Suppressor T cells in allogeneic bone marrow chimeras

Immunosuppression is believed to play a role in the maintenance of stable bone marrow (BM) chimeras. This study investigates the nature and specificity of the suppression that lymphocytes from allogeneic BM chimeras exert upon the alloreactivity of donor and recipient lymphocytes. Lethally irradiated CBA/J (H-2k) mice were infused with 10(7) unseparated (WBM) or T cell-depleted BM (TDBM) cells of B10.BR mice (H-2k, disparate at minor histocompatibility antigens). Mixtures consisting of spleen cells (SC) from BM chimeras and SC from either normal donor, recipient, or third party (C3H, H-2k) mice, were sensitized with irradiated BALB/c (H-2d) leukocytes, then assayed for proliferative and anti-H-2d cytotoxic activity and compared with those of appropriate control cultures. The alloreactivity of all three types of normal SC was non-specifically suppressed by SC from both WBM and TDBM chimeras taken 2 weeks post-BM transplantation (BMT). In contrast, at 4 weeks post-BMT, SC from both chimeras suppressed the alloreactivity of recipient-type cells whereas only SC from WBM, but not from TDBM chimeras, suppressed normal donor-type response, and neither could suppress the response of normal third party cells. The suppression of donor-type alloreactivity diminished with time, while that exerted on recipient-type lasted for at least 10 weeks post-BMT. The suppression of donor alloreactivity was mediated by radioresistant Thy1.2+, Lyt1+2+ cells while that exerted upon recipient's alloreactivity was mediated by radiosensitive Thy1.2+, Lyt1+2- cells. Both types of suppressor cells were of donor origin. The potential biological role of the suppressive activity in the engraftment of allogeneic BM is discussed.     

Increased apoptosis in bone marrow B lymphocytes but not T lymphocytes in myelodysplastic syndrome

The hallmark of myelodysplastic syndrome (MDS) is enhanced apoptosis in myeloid, erythroid, and megakaryocytic cells in the bone marrow leading to ineffective hematopoiesis. Recent studies suggested that immunological and microenvironmental factors play a role in the pathophysiology of this disease. We report a significant increase in apoptosis in bone marrow B lymphocytes in MDS as compared to that found in acute myeloid leukemia and healthy controls. Furthermore, we demonstrate that patients with refractory anemia with excess blasts in transformation (RAEB-T) had apoptosis levels in lymphocytes similar to those seen in other subtypes of MDS. Our findings suggest that the alterations in B lymphocytes in the form of increased apoptosis can be seen in MDS and support the concept that immune modulation plays a role in the pathophysiology of MDS.     

Assay of lymphokine-activated killer activity generated from bone marrow cells of children with acute lymphoblastic leukemia

We recently reported that low molecular weight B-cell growth factor (LMW-BCGF) plus recombinant interleukin-2 (rIL-2) synergistically induced lymphokine-activated killer (LAK) activity from the bone marrow (BM) cells of children with acute lymphoblastic leukemia (ALL). The kinetics of cell growth, antigenic phenotype, and lytic activity of the generated effector cells were further analyzed in this study. BM cells from ALL patients with active disease and in complete remission (CR) were cultured with a combination of LMW-BCGF and rIL-2. Monoclonal antibodies (anti-CD3 and anti-Leu 19) and immunomagnetic beads were used to separate LAK cells into three subsets: CD3+/Leu 19-, CD3+/Leu 19+, and CD3-/Leu 19+. Cytotoxicity assays with different subsets were performed versus K562, Raji, and autologous leukemic cells, using a 3- hour 51Cr release test. There was a significant cell expansion of 54- fold (mean value) for CD3+ cells and 15-fold for Leu 19+ cells in culture with LMW-BCGF plus rIL-2 for 7 to 14 days, whereas no cell expansion was observed in culture with rIL-2 alone. Although NK activity (K562) was generated from leukemic BM cells in culture with rIL-2 alone, it is only about one third of that generated in culture with rIL-2 plus LMW-BCGF. Analysis of lytic activity of cells generated in the latter cultures demonstrated that CD3-/Leu 19+ cells expressed highest lytic activity against NK-sensitive K562 cells as well as against NK-resistant Raji cells. CD3+/Leu 19+ cells showed median cytotoxicity, and CD3+Leu 19- cells mediated only minimal cytotoxic activity. Also, lytic activity of CD3-/Leu 19+ cells against autologous leukemic blasts was noted in patients with active disease. Our results demonstrate that LAK activity generated from BM cells by LMW-BCGF and r- IL2 is mediated mainly by two types of Leu 19+ cells: CD3-/Leu 19+ NK cells and CD3-/Leu 19+ T cells. Although CD3+ T cells (both Leu 19+ and Leu 19-) mediated less antitumor cytotoxicity than CD3-/Leu 19+ cells, the former cells were the major expanding cell population in culture with LMW-BCGF and rIL-2. The new culture system may be effective in generation of cells with LAK activity for therapeutic use.     

