Adult bone marrow contains precursors for CD5+ B cells

To determine directly whether B cell precursors of adult origin are capable of generating CD5⁺ B cells, we reconstituted neonatal C3H.SCID mice with adult C57BL/6 bone marrow and analyzed splenic B cells 10 months later. Surface staining and flow cytometry revealed that the B cells were of donor origin and that 30% were CD5⁺. This confirms that in vivo generated CD5⁺ B cells can be adult derived. After anti-IgM (but not lipopolysaccharide) stimulation in vitro, virtually all of the B cells from the bone marrow-reconstituted mice expressed surface CD5. Sequence analysis of expressed V_HDJ_H genes from the CD5⁺ B cells present after anti-IgM stimulation revealed a high frequency of N nucleotide addition in CDR3 regions. The presence of N nucleotides indicates that these sequences were derived from CD5⁺ B cells of adult origin rather than from long-lived fetal precursor B cells present in either the adult bone marrow at the time of transfer or adult spleen. These experiments demonstrate conclusively that adult bone marrow contains precursors for CD5⁺ B cells and that unlike fetal liver-derived precursors these express terminal deoxynucleotidyl transferase.     

Old mice retain bone marrow B1 progenitors, but lose B2 precursors, and exhibit altered immature B cell phenotype and light chain usage

The bone marrow of old adult mice (∼2 years old) has reduced B lymphopoiesis; however, whether the B1 pathway in adult bone marrow is also compromised in senescence is not known. Herein, we show that phenotypic (IgM⁻Lin⁻CD93⁺[AA4.1⁺] CD19⁺B220^low/−) B1 progenitors are retained in old bone marrow even as B2 B cell precursors are reduced. Moreover, B1 progenitors from both young adult and old mice generated new B cells in vitro enriched for CD43 expression, likely due to their activation, and exhibited increased λ light chain usage and diminished levels of κ light chain expression. B1 progenitors were shown to have lower surrogate light chain (λ5) protein levels than did B2 pro-B cells in young mice and these levels decreased in both B1 and B2 precursor pools in old age. These results indicate that the B1 B cell pathway persists during old age in contrast to the B2 pathway. Moreover, B1 B cell progenitors generated new B cells in the adult bone marrow that have distinct surface phenotype and light chain usage. This is associated with decreased surrogate light chain expression, a characteristic held in common by B1 progenitors as well as B2 precursors in old mice.     

High expression of NKG2A/CD94 and low expression of granzyme B are associated with reduced cord blood NK cell activity

Human umbilical cord blood （CB） has recently been used as a source of stem cells in transplantation. NK cells derived from CB are the key effector cells involved in graft-versus-host disease （GVHD） and graft-versus-leukemia （GVL）. It was reported that the activity of CB NK cells was lower than that of adult peripheral blood （PB） NK cells. In this study, we analyzed the expression of some NK cell receptors and cytotoxicity-related molecules in CB and PB NK cells. The expressions of activating NK receptors, CD16, NKG2D and NKp46, did not show significant difference between CB and PB NK cells. But the expression of inhibitory receptor NKG2A/CD94 was significantly higher on CB NK cells. As to the effector function molecules, granzyme B was expressed significantly lower in CB NK cells, but the expressions of intracellular perforin, IFN-γ, TNF-α and cell surface FasL and TRAIL did not show difference between CB and PB NK cells. The results indicated that the high expression of NKG2A/CD94 and low expression of granzyme B may be related with the reduced activity of CB NK cells.     

Analysis of the activating receptors and cytolytic function of human natural killer cells undergoing in vivo differentiation after allogeneic bone marrow transplantation

Patients undergoing allogeneic bone marrow transplantation offer a unique system to analyze NK cell development in vivo. We analyzed NK cells from 23 such patients to assess the acquisition of activating receptors. Four patients displayed an immature NK cell surface phenotype at engraftment, as their cells were CD16(-)KIR(-) and NKG2D(-) but expressed low levels of NKp46, NKp30, 2B4 and NKG2A. These NK cells had particularly low cytolytic activity against the HLA-class-I(-) melanoma F01 cell line and the 721-221 EBV-infected B cell line. Moreover, cytoxicity was inhibited upon mAb-mediated crosslinking of 2B4. Analysis of NK cells at day 30 after bone marrow transplantation revealed the occurrence of both phenotypic and functional maturation. These data are in agreement with a previous in vitro study showing that immature NK cell precursors express CD16, NKG2D and KIR only at a late stage of differentiation and also express inhibitory 2B4. Our present study allows a better understanding of the NK cell differentiation in vivo.     

