Immunoregulatory influence of thymic epithelium and thymopoietin on human B-lymphocyte differentiation

Thymic influence upon immunoregulation of B-lymphocyte differentiation in human bone marrow was investigated. Mononuclear cells isolated from marrow of normal adult volunteers were incubated with thymic epithelial monolayers or with the polypeptide thymopoietin. Generation of pokeweed mitogen-stimulated anti-sheep red blood cell antibody-secreting direct plaque-forming cells (PFC) was found to be inhibited following incubation of marrow mononuclear cells with thymic epithelial monolayers. Addition of 50 ng/ml thymopoietin to pokeweed mitogen-stimulated cultures resulted in enhanced marrow PFC responses, whereas higher doses of thymopoietin were inhibitory for the generation of PFC in this assay system. The data suggest that both helper and suppressor T cells are recruited from their precursors in human bone marrow by thymic influences. Generation of helper or suppressor cells may be dependent upon (a) the stage of differentation of precursor T cells and (b) upon the specific action and intensity of the thymic influence.     

Participation of the cyclase system in the molecular mechanisms of differentiation control of immunocompetent cells

The effects of polypeptide thymic and bone marrow factors on the cyclase system of rat thymus and spleen lymphocytes were studied. It was shown that the induction of maturation of rat T-lymphocyte precursors into immunocompetent T-cells under effects of the thymic factor is accompanied by the activation of the adenylate cyclase system and the elevation of the cAMP/cGMP ratio. The observed increase in the cGMP level in splenic lymphocytes suggests the presence of cAMP- and cGMP-dependent steps of in the reaction of T-lymphocyte maturation. The bone marrow factor causes an elevation of the cAMP level only in splenic lymphocytes, which points to differences in the lymphocyte subpopulations that are sensitive to thymus and bone marrow factors. Impairments in T-lymphocyte maturation in patients with immunodeficiencies are concomitant with shifts in the isoenzyme spectrum of lactate dehydrogenase in peripheral blood lymphocytes as well as with changes in the sensitivity of the cAMP system to the thymic factor and isoproterenol. These disturbances are relieved by administration of the thymic factor. The roles of the cyclase system and polypeptide thymic and bone marrow factors in the molecular mechanisms of immunocompetent cell maturation are discussed.     

Enhancement of the immune system of chemotherapy-treated cancer patients by simultaneous treatment with thymic extract,TP-1

Summary Thirty patients with incurable gastrointestinal cancer were treated in a randomized clinical trial, either with combination chemotherapy (5-fluorouracil, vincristine, and methyl-CCNU) or with the same chemotherapy and the thymic extract TP-1. T-lymphocyte counts slowly and steadily, increased in the TP-1-treated patients over a period of 4–5 months, in contrast to decreasing counts in the patients treated by chemotherapy only. The changes in both groups were significantly different from pretreatment counts (P<0.01). Skin tests for primary sensitization with DNCB were converted to positive or improved after TP-1 treatment (P<0.05 against control patients). In conclusion, TP-1 had a progressively accumulating effect on T-cell lymphopoiesis and activity (skin tests), which was strong enough to overcome chemotherapy-induced immunosuppression without any appreciable side effects.     

Early appearance of donor-type antigen-presenting cells in the thymuses of 1200 R radiation-induced bone marrow chimeras correlates with self-recognition of donor I region gene products

The thymus exerts a potent influence on the development of I region self-recognition and antigen recognition by T cells. The mechanism by which the thymus acts on nascent T cells is unknown. It is assumed, however, that a cell interaction between the developing T cell and an la antigen-bearing cell in the thymus is involved. There are several candidates for the critical thymic cell; thymic epithelial, nurse, and antigen-presenting cells (APC) or dendritic cells. Because thymic epithelial cells derive from the third pharyngeal pouch and thymic APC derive from bone marrow, radiation-induced bone marrow chimeras allow the artificial creation of a chimeric thymus gland in which thymic epithelial cells and APC can be genetically different. We made radiation-induced bone marrow chimeras (F1 leads to P) using supralethal radiation doses (1200 R) and found bone marrow donor- (F1) type APC in the thymuses 3 wk after radiation. When such mice fully reconstitute their immune systems, their T cells behave as donor F1 phenotype T cells. Thus, the I region self-restriction and antigen-recognition repertoire of the T cells correlates with the genotype of the bone marrow-derived thymic APC, not the thymic epithelial cell.     

