Induction of a T-cell specific antigen on bone marrow lymphocytes with thymus RNA.

Expression of a rabbit T-cell specific antigen can be induced on bone marrow lymphocytes following exposure to an RNA extract obtained from the thymuses of young rabbits. The presence of the antigen was demonstrated using goat anti-rabbit T-cell serum in a complement-dependent cytotoxicity assay. The T-cell antigen first appeared 3 h after addition of the thymus RNA to bone marrow cell cultures and the maximum number of cells expressing the T-cell antigen was observed within 24 h. RNA obtained from a source other than the thymus was found to be ineffective in inducing expression of the T-cell antigen. The induction of the antigen appears to be dependent on the presence of intact thymus RNA, as RNase treatment but not trypsin treatment, destroyed the ability of the RNA to induce the T-cell antigen.     

The humoral immune response of mouse bone marrow lymphocytes in vitro.

Mouse bone marrow (BM) small lymphocytes are shown to contain competent precursors for a primary haemolytic plaque forming cell (PFC) response to heterologous red blood cells and TNP in an in vitro culture system. Their response is dependent on T co-operative factors, which can be provided by irradiated spleen cells activated by concanavalin A or the supernatant of an allogeneic culture, added at the beginning or after 24 h of culture. The frequency of PFC precursors for the response to SRBC is found to be equal or higher in BM than spleen cultures. However, BM lymphocyte cultures stimulated by E. coli lipopolysaccharide show an increase of DNA synthesis but contain only few polyclonal PFC, in contrast to spleen.     

Hidden Thy-1 antigen in a subpopulation of mouse bone marrow cells.

Immunofluorescence and cytolysis studies using antisera against the xeno- and alloantigenic components of the Thy-1 molecule have shown that in adult mouse bone marrow more cells bear the xeno than the alloantigen. Allo+ cells form a subset of a larger, xeno+ population. Xeno+ allo- 'prothymocytes' can be induced to express the alloantigen in vitro; this could result from the unmasking of cryptic antigen, the presence of which is demonstrated, rather than de novo antigen synthesis. Some xeno+ allo- cells are involved in splenic haemopoiesis, either as a subpopulation of precursors, or as accessory helper cells.     

Antibody formation in mouse bone marrow. V. The response to the thymus-independent antigen Ecsherichia coli lipopolysaccharide.

The occurrence of plaque-forming cells (PFC) in mouse bone marrow was studied during primary and secondary responses to the thymus-independent antigen Escherichia coli lipopolysaccharide (LPS). Anti-LPS responses were induced by various doses of LPS. During the primary response, doses of 1 and 10 mug LPS intravenously (i.v.) were found to evoke a distinct PFC response in both spleen and bone marrow. The spleen contained the majority of PFC until about 5 days after immunization. During the course of the reaction the number of PFC in the bone marrow rose to a level which equalled or surpassed the level in the spleen. LPS doses of 0-001, 0-01 and 0-1 mug i.v. only induced a PFC response in the spleen. Apparently there is a minimal threshold dose of LPS of about 1 mug for PFC to appear in the bone marrow. The secondary response was studied in mice primed with 1 mug LPS i.v. and boosted with either 0-001, 0-1 or 10 mug LPS i.v. 3 months later. After each dose tested the PFC activity in the spleen was several times higher than during the primary response. As was observed in the primary response doses of 0-001 and 0-1 mug LPS i.v. did not evoke a PFC response in the bone marrow. After boosting with 10 mug of LPS i.v. a significant PFC response was found in spleen, bone marrow, thymus, lymph nodes, Peyer's patches and blood. From about 5 days after the booster injection the number of PFC in the bone marrow exceeded the total number found in all other lymphoid organs. The results are discussed in relation to the bone marrow PFC response to the thymus-dependent antigen sheep red blood cells. To this antigen a clear PFC response in the bone marrow is found only during the secondary response.     

Effect of age on the proportion of mouse bone marrow cells migrating in response to newborn thymus supernatant. Cell migration and thymus evolution in mice.