Ly-49 G2+ NK cells are responsible for mediating the rejection of H-2b bone marrow allog rafts in mice

NK cells can mediate the specific rejection of bone marrow butnot solid tissue allografts in lethally irradiated mice. NKcells are also responsible for the phenomenon of ‘hybridresistance’ in which F₁ hybrid H-2 heterozygous mice canreject parental H-2 homozygous bone marrow grafts. Ly-49C andLy-49 G2 are markers identified on subsets of NK cells. WhileLy-49C⁺ NK cells have been demonstrated to mediate the specificrejection of H-2^d bone marrow allografts, the role of the Ly-49G2⁺ NK subset is unclear because depletion of this subset invivo did not affect splenic NK activity against tumor targets.Through bone marrow transplantation typing studies, we demonstratethat Ly-49 G2⁺ NK cells complement Ly-49C⁺ NK cells in thatthey specifically mediate the rejection of H-2^b bone marrowallografts in lethally irradiated mice. In support of this,depletion of the Ly-49C⁺ NK subset in vivo also enhanced theability of the mice to reject H-2^b bone marrow cells suggestingthat the depletion was augmenting the ability of the Ly-49 G2⁺NK cells to reject the marrow allografts. Depletion of Ly-49G2⁺ NK cells in F₁ hybrid mice abrogated their ability to rejectparental H-2^b but not H-2^d bone marrow grafts. Therefore, Ly-49G2 denotes, a subset of NK cells that appears to play a criticalrole in the recognition of H-2^b bone marrow cells in allogeneicand F₁ hybrid mice.     

Impaired V(D)J recombination and increased apoptosis among B cell precursors in the bone marrow of c-Abl-deficient mice

Previous studies on c-Abl-deficient mice have shown high post-natal mortality and lymphopenia. However, the mechanisms by which c-Abl may influence B lymphopoiesis remain obscure. In this study, we analyzed B cell sub-populations at various differentiation stages in the bone marrow (BM) of c-Abl-deficient mice. Phenotypic analyses revealed that c-Abl(-/-) pro-B cells were reduced to half of normal incidence and absolute number, while pre-B cells showed an even greater reduction. Both c-Abl(-/-) pro-B and pre-B cell populations showed considerably elevated apoptosis ex vivo and in short-term culture but their cell cycle progression was not impaired. In contrast, apoptosis of immature IgM(+)IgD(-) B lymphocytes remained at normal control levels. Inhibition of c-Abl activity by STI571 in normal BM cultures significantly increased apoptosis in B cell precursors while the survival of immature B cells was not affected. To determine whether c-Abl deficiency affects Ig heavy-chain rearrangement, we found that the frequency of V(D)J recombination was markedly reduced by 15-fold in c-Abl(-/-) pro-B cells compared with the control values. However, no perturbation in the levels of signal-end recombination intermediates was found. Taken together, we propose that c-Abl mediates a stage-specific anti-apoptotic response in precursor B cells and is required for efficient V(D)J recombination during B cell development.     

Double negative Treg cells promote nonmyeloablative bone marrow chimerism by inducing T-cell clonal deletion and suppressing NK cell function

The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft-versus-host-disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation-free condition. Our data indicate that adoptive transfer of donor-derived T-cell receptor (TCR) αβ(+) CD3(+) CD4(-) CD8(-) NK1.1(-) (double negative, DN) Treg cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable-mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third-party C3H (H-2k) skin grafts. Adoptive transfer of CD4(+) and CD8(+) T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T-cell alloreactive responsiveness was reduced in the DN Treg cell-treated group and clonal deletions of TCRVβ2, 7, 8.1/2, and 8.3 were observed in both CD4(+) and CD8(+) T cells. Furthermore, DN Treg-cell treatment suppressed NK cell-mediated BM rejection in a perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable-mixed chimerism and donor-specific tolerance in the irradiation-free regimen.     