Inhibition of antigen-induced proliferation of T cells from radiation-induced bone marrow chimeras by a monoclonal antibody directed against an Ia determinant on the antigen-presenting cell.

Chimeric B10.A T cells that had matured in a (B10.A X B10.Q)F1 environment acquired the ability to respond to poly(Glu56Lys35Phe9) (GL pi), an antigen to which the B10.A mouse is a nonresponder. The response of the chimeric B10.A T cells was initiated by GL phi on responder B10.Q antigen-presenting cells (APC) but not by GL phi on nonresponder B10.A APC. Similarly, chimeric B10.Q T cells that had matured in a (B10.A X B10.Q)F1 environment acquired the ability to respond to poly(Glu60Ala30Tyr10) (GAT) when the antigen was presented on responder B10.A APC, but not when GAT was presented on nonresponder B10.Q APC. No syngeneic haplotype preference was observed for either antigen. These interactions between H-2 nonidentical T cells and APC were inhibited by anti-H-2 antisera and a monoclonal anti-Ia antibody directed against the APC but not by such antibodies when they were directed against the T cell. These data suggest that, when they develop in a responder chimeric environment, genotypic nonresponder T cells become responders by acquiring receptors that allow them to recognize responder I region products on the surface of APC. Furthermore, the data demonstrate that the site of action of the blocking effects of the anti-Ia antibodies is the APC, thus providing strong evidence in support of the idea that Ia antigens on APC are the Ir gene products.     

Myelopoiesis and erythropoiesis of bone marrow cells cultured in vitro in patients recovered from aplastic anaemia

Methylcellulose culture assay was used to detect committed haemopoietic stem cells, CFU-C and CFU-E, in aplastic anaemia patients with autologous haemopoietic reconstitution. Severe diminution of CFU-C was found in all the patients studied and the absence of a dose-response to colony stimulating factor (CSF) was demonstrated. A reduced number of CFU-E and lower erythropoietin (Ep) sensitivity of those progenitors was detected as well. Autologous serum added to the bone marrow cultures of these patients enhanced the growth of CFU-C but inhibited CFU-E growth. According to the results presented, some residual damage at the stem cell level is suggested.     

Recipient lymphocyte infusion in MHC-matched bone marrow chimeras induces a limited lymphohematopoietic host-versus-graft reactivity but a significant antileukemic effect mediated by CD8+ T cells and natural killer cells

Background

Challenge of MHC-mismatched murine bone marrow chimeras with recipient-type lymphocytes (recipient lymphocyte infusion) produces antileukemic responses in association with rejection of donor chimerism. In contrast, MHC-matched chimeras resist eradication of donor chimerism by recipient lymphocyte infusion. Here, we investigated lymphohematopoietic host-versus-graft reactivity and antileukemic responses in the MHC-matched setting, which is reminiscent of the majority of clinical transplants.