The effect of mouse age on the in vitro migration of hemopoietic precursor cells from bone marrow of C57BL/6 to thymic supernatant from newborn mice was studied to determine whether a reduction in the migratory readiness or number of precursor cells might at lest partly explain thymic involution due to aging. The percentage of bone marrow cells migrating to the thymic supernatants increased to age of 7 weeks, and then decreased progressively. Thymus weight underwent almost exactly the same evolution. The decline of the population of migration-ready T-precursor in adult mice may this explain age-related thymic involution.     

The initial characterization of a thymus factor chemotactic to bone marrow cells

Thymus supernatants were produced by cluturing minced newborn CBA/J mouse thymuses in serum-free media for 48 h. Supernatants thus obtained were chemotactic to a subset of bone marrow cells as assessed in blind well chambers, and enriched for immature lymphoid cells in the migrating cell population. The enriched population of cells was shown to be capable of homing to the thymus of an irradiated mouse in vivo in a significantly higher percentage than nonmigrated bone marrow cells. In this report, initial characterization of the factor(s) responsible for this in vitro migration is presented. Several well studied thymic factors were compared with the thymus supernatants for their ability to induce migration of bone marrow cells in vitro. Thymulin (FTS-Zn), FTS, and TP-5 (the pentapeptide fragment of thymopoietin) were used. None of these factors demonstrated chemotactic properties in the migration assay using concentration ranges in which other in vitro activities have been observed. The chemoattractive activity of the supernatant was unaltered by ultracentrifugation. The effects of temperature on the chemotactic properties of thymus supernatant were examined, and a fifty percent decrease in observed migration occurred with thymus supernatant heated to 100 degrees C for 1 h. In addition, incubation of the supernatant for 1 h at 37 degrees C with chymotrypsin, but not with trypsin, inhibited migration, presumably by inactivation of the active factor. Using Amicon microconcentrators, the supernatant was separated into several fractions based on molecular weight. Initial data suggest that the active fraction is in the less than 10,000 mw range.     

Differentiation of functionally active mouse T lymphocytes from functionally inactive bone marrow precursors. III. Induction of T-cell activities by growth of bone marrow on feeder layers prepared from mouse thymocytes.

Four patients with Wiskott-Aldrich syndrome were treated with transfer factor (TF), in an attempt to improve their clinical condition. Before and during treatment, cellular reactivity was followed in vivo (delayed type skin reactivity) and in vitro (lymphocyte transformation). In all patients positive skin reactions were occasionally observed during TF treatment. The lymphocyte reactivity in vitro to phytohaemagglutinin (PHA) was slightly diminished, the responding capacity of the mixed lymphocyte culture (MLC) severely impaired and the response to bacterial, viral and fungal antigens absent before and during TF treatment. No clinical improvement was observed after large doses of TF. No correlation between skin test reversal and TF administration was found, and lymphocyte transformation in vitro did not improve. Subsequently, a double blind trial, in which we compared the effect of TF and placebo, was carried out in these patients, which revealed no effect of TF therapy. It is concluded that the results of treatment with TF in these patients are highly controversial.     

Antibody formation in mouse bone marrow: III. Effects of route of priming and antigen dose

The influence of the route of priming and the dose of sheep red blood cells (SRBC) on the IgM-, IgG- and IgA-plaque-forming cell (PFC) activity in mouse bone marrow during the secondary response to SRBC was studied. After intraperitoneal and subcutaneous priming the number of IgM-, IgG- and IgA-PFC during the secondary response increased with the number of SRBC used for priming. After intravenous (i.v.) priming the priming dose—secondary response relationship was found to be an optimum curve for IgM-PFC as well as for IgG- and IgA-PFC. A peak secondary response in the bone marrow was found after priming with 10⁷ and with 10⁸ SRBC i.v.

The appearance of IgG- and IgA-memory cells in the bone marrow after i.v. immunization with SRBC was shown to be dependent on the priming dose: the appearance of IgG-memory cells required a higher dose of SRBC than the appearance of IgM-memory cells, and the appearance of IgA-memory cells required even more antigen.