CD73对骨髓间充质干细胞增殖与衰老的影响

目的探讨CD73对骨髓间充质干细胞(BMSCs)增殖与衰老的影响。方法培养小鼠BMSCs,构建慢病毒CD73过表达载体和CD73 RNAi干扰载体,分别转染BMSCs,实验分组:CD73过表达组(OE组)、CD73RNAi干扰组(RNAi组)和对照组(CON组)。衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测细胞的衰老情况;MTT法检测细胞增殖能力变化;流式细胞术检测细胞周期变化;透射电子显微镜观测转染后细胞的超微结构;非损伤细胞功能分析仪检测细胞生长曲线。结果慢病毒转染BMSCs后72 h和168 h,衰老细胞数量RNAi组CON组OE组(P0.05);转染后3~4d BMSCs的增殖能力为OE组较RNAi组和CON组细胞增殖速度快(P0.05);转染后3d各组细胞周期的变化是:OE组、RNAi组和CON组的G1期细胞所占比例分别为25.9%、44.1%和51.7%;S期细胞所占比例分别为48.8%、30.9%和26.9%;G2期细胞所占比例分别为25.2%、25.0%和21.4%;透射电子显微镜观察结果显示,OE组细胞质内粗面内质网和核糖体增多;细胞生长曲线显示OE组高于CON组和RNAi组。结论 CD73促进BMSCs增殖且抑制BMSCs衰老。     

转IL—2基因的骨髓基质细胞对骨髓移植后免疫功能重建的促进作用

目的探讨了用逆转录病毒载体转入小鼠IL-2基因的基质细胞系QXMSC1对异基因骨髓移植后免疫功能重建的促进作用.方法将小鼠IL-2cDNA片段连接到逆转录病毒载体PLXSN上,构建重组逆转录病毒载体PL2SN(含小鼠IL-2cDNA).用磷酸钙共沉淀法将PL2SN转入单嗜性包装细胞系CRE,获得G418抵抗细胞株后,以上清感染双嗜性包装细胞系CRIP,G418筛选后获得高滴度的包装细胞系CRIPIL-2.感染NIH3T3细胞测定CRIPIL-2培养上清病毒滴度为3.4×105cfu/ml.感染骨髓基质细胞系QXMSC1(H-2d),G418筛选,有限稀释法得10个单克隆细胞株,选择表达IL-2最高的细胞株为实验用细胞QXMSC1IL-2,用于以后的实验.供体小鼠BALB/c(H-2d)骨髓以抗T细胞单抗anti-Thy1.2加补体去除骨髓中T细胞,受体小鼠C57BL/6(H-2b)经γ射线致死剂量照射后,输入供体骨髓1×107/只,同时输入基质细胞QXMSC1IL-25×105/只.在第30天、60天检测BMT小鼠脾细胞对LPS、ConA的反应,脾细胞产生IL-2的能力,BMT小鼠产生抗体生成细胞(PFC)的能力及产生DTH的能力.流式细胞仪检测了QXMSC1IL-2对BMT小鼠T细胞亚群的影响.结果电泳及酶切鉴定证实构建的PL2SN质粒.QXMSC1IL-2细胞系IL-2的分泌量为857U(1×106*24h).异基因骨髓移植,共输入QXMSC1IL-2能明显增加BMT小鼠脾细胞对LPS、ConA的反应.脾细胞产生IL-2的能力增强.PFC数目增加,DTH反应增强.T细胞亚群中CD4+/CD8+的比例有所恢复.结论转入IL-2的骨髓基质细胞系可促进骨髓移植后免疫功能重建.     

CD8+ T cells drive autoimmune hematopoietic stem cell dysfunction and bone marrow failure

Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction, resulting in varying degrees of hypoplasia and blood pancytopenia, and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4⁺ T cells in driving BM failure has been clearly established in several models. However, animal model data demonstrating a functional role for CD8⁺ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8⁺ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8⁺ T cells in BM failure and a beneficial role for Foxp3⁺ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice, which have a deficit in functional Tregs, develop spontaneous BM failure. Furthermore, we demonstrate a critical role for CD8⁺ T cells in the development of BM failure, which is dependent on the cytokine, IFNγ. CD8⁺ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells, resulting in anemia. Adoptive transfer experiments demonstrate that CD8⁺ T cells dramatically expedite disease progression and promote CD4⁺ T cell accumulation in the BM. Thus, BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNγ-producing CD8⁺ T cell (Tc1) response.     

Immature B cells from human and mouse bone marrow can change their surface light chain expression

The capacity of bone marrow-derived surface immunoglobulin-positive (sIg⁺) human and mouse immature B cells, generated either in vitro or in vivo, to change their light (L) chain expression, has been assayed by the number of cells which change in vitro from one type of L chain to the other type, or to no sIg at all. Immature sIg⁺ B cells were generated in vitro from sIg^? precursor cells from human or mouse bone marrow. The immature sIg⁺ cells expressed RAG-1. Human sIg⁺ cells expressed xfr; and λ L chains in ratios between 1:1 and 3:1, whereas in mouse cells, this ratio ranged from 10:1 to 20:1. Upon reculture of the human and mouse xfr;⁺sIg⁺ cells, about half of them remained xfr;⁺, a quarter became λ⁺, and another quarter became sIg^?. Between 1 and 3% expressed both xfr; and λ chains. Of the human λ⁺ cells, about two-thirds remained λ⁺, only 1 to 2% became xfr;⁺, while the other third became sIg^?. Again, between 1 and 3% expressed both xfr; and λ L chains. These results indicate that expression of sIgM in the B cell membrane does not terminate L chain gene rearrangement, and that some order exists in xfr; versus λ gene rearrangements. Hence, human and mouse xfr;⁺ immature B cells can become λ⁺, but very few of the λ⁺ cells can become xfr;⁺, and both can become sIg^?. Further, human CD10⁺/sIg⁺ xfr;⁺ and λ⁺ cells and mouse B220^low/sIg^low xfr;⁺ cells enriched from bone marrow, i.e. immature B cells differentiated in vivo, changed their Ig phenotype upon in vitro culture, but in lower frequencies. By contrast, human and mouse mature B cells did not change their L chain or Ig phenotype. Hence, at least a part of the sIg⁺ immature B cells in bone marrow retain the capacity to change their L chain and Ig phenotype, and this capacity is lost when they become mature, peripheral B cells.     

CD40 expression and function in murine B cell ontogeny

The CD40 antigen, a member of the nerve growth factor/tumornecrosis factor receptor family, is expressed on all matureB lymphocytes and plays a crucial role in B cell activation,T cell- dependent antigen-driven isotype switching and germinalcenter formation. We have analyzed C040 expression and functionduring mouse B cell development by examining B cell precursorsin normal mice and in transgenic animals in which B cell developmentis frozen at discrete stages. These models included RAG-2 ^-/-mice, and transgenlc littermates that express µ heavychain and/ or the bcl-2 proto-oncogene transgene. CD40 was undetectableat the pro-B cell stage, but was expressed, although at lowlevels, on pre-B cells. However, pre-B cells failed to respondto CD40 triggering either by expression of CD23 or by proliferationin the presence of lL-4. Overexpression of bcl-2 increased thedensity of CD40 expression on pre-B cells: these cells respondto CD40 ligation by expressing CD23 and by proliferating inthe presence of IL-4.     

Discrete early pro-B and pre-B stages in normal human bone marrow as defined by surface pseudo-light chain expression

Vpre-B and δ-like genes are selectively expressed in B cell precursors and encode polypeptide chains associated in a m?-pseudo light chain (m?-PSL) complex which is thought to regulate some early steps of B cell differentiation. We have generated anti-Vpre-B monoclonal antibodies which allowed us to identify different steps of differentiation from the pro-B to the immature B cells by following surface expression of Vpre-B, m? and light chains in normal adult human bone marrow. Already present at the surface of a small fraction of B cell progenitors (CD34⁺/CD19⁺) the Vpre-B molecule was consistently found coexpressed with CD19 and was also found with the sequentially occurring CD10, CD20, CD21, CD22 and CD5 markers. Three discrete cell types were identified: (i) a subpopulation expressing Vpre-B without m? and which represented an early stage of differentiation, (ii) a minor subpopulation co-expressing Vpre-B and m? without the conventional light chains and (iii) a major subpopulation co-expressing Vpre-B, m? and χ or δ chains, considered an intermediate pre-B/B stage. The presence of the PSL chain in various cell subpopulations, in possible association with discrete molecules and/or different contexts, suggests its involvement at several steps of early B cell differentiation.     

Precursor B cells of mouse bone marrow express two different complexes with the surrogate light chain on the surface

Two monoclonal antibodies raised against the complex of μ heavy (H) chain and V_pre-B/λ5 surrogate light (L) chains recognize surrogate L chain in different conformations on normal pre-B cells. One, LM34 recognizes free λ5 protein and free λ5/V_pre-B surrogate L chains and binds to surrogate L chains on the surface of early, pro-B and pre-B-I cells where the surrogate L chain is associated with a gp130/gp35–65 complex of proteins. It also recognizes the surrogate L chain associated with the μH chain on pre-B-II cells. The other monoclonal antibody, SL156, does not recognize free surrogate L chain or its components, nor its complex with gp130/gp35–65 on pro-B and pre-B-I cells. However, it does bind to a conformational epitope on the surrogate light chain/μH chain complex on a subpopulation of pre-B-II cells and on μH chain-positive pre-B cell lines. On mouse precursor B cells prepared ex vivo on ice, expression of the surrogate L chain is very low and almost undetectable. Incubation of the precursor cells for 1 h at 37 °C up-regulates the surface expression of surrogate L