Design and Methods

We challenged C3H→AKR radiation chimeras with AKR-type splenocytes (i.e. recipient lymphocyte infusion) and BW5147.3 leukemia cells. We studied the kinetics of chimerism using flowcytometry and the mechanisms involved in antileukemic effects using in vivo antibody-mediated depletion of CD8⁺ T and NK cells, and intracellular cytokine staining.

Results

Whereas control chimeras showed progressive evolution towards high-level donor T-cell chimerism, recipient lymphocyte infusion chimeras showed a limited reduction of donor chimerism with delayed onset and long-term preservation of lower-level mixed chimerism. Recipient lymphocyte infusion chimeras nevertheless showed a significant survival benefit after leukemia challenge. In vivo antibody-mediated depletion experiments showed that both CD8⁺ T cells and NK cells contribute to the antileukemic effect. Consistent with a role for NK cells, the proportion of IFN-γ producing NK cells in recipient lymphocyte infusion chimeras was significantly higher than in control chimeras.

Conclusions

In the MHC-matched setting, recipient lymphocyte infusion elicits lymphohematopoietic host-versus-graft reactivity that is limited but sufficient to provide an antileukemic effect, and this is dependent on CD8⁺ T cells and NK cells. The data indicate that NK cells are activated as a bystander phenomenon during lymphohematopoietic T-cell alloreactivity and thus support a novel type of NK involvement in anti-tumor responses after post-transplant adoptive cell therapy.     

A recombinant bcl-x s adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.     

Depletion of T cells from bone marrow for allogeneic transplantation: method for treatment of bone marrow in bulk

T lymphocytes were depleted from donor marrow for 23 patients undergoing allogeneic bone marrow transplantation using an anti-T-cell antibody, CT-2, and complement. The methodology is described in detail for in vitro depletion of large quantities of bone marrow. The extent of T-lymphocyte depletion using various T-cell markers, the percent of marrow lost in the processing and quantity of antibody, and complement needed are presented. These techniques for in vitro T-lymphocyte depletion were reproducible and did result in an average final yield of 47% of the harvested donor marrow.     

阿托伐他汀主要经AMP蛋白激酶而非PI3K/Akt信号通路抑制猪骨髓间充质干细胞凋亡

目的 骨髓间充质干细胞移植治疗急性心肌梗死的效果受梗死后局部恶劣微环境限制,前期研究已证实他汀类药物可通过改良心肌微环境提高移植疗效,但具体机制不详.本研究旨在观察阿托伐他汀( Ator)对体外缺氧无血清(H/SF)诱导的猪骨髓间充质干细胞凋亡的影响并探索其作用机制及通路.方法 猪骨髓间充质干细胞分正常对照组、H/SF组、浓度梯度(0.001～10 μmol/L)Ator处理组、AMP蛋白激酶(AMPK)通路抑制剂compound C组(CC组),Ator+ CC组、磷脂酰肌醇3激酶(PI3K)/丝氨酸苏氨酸激酶(AKt)通路拮抗剂LY294002组(LY组)、Ator+ LY组.流式细胞仪检测各组细胞凋亡比例,Western blot检测AMPK、Akt、内皮型一氧化氮合酶(eNOS)蛋白及其磷酸化水平,实时聚合酶链反应(Real Time-PCR)检测AMPK、Akt和eNOS基因表达水平.结果 Ator 0.01～10 μmol/L组间充质干细胞凋亡比例显著低于H/SF对照组(1.94％ ～6.10％比10.94％,P＜0.01或0.05),Ator+ CC组间充质干细胞凋亡比例显著高于1μmol/L Ator组(4.94％±0.98％比2.59％ +0.84％,P＜0.01),而Ator+ LY组细胞凋亡比例与1μmol/L Ator组比较差异无统计学意义(2.02％±0.45％比2.59％ +0.84％,P＞0.05).Ator浓度梯度组AMPK、Akt和eNOS基因表达增加伴AMPK和eNOS磷酸化水平提高(P＜0.01或0.05).eNOS的磷酸化水平与AMPK磷酸化显著相关(r =0.599,P=0.004),而与Akt磷酸化水平无显著相关(P =0.263).结论 阿托伐他汀可抑制H/SF诱导的猪骨髓间充质于细胞凋亡,该效应主要与AMPK信号通路介导的eNOS活化有关,本实验条件下PI3K/Akt信号通路不起主要作用.     

Suppression by bone marrow but not by blood T lymphocytes of autologous and allogeneic granulocytic stem cells in a case of lymphoproliferative T-cell syndrome

The bone marrow T lymphocytes of a patient suffering from a lymphoproliferative T-cell syndrome suppressed the in vitro growth of autologous and allogeneic CFU-GM. The patient's blood T lymphocytes had no suppressor effect on these same stem cells.     

Subpopulations of common acute lymphoblastic leukemia antigen-positive lymphoid cells in normal bone marrow identified by hematopoietic differentiation antigens

Bone marrow samples from normal adults and children with nonhematologic malignancies not involving the marrow, acute lymphoblastic leukemia (ALL) in continued remission, and immune cytopenias were studied by two- color immunofluorescence (IF) and flow cytometry to characterize common acute lymphoblastic leukemia antigen (CALLA)-positive marrow lymphoid cells. Marrow was separated by Ficoll/Hypaque centrifugation followed by passage over a monoclonal antibody affinity column to remove myeloid cells prior to IF staining. A higher proportion of CALLA-positive cells was found in the pediatric marrows (mean, 33.7% +/- 6.3% SEM) than in the adult marrows (mean, 4.5% +/- 1.6% SEM). Two subpopulations of CALLA-positive cells identified by cell sorting to be of lymphoid morphology were found in both adult and pediatric marrows. A small subpopulation comprising 12.3% of the total CALLA-positive cells was characterized by a high intensity of CALLA, terminal deoxynucleotidyl transferase (TdT), and MY10 expression, but low B1, common leukocyte antigen, and peanut agglutinin receptor expression. The remainder of the CALLA-positive cells displayed low intensity of CALLA expression, positivity for the common leukocyte antigen, B1 and peanut agglutinin, and negative reaction with TdT and MY10. Both CALLA-positive subpopulations were positive for HLA-DR and the pan-B cell marker B4, but negative for cytoplasmic immunoglobulin, B2 and Leu 1. BrdU labelling studies showed that a similar proportion of cells in each subpopulation was in S phase. A slightly higher proportion of the strongly CALLA-positive cells possessed the morphologic features of high nuclear/cytoplasmic (N/C) ratio and prominent nucleoli. These studies suggest that a discrete maturation step occurs among CALLA- positive marrow lymphoid cells, resulting in the loss of TdT and MY10 expression, but gradual acquisition of the B cell marker B1 and the common leukocyte antigen. The presence of B4 antigen in nearly all CALLA-positive cells suggests that both subpopulations of normal CALLA- positive marrow cells are committed to the B cell lineage.     

Reversal of hyperglycemia in diabetic rats by portal vein transplantation of islet-like cells generated from bone marrow mesenchymal stem cells

AIM： To study the capacity of bone marrow mesenchymal stem cells （BM-MSCs） trans-differentiating into islet-like cells and to observe the effect of portal vein transplantation of islet-like cells in the treatment of streptozotocin-induced diabetic rat.
METHODS： BM-MSCs were isolated from SD rats and induced to differentiate into islet-like cells under defined conditions. Differentiation was evaluated with electron microscopy, RT-PCR, immunofluorescence and flow cytometry. Insulin release after glucose challenge was tested with ELISA. Then allogeneic islet-like cells were transplanted into diabetic rats via portal vein. Blood glucose levels were monitored and islet hormones were detected in the liver and pancreas of the recipient by immunohistochemistry.
RESULTS： BM-MSCs were spheroid adherent monolayers with high CD90, CD29 and very low CD45 expression. Typical islet-like cells clusters were formed after induction. Electron microscopy revealed that secretory granules were densely packed within the cytoplasm of the differentiated cells. The spheroid cells expressed islet related genes and hormones. The insulin-positive cells accounted for 19.8% and mean fluorescence intensity increased by 2.6 fold after induction. The cells secreted a small amount of insulin that was increased 1.5 fold after glucose challenge. After transplantation, islet-like cells could locate in the liver expressing islet hormones and lower the glucose levels of diabetic rats during d 6 to d 20.
CONCLUSION： Rat BM-MSCs could be transdifferentiated into islet-like cells in vitro. Portal vein transplantation of islet-like cells could alleviate the hyperglycemia of diabetic rats.     

Adult bone marrow cells can differentiate into hemopoietic cells and endothelial cells but not into other lineage cells in normal growth and normal life

There have been reports that bone marrow cells (BMCs) can differentiate into various cells and tissues and that BMCs improve the function of the injured organs or reduce the organ damage, thereby rescuing the individuals from death. However, these reports also noted that injuries were induced in the organs. Therefore, it is not clear whether BMCs can differentiate into parenchymal cells in organs in normal life or whether BMCs can supply organ-specific stem cells. In this paper, we examine whether adult BMCs could contribute to the development of various organs in normal development after birth and in normal life. BMCs from adult eGFP mice (8 weeks old) were injected into the liver of newborn C57BL/6 mice. The existence of donor-derived cells in various organs was examined 1 year after the injection. In the organs of recipient mice, some of the CD45⁺ hemopoietic cells (1.4–13.2%) and CD31⁺ endothelial cells (0–2.2%) expressed eGFP, though no other lineage cells did so. These results suggest that adult BMCs can differentiate into not only hemopoietic cells but also vascular endothelial cells, but cannot differentiate into other lineage cells in normal growth and normal life.     

Granulocyte-colony stimulating factor increases donor mesenchymal stem cells in bone marrow and their mobilization into peripheral circulation but does not repair dystrophic heart after bone marrow transplantation

BACKGROUND: Hereditary disordered cardiac muscle could be replaced with intact cardiomyocytes derived from genetically intact bone marrow (BM)-derived stem cells. METHODS AND RESULTS: Cardiomyopathic mice with targeted mutation of delta-sarcoglycan gene underwent intra-BM-BM transplantation (IBM-BMT) from transgenic mice expressing green fluorescence protein. The host BM and the peripheral blood were completely reconstituted by donor-derived hematopoietic cells by IBM-BMT. Treatment with granulocyte-colony stimulating factor (G-CSF) markedly increased donor-derived mesenchymal stem cells (MSC) in the BM and their mobilization into the peripheral blood after IBM-BMT. Treatment with isoproterenol (iso) for 7 days caused myocardial damage and left ventricular (LV) dysfunction in the cardiomyopathic mice. Co-treatment with iso and G-CSF increased donor BM cell recruitment to the heart and temporarily improved LV function in the cardiomyopathic mice with or without IBM-BMT. However, the cardiac muscle was not replaced with donor BM-derived cardiomyocytes in the cardiomyopathic mice with or without IBM-BMT, and this was associated with no improvement of LV function of mice aged 20 weeks. CONCLUSIONS: These results suggest that G-CSF enhances engraftment of donor MSC in the BM and their mobilization into the peripheral circulation after IBM-BMT but MSC recruited to the heart do not differentiate into cardiomyocytes and do not repair the dystrophic heart.     

Direct growth suppression of myeloid bone marrow progenitor cells but not cord blood progenitors by human cytomegalovirus in vitro

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte- CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.