The effect of the booster dose upon the PFC activity was studied in mice primed with 10⁷ SRBC i.v. and boosted with 10⁴, 10⁶, or 10⁹ SRBC i.v. 2 months later. IgM-PFC as well as IgG- and IgA-PFC were present in spleen and bone marrow at each booster dose tested. In each group of mice the PFC activity in the bone marrow rose to a level which surpassed the level in the other lymphoid organs between the 7th and 10th day after the booster injection. Thus, independently of the booster dose, mouse bone marrow is the major source of PFC during the late phase of the secondary response to SRBC. Mice boosted with 10⁹ SRBC i.v. showed a prolonged presence of IgM-, IgG- and IgA-PFC in the thymus. Light and electron microscopic studies revealed the presence of plasma cells and macrophages studded with phagocytosed material in the thymic medulla of these mice, thus providing evidence for a real PFC response within the thymus. A thymic PFC response was absent in mice boosted with either 10⁴ or 10⁶ SRBC i.v. Therefore the thymic PFC activity during the secondary response to SRBC does depend on the booster dose.

     

骨髓间充质干细胞移植后在胸腺内的定居

BACKGROUND: Bone marrow mesenchymal stem cells have low immunogenicity and can induce immune tolerance. At present, the mechanism of immune regulation of bone marrow mesenchymal stem cells is not completely understood. It has been rarely reported whether the bone marrow mesenchymal stem cells can migrate to the thymus after transplantation.OBJECTIVE: To observe the distribution and survival of bone marrow mesenchymal stem cells in the thymus of aging rats after transplantation.METHODS:Bone marrow mesenchymal stem cells cultured in vitro were transfected by adenovirus vectors expressing green fluorescent protein. Transfected bone marrow mesenchymal stem cells were injected into the portal vein of aging rats. At days 3, 7, 14, 21 after transplantation, the survival of bone marrow mesenchymal stem cells homing to the thymus was observed under fluorescence microscope. At day 3 after transplantation, thymus tissues were taken and stained with hematoxylin-eosin for pathological observation. RESULTS AND CONCLUSION:Green fluorescent protein-labeled bone marrow mesenchymal stem cells had a strong green fluorescence at days 3 and 7 after transplantation, and the cell contour was clear. There was no significant difference in the mean absorbance values at days 3 and 7 (P > 0.05). Expression of green fluorescent protein was weakened significantly at days 14 and 21 compared with that at day 3 (P < 0.05). At 3 days after transplantation, the transplanted bone marrow mesenchymal stem cells were clearly visible in the thymus, and acute rejection was not observed. The results show that bone marrow mesenchymal stem cells can migrate to the damaged thymus tissue through the blood circulation, and can survive at least 1 week.  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

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.     

Differentiation of functionally active mouse T lymphocytes from functionally inactive bone marrow precursors II. Limited recovery of T-cell responses from mouse bone marrow in tissue culture.

The limited differentiation of mature T cell function from mouse bone marrow in tissue culture is described and compared with similar differentiation occuring in vivo in irradiated bone marrow protected mice. Data are presented to show that a pool of precursors, similar in size to that able to produce early (transient?) regeneration in thymectomized recipients, is responsible for the development of mitogen responsive T cells active in MLC (proliferation) and CML (development of cytotoxic cells) assays. In contrast, a helper cell population which augments antibody formation from T-depleted normal spleen cells derives from a pool of similar precursors yet does not seem to be theta positive. Similarly, larger cells (perhaps typical of those giving rise to suppressor T cells in vivo) give rise to a suppressor cell pool after 4 days of culture, though again only a fraction of this suppressor activity could be attributed to theta positive cells. It is suggested that much of the data for regenration of T lymphocytes in vitro from T-depleted sources needs to be re-interpreted in terms of this evidence for a pool of post-thymic precursors of T cells in such T-deficient cell populations.     

The effects of thymus supernatant on the thymus homing ability of aged murine bone marrow cells.

This study examined the effect of thymus supernatant on the ability of bone marrow cells from senescent mice to home to the thymus. An in vitro and an in vivo assay were employed. The in vitro assay used a blind well migration of aged bone marrow cells to thymus supernatant across a membrane with 5-microns pores. The in vitro assay measured the ability of aged bone marrow to repopulate the thymus of an irradiated host. Our results support previous reports that the bone marrow from old mice has a greatly reduced ability to migrate to thymus supernatant and to repopulate the thymus of an irradiated host. Further, we found that a brief treatment of the old bone marrow with thymus supernatant significantly improved its thymus homing ability both in vitro and in vivo.     

