中KLF17表达后,其裸鼠移植瘤生长速率及移植瘤重量均显著高于空载体对照组(P0.01,P0.05)。在裸鼠移植瘤组织中,过表达组KLF17 mRNA及蛋白显著高于对照空载体组。转录组测序结果显示KLF17可能调控的基因有ras基因同源家族成员V(ras homolog family member V,RHOV)和冠蛋白1C(coronin 1C,CORO1C)等。TCGA数据库中肺腺癌患者10年累计生存时间在RHOV和CORO1C mRNA不同表达组明显不同,高表达RHOV及CORO1C的肺腺癌患者生存时间显著低于其低表达组。Gene Ontology和KEGG PATHWAY富集分析提示KLF17调控的靶基因(差异基因)可能与肿瘤细胞的刺激应答、生长及黏附有关,并且参与了细胞趋化性、黏附及细胞外基质受体相关的信号通路。结论:KLF17抑制裸鼠移植瘤的生长,并下调RHOV和CORO1C等癌基因,参与调控肿瘤黏附及生长相关信号通路。     

Fibroblast growth factor-2 maintains a niche-dependent population of self-renewing highly potent non-adherent mesenchymal progenitors through FGFR2c

Bone marrow (BM) mesenchymal stem/stromal cells (MSC) are a heterogeneous population of multipotent progenitors currently under investigation for a variety of applications in regenerative medicine. While self-renewal of stem cells in different tissues has been demonstrated to be regulated by specialized microenvironments called niches, it is still unclear whether a self-renewing niche also exists for MSC. Here, we show that primary human BM cultures contain a population of intrinsically non-adherent mesenchymal progenitors (NAMP) with features of more primitive progenitors than the initially adhering colony-forming units-fibroblast (CFU-f). In fact, NAMP could generate an adherent progeny: (a) enriched with early mesenchymal populations (CD146+, SSEA-1+, and SSEA-4+); (b) with significantly greater proliferation and multilineage differentiation potential in vitro; and (c) capable of threefold greater bone formation in vivo than the corresponding CFU-f. Upon serial replating, NAMP were able to regenerate and expand in suspension as non-adherent clonogenic progenitors, while also giving rise to an adherent progeny. This took place at the cost of a gradual loss of proliferative potential, shown by a reduction in colony size, which could be completely prevented when NAMP were expanded on the initially adhering BM fraction. Mechanistically, we found that NAMP crucially depend on fibroblast growth factor (FGF)-2 signaling through FGFR2c for their survival and expansion. Furthermore, NAMP maintenance depends at least in part on humoral signals distinct from FGF-2. In conclusion, our data show a niche/progenitor organization in vitro, in which the BM adherent fraction provides a self-renewing microenvironment for primitive NAMP.     

Rearrangements of T-cell antigen receptor gamma and delta chain genes are detected in the long-term cultured bone marrow cells of athymic nude mice but not in those of euthymic mice.

We have previously shown that extrathymic rearrangements of T-cell receptor (TcR) gamma and delta chain genes occur in the peripheral lymphoid tissues of athymic nude mice. To further determine where the TcR gene rearrangements occur in nude mice, we investigated the rearrangement and expression of the TcR genes in the long-term cultured bone marrow (LTBM) cells which were homogenous in developments without mature T cells as assessed by FACS analysis. The LTBM derived from euthymic mice contained TcR gamma and delta chain genes in germline configuration, while gene rearrangements of both locus were detected in the LTBM cells from nude mice. These results suggested that gamma and delta gene rearrangements do occur in the bone marrow cells of nude mice and that the T-cell precursors in bone marrow may be increased in frequency in such animals.     

The generation and fate of thymocytes

In the adult thymus the majority of thymocytes are at a non-proliferating end stage, during which 3% of all CD4+CD8+ cortical thymocytes are selected on the basis of appropriate T-cell antigen-receptor (TcR) specificity to become CD4-CD8+ or CD4+CD8- mature thymocytes. The selected mature cells gradually emigrate to the periphery. The 97% unselected, or positively rejected, CD4+CD8+ thymocytes die in the thymus. This 3-day end stage is, however, the product of around 2 weeks of proliferation by less mature thymocytes, which gives about a 10(5)-fold expansion from the hundred prothymocytes estimated to seed the thymus each day. Rearrangement and expression of TcR genes and a sequence of changes in surface antigens occurs during this prolonged period. Two sequential waves of expansion and differentiation appear to be involved. The first, about 1 week long, involves the less than 0.1% of thymocytes which still resemble the prothymocyte; this leads via a non-dividing interval to the second 1-week stage involving the 3% of CD4-CD8- thymocytes and then the CD4+CD8+ blasts.     

Effect of a Trichinella spiralis infection on the distribution of mast cell precursors in tissues of thymus-bearing and non-thymus-bearing (nude) mice determined by an in vitro assay.

The frequency of precursor cells capable of giving rise to cells with characteristics of mucosal mast cells in tissues from thymus-bearing and non-thymus-bearing (nude) mice orally infected with Trichinella spiralis was determined with an in vitro assay. Analysis of the frequency of mast cell precursors in bone marrow, blood, spleen and small intestinal tissue revealed similar frequencies of mast cell precursors in bone marrow from both thymus-bearing and athymic mice. These frequencies in bone marrow were not affected by infection. However, in blood and spleen from thymus-bearing mice at Day 7 post-infection (p.i.), and in the gut at Day 14 p.i., significant increases of mast cell precursor frequencies were detected. In contrast, no significant increase was observed in the tissues of infected nude mice. These data are in accordance with in vivo findings, indicating that a mucosal mast cell response in the gut is both thymus and antigen dependent. It was concluded that a mucosal mast cell response to infection with T. spiralis is probably due to local proliferation and maturation of residing mast cell precursors, that this response might be amplified by an influx of precursor cells from the blood into the gut, and that both phenomena are T-cell dependent.