Normal human bone marrow suppressor cells and in liver cirrhosis

Suppressor properties of bone marrow cells were studied in healthy donors and patients with hepatocirrhosis using the technique registrating the activity of bone marrow B-suppressors by the inhibition of xenogenic target cell proliferation. The activity of bone marrow suppressor cells in patients with various types of hepatocirrhosis was reduced as compared to healthy subjects. In addition, the in vitro spontaneous proliferation level of bone marrow cells in hepatocirrhosis was considerably higher than that of healthy donors. This fact can be possibly attributed to the decline in the number of bone marrow B-suppressors or inhibition of their functional activity in hepatocirrhosis. Peripheral blood lymphocytes of these patients, like the lymphocytes of healthy donors, showed practically no suppressive effect in vitro.     

Effect of a glucan,sizofiran, on natural-killer activity of 5-fluorouracil-treated murine bone marrow cells

Summary The number of bone marrow cells in C3H/He mice was reduced 3–4 days after treatment with 130 mg/kg intraperitoneal 5-fluorouracil (5-FU). Higher rates of spontaneous proliferation and natural killer (NK) activity, accompanied by an increase in asialoGM1-positive cells, were observed in treated mice. When sizofiran at a dose of 200 µg/animal was intramuscularly injected after 5-FU treatment, the rates of proliferation and NK activity of bone marrow cells were higher than with 5-FU alone. The cell number was not influenced by sizofiran alone. These results indicate that all precursors of the various mature cell types (including NK cells) differentiate and regenerate rapidly to replace cells damaged by 5-FU treatment, and that sizofiran has the potential to assist this recovery. These results suggest that administration of sizofiran after chemotherapy may be useful in cancer patients.     

不同化疗方案对晚期非小细胞肺癌患者骨髓抑制及免疫功能的影响

目的:探讨不同化疗方案对晚期非小细胞肺癌患者骨髓抑制及免疫功能的影响。方法:选取2011年1月至2013年12月收治的130例晚期非小细胞肺癌患者,按照知情同意原则随机分为三组:NP(长春瑞滨+顺铂)组45例、GP(吉西他滨+顺铂)组43例、TP(紫杉醇+顺铂)组42例,分别于化疗前及化疗2个周期后检测患者的骨髓抑制及免疫水平。结果:三种化疗方案进行治疗后骨髓抑制水平由高到低排列为GP组、TP组、NP组,差异有统计学意义(P0.05);GP组血小板减少发生率高于其他两组,TP组白细胞下降发生率高于其他两组,差异有统计学意义(P0.05);三组患者化疗后的免疫功能指标均较化疗前低,差异有统计学意义(P0.05)。三种化疗方案进行治疗后免疫功能抑制水平由高到低排列为GP组、TP组、NP组,差异有统计学意义(P0.05)。结论:GP组患者血小板下降更明显,TP组白细胞下降更为明显,NP组对骨髓抑制及免疫功能抑制较缓和,更适于老年人,因此临床选择化疗方案时要综合考虑患者骨髓状况、免疫功能情况及年龄等。     

Spontaneous growth of colonies with mature T lineage phenotype from T-cell-depleted bone marrow precursors in a patient with a lymphoproliferative disorder of the large granular lymphocytes

Bone marrow cells from a patient with pancytopenia and a lymphoproliferative disorder of large granular lymphocytes (LGL) were cultured and tested for their hemopoietic colony-forming potential. Neither erythroid nor granulocyte-macrophage colony formation could be obtained from unfractionated, or LGL-depleted bone marrow cell preparations. However, a spontaneous growth of lymphoid colonies was observed after culturing LGL-depleted (T3-) bone marrow cell suspensions for 25 days. Pooled colonies expanded with recombinant interleukin-2 yielded a population composed predominantly of mature T cells (T3+, Leu 6-). These findings suggest that some (T3-) T cell precursors may mature in the bone marrow and that, in our patient, LGL may have exerted a suppressor effect on this maturational process.     

Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.     

Differential recovery of polymorphonuclear neutrophils,B and T cell subpopulations in the thymus,bone marrow,spleen and blood of mice following split-dose polychemotherapy

In these studies, we examined the effect of a maximum-tolerated, split-dose chemotherapy protocol of cyclophosphamide, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea carmustine on neutrophil and lymphocyte subpopulations in the peripheral blood (PBL), thymus, bone marrow and spleen. It was found that this protocol of polychemotherapy, modeled after the induction protocol used with autologous bone marrow transplantation for breast cancer, suppressed both B and T cell populations and T cell function at times when the absolute neutrophil count had returned to normal or supernormal numbers. In the peripheral blood, 7 days following initiation of chemotherapy, there was a twofold increase in the percentage of granulocytes as compared to the level in control animals on the basis of a differential count. The polymorphonuclear neutrophil (PMN) frequency in the bone marrow was increased on day 14 and statistically identical to that in control mice on all other days analyzed. In contrast to the bone marrow cells and PBL on day 7, the frequency of PMN in the spleen and thymus was depressed. B cells (B220+) were depressed in the PBL, spleen and bone marrow and took 18–32 days to return to their normal frequency, while the frequency of B cells in the thymus was increased owing to a loss of immature T cells. The percentage of CD3+ cells in the thymus, spleen and bone marrow was significantly increased and required 10–18 days to return to normal levels, while the absolute number of CD3+ cells in the blood varied around the normal value. The ratio of CD4+ to CD8+ cells in all the organs studied varied only slightly owing to a similar reconstitution of CD4+ and CD8+ cells. In contrast to the phenotypic recovery of the CD3+, CD4+ and CD8+ cells, the ability of the splenic lymphocytes to respond to concanavalin-A was depressed and remained depressed, despite the phenotypic reconstitution of the T cell subsets, on the basis of both percentage and absolute cell number. These results show a selective T and B cell depression following multi-drug, split-dose chemotherapy in tissue and blood leukocyte populations and a chronic depression in T cell function.     

Effect of thymic humoral factor in vitro on human bone marrow and blood cells from B-, T- and null-cell acute lymphatic leukemia.

The nature of null-cell acute lymphatic leukemia (ALL) was investigated with the aid of a thymic humoral factor (THF), bone marrow cells, and a local xenogeneic graft-versus-host reaction (GVHR). Lymphocytes obtained from the blood and bone marrow of six children with T-cell ALL, five with null-cell ALL, one with perinatal B-cell ALL, one with acute myelocytic leukemia, and one with erythroleukemia were tested for membrane surface markers (E, EAC, and SM Ig); functional activity of T cells was tested by a local GVHR. All of the specimens obtained at the initial presentation showed a lack of functional activity of the lymphocytes. Incubation of null cell and acute myelocytic leukemia (AML) bone marrow with THF led to the acquisition of the characteristics of functional, immunocompetent T cells. No such effect was seen when the bone marrow of T-cell ALL and peripheral blood lymphocytes of B-cell perinatal ALL were incubated with THF. This study demonstrates that the null cell in ALL bone marrow can be differentiated into a T cell whereas the stem cell in AML bone marrow constitutes a pluripotential undifferentiated cell which also can mature into a T cell.     

Medullary epithelial cell lines from murine thymus constitutively secrete IL-1 and hematopoietic growth factors and express class II antigens in response to recombinant interferon-gamma

In this report, we describe the generation of two cloned epithelial cell lines, TE-71 and TE-75, from murine thymus. These cell lines resemble medullary thymic epithelium by a number of criteria, including reactivity with the monoclonal antibodies A2B5 and ER-TR5, the fucose-specific lectin derived from Ulex europeus, and the expression of keratins normally expressed by medullary thymic epithelial cells in situ. Constitutive Class II antigen expression by these cells is not detectable at the light or electron microscopic level or with flow cytometry. Following exposure to recombinant interferon-gamma or supernatants from mitogen-stimulated spleen cells, expression of Class II antigens by these thymic epithelial cell lines is increased, although less than the levels expressed by spleen cells. Medium conditioned by TE-71 and TE-75 cells exhibited colony-stimulating activity for bone marrow cells. In addition, TE-71-conditioned medium exhibited IL-1-like activity which could be neutralized with anti-IL-1 antibodies.     

Ultrastructural study of spontaneous bone marrow rosette-forming cells

Mouse bone marrow contains spontaneous rosette-forming cells (RFC) which include more than 70% T-cell precursors, as assessed by their transformation into theta-positive cells after incubation with thymic hormone. Such spontaneous RFC, examined in C57B1/6 mouse bone marrow by electron and scanning electron microscopy, have consistently been shown to be small, inactive mouse lymphocytes when macrophages have been eliminated by cell preincubation. These data suggest that thymic hormone target cells include small quiescent lymphocytes.     