巨噬细胞型骨髓基质细胞对肿瘤抗原提呈的体外实验研究

目的　探讨骨髓基质细胞对肿瘤抗原的提呈功能。方法　小鼠骨髓贴壁细胞经 Ｇ Ｍ Ｃ Ｓ Ｆ 诱导,形成以成熟巨噬细胞为主的基质细胞,用小鼠红白血病细胞 Ｆ Ｂ Ｌ３ 肿瘤抗原刺激,然后再与 Ｆ Ｂ Ｌ３ 肿瘤抗原致敏的 Ｔ 淋巴细胞混合培养。结果　骨髓基质细胞经 Ｆ Ｂ Ｌ３ 肿瘤抗原刺激后, Ｔ Ｎ Ｆα和 Ｉ Ｌ１β的分泌水平明显升高,经抗原预激的骨髓基质细胞能特异性地刺激同种抗原致敏的 Ｔ 淋巴细胞增殖和分泌高水平的 Ｉ Ｌ２ 。单抗阻断试验发现, Ｍ Ｈ ＣⅡ类分子和 Ｂ７２ 分子的联合阻断能有效地抑制致敏 Ｔ 淋巴细胞分泌 Ｉ Ｌ２ 。结论　本实验证实骨髓基质细胞具有抗原提呈功能, Ｍ Ｈ ＣⅡ类分子和 Ｂ７２ 分子在其抗原提呈中发挥了重要作用。     

Differentiation of functionally active mouse T lymphocytes from functionally inactive bone marrow precursors. IV. Recovery of T-cell function from bone marrow precursors in a histo-incompatible environment.

Regeneration of T-cell activities in vivo or in vitro from mouse bone marrow precursors differentiating in the presence of an allogeneic thymus was investigated. The data indicated that T-depleted bone marrow cells fail to affect long-term reconstitution of allogeneic recipients unless a pool of rapidly maturing T-precursor cells is also removed (post-thymic pool). Animals reconstituted with pre-thymic bone marrow stem cells become stable chimaeras in which cells capable of generating an in vitro CML response to host antigens, as well as cells capable of suppressing that response, could be demonstrated. Similar data (CML directed against the H-2 antigens of the 'host' thymus feeder layer and cells capable of inhibiting that response) were obtained when pre-thymic bone marrow cells were grown in vitro on allogeneic thymus feeder cells. When cytotoxic T-lymphocyte precursor (CTLp) and helper (CTLh) cells were separately investigated, a restriction in their co-operation for an anti-host response was observed when precursor cells differentiated in an allogeneic environment. Only CTLp and CTLh differentiating in the presence of the same allogeneic thymus source (whether in vivo or in vitro) could co-operate to generate CTL directed to H-2 antigens of that thymus source.     

Blastogenic response of lymphocytes separated from bone marrow to allogeneic lymphoid cells in vitro

Fractions of cells were separated from the bone marrow and spleen of Lewis rats by brief centrifugation in linear sucrose-serum density gradients and were cultured in vitro either alone or mixed with F₁ (Lewis × Brown Norway) hybrid rat lymphoid cells. After 4 days the incorporation of [³H]thymidine into DNA was measured by scintillation counting, and the morphology and incidence of [³H]thymidine-labelled cells were determined in radioautographs.

Lymphocyte-rich, slowly-sedimenting fractions of Lewis rat bone marrow cells showed increased incorporation of [³H]thymidine and the development of many large proliferating blast-like cells when cultured with F₁ hybrid cells. This blastogenic response was greater than could be ascribed to contaminating intravascular blood lymphocytes. Slowly-sedimenting fractions of spleen cells, consisting mainly of small lymphocytes, showed a greater blastogenic responsiveness to F₁ hybrid cells than that of whole spleen, while rapidly-sedimenting spleen cell fractions, containing many large cells, showed a reduced responsiveness. Paradoxically, rapidly-sedimenting marrow cell fractions containing few small lymphocytes, showed an increment in [³H]thymidine incorporation when cultured with F₁ cells but this was due to active macrophage proliferation as well as to blastogenesis.

The results demonstrate that some parenchymal bone marrow lymphocytes undergo blastogenic transformation in response to allogeneic lymphocytes in vitro.

     

Antibody formation in mouse bone marrow: VI. The regulating influence of the spleen on the bone marrow plaque-forming cell response to Escherichia coli lipopolysaccharide

Mouse bone marrow is capable of a distinct plaque-forming cell (PFC) response after i.v. immunization with the thymus-independent antigen E. coli lipopolysaccharide (LPS). Both during the primary and secondary response to i.v. administered LPS the spleen contained the majority of PFC until about 5 days after immunization. In the course of the reaction the number of PFC in the bone marrow rose to a level which surpassed the level in the spleen. This paper deals with the regulating influence of the spleen on the primary and secondary anti-LPS PFC response in the bone marrow.

Splenectomy prior to the first injection of 5 μg LPS i.v. initially did not affect the bone marrow PFC response. However, at the 7th day after immunization the PFC response in the bone marrow fell to only 10 per cent of the bone marrow PFC activity in sham-splenectomized mice. In contrast to the primary response no regulating influence of the spleen on the bone marrow PFC activity could be demonstrated during the secondary response. The influence of splenectomy on the appearance of B-memory cells in the bone marrow depended on the priming dose. The appearance of LPS-specific B-memory cells in the bone marrow was not affected by splenectomy at priming doses of LPS as high as 1 and 0.1 μg. On the other hand splenectomy before 0.01 μg LPS i.v. reduced, and splenectomy prior to 0.001 μg LPS i.v. completely prevented the appearance of B-memory cells in the bone marrow.

     

Subpopulations of mouse spleen lymphocytes. III. Cellular interactions in the response to concanavalin A.

The profile of response to concanavalin A (con A) of purified mouse T cells was found to differ appreciably from that of non-fractionated spleen cells, in agreement with results previously published by other investigators. Experiments designed to elucidate the reasons underlying these differences have revealed that the response of the spleen cells to con A is determined by a complex interplay between several cell types. (a) B cells contribute to the overall incorporation of thymidine in the presence of con A-stimulated T cells. However, the B cells participate in the response only if the T cells are dividing. (b) A population of 'adherent cells' is present in the spleen, which enhances the stimulation of the spleen cells by low doses of con A but suppresses the response to high doses of mitogen. These adherent cells include most likely the conventional macrophages, but probably also a population of 'suppressor T cells'. (c) Such 'suppressor T' cells can be readily detected among the peritoneal exudate cells. Addition of the exudate cells to cultures of purified T cells enhances the response to low doses of con A. This effect can be further increased by treating the peritoneal cells with a cell T-specific antiserum and complement, i.e. by eliminating the T cells.     

The thymus in patients with allogeneic bone marrow transplants.

The thymus glands from 11 patients with aplastic anemia or acute leukemia who received allogeneic bone marrow transplants were studied at autopsy. All showed marked cortical involution. In the short-term survivors the medulla and perivascular spaces were lymphocyte-depleted and the epithelial cells formed pseudorosettes. In those surviving over 2 months, increasing numbers of small lymphocytes were present, presumably reconstituted with donor lymphocytes. Phagocytosis of cellular debris was frequent, especially in patients with graft-versus-host reaction (GVHR) or treated with anithymocyte globulin (ATG). Plasma cells were numerous in perilobular tissue and were occasionally found within the medulla. The findings are compatible with the concept that the thymus plays an important role in the immune deficiency experienced after allogeneic bone marrow transplantation and in the subsequent lymphoid reconstitution.     

Trafficking from the bone marrow to the thymus: a prerequisite for thymopoiesis

Summary:  T-cell development in the thymus requires periodic importation of hematopoietic progenitors from the bone marrow. Such thymus settling progenitors arise from hematopoietic stem cells (HSCs) that are retained in a specific bone marrow microenvironmental niche. Vacation of this niche is required for HSC proliferation and differentiation into downstream progenitors. In order to reach the thymus, progenitors must then be mobilized from bone marrow to blood. Finally, progenitors in blood must settle in the thymus. Here we review signals and molecular interactions that are likely to play a role in trafficking from the bone marrow to the thymus, focusing on how these interactions may regulate which progenitors physiologically contribute to thymopoiesis.