Effect of syngeneic lymphocytes, T immunodeficiency and the genotype of bone marrow donors on the differentiation of early and late splenic colonies

The formation of "early" (5-8 days) and "late" (12-14 days) colonies in spleen of lethally irradiated syngeneic or hybrid recipients after transplantation of bone marrow cells has been studied. The differentiation pattern did not depend on bone marrow cell donor's genotype and the donor-recipient combination. Erythroid to granulocyte colonies ratio (E/G) equals 2. Change of direction of bone marrow colony-forming units (CFU) differentiation has the same pattern at different stages of colony-formation. Under the influence of antigen-stimulated lymphocytes the granulopoiesis (E/G 0.3-0.5) dominanted. The thymectomy of adult animals leads to a predominant formation of erythroid colonies (E/G 3.5-5.1). When T-immunodeficiency is reversed with syngeneic lymphocytes, the differentiation of CFU is normalized at all stages of colony-formation. The process of differentiation of haemopoietic precursors, that form "early" and "late" colonies, is under T-lymphocyte control.     

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1-3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.     

Phytohemagglutinin-lnduced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment

Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25? and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25? or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunoflu-orescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.     

An epithelial progenitor pool regulates thymus growth

Thymic epithelium provides an essential cellular substrate for T cell development and selection. Gradual age-associated thymic atrophy leads to a reduction in functional thymic tissue and a decline in de novo T cell generation. Development of strategies tailored toward regeneration of thymic tissue provides an important possibility to improve immune function in elderly individuals and increase the capacity for immune recovery in patients having undergone bone marrow transfer following immunoablative therapies. In this study we show that restriction of the size of the functional thymic epithelial progenitor pool affects the number of mature thymic epithelial cells. Using an embryo fusion chimera-based approach, we demonstrate a reduction in the total number of both embryonic and adult thymic epithelium, which relates to the initial size of the progenitor cell pool. The inability of thymic epithelial progenitor cells to undergo sufficient compensatory proliferation to rescue the deficit in progenitor numbers suggests that in addition to extrinsic regulation of thymus growth by provision of growth factors, intrinsic factors such as a proliferative restriction of thymic epithelial progenitors and availability of progenitor cell niches may limit thymic epithelial recovery. Collectively, our data demonstrate an important level of regulation of thymic growth and recovery at the thymic epithelial progenitor level, providing an important consideration for developing methods targeted toward inducing thymic regeneration.     

Nature of the target cells in bone marrow B-suppressor action studied on a simplified model of suppression activity registration

The addition of bone marrow cells to spleen cells and lymph node cells stimulates by mitogents, but not to fibroblast-like cells, leads to a significant reduction of DNA synthesis in mixed cultures in vitro. The suppression effects appears only in two days and the suppressor cell activity is the stronger, the intensive is the target cell proliferation. It is shown that intact bone marrow cells can suppress the lipopolysaccharide-activated bone marrow cell proliferation in vitro. A conclusion may be draw that cells of the lymphoid system serve as target cells for the bone marrow suppressor cells, and the role of these lymphoid system cells is to control immunogenesis processes by suppressing the target cell proliferation activity in the bone marrow.     

Immune mechanisms in leukemia: protective capacity of the major lymphoid cell compartments.

The capacity of immune spleen, lymph node, peritoneal, bone marrow, and thymic cells to protect C58/wm mice from syngeneic transplanted line Ib leukemia was quantified. Cells harvested 14 to 15 days after primary immunization were used for adoptive protection tests. Regression curves were computer analyzed and log10, PD50 values compared. For immune spleen, lymph node, peritoneal, bone marrow, and thymic cells the PD50 values were 4.53, 5.92, 4.88, 5.51, and 5.59, respectively. When immune spleen cells were treated with anti-Thy 1.2 serum the PD50 value was increased from 4.73 to 6.09, i.e., protection was reduced greater than or equal to 95%. Similar treatment of immune thymic cells reduced protection below measureable values. Anti-B cell sera (anti-IgM and anti-Ly 4.2) did not reduce the protective effect of immune spleen or marrow cells. These results indicate that a major protective cell population in each of these compartments was theta-positive. Experiments were carried out to characterize the cortisone (CS) and x-ray sensitivity of immune spleen, thymic, and marrow cells .When donor mice were treated with 12.5 mg of cortisone acetate/day for 2 days before lymphoid cells were harvested, the orotective effects of immune spleen cells, but not immune thymic or marrow cells, was reduced. When immune spleen cells were x-irrated in vitro, their protective effect was reduced by 350 R and abolished by 1000 R. When mice received whole boyd x-irradiation 24 hr before immune spleen cells were transferred their protective effect was reduced by 1000 R but only slightly lowered by 350 R. The possible significance of the multicompartmental nature of immunity to leukemia was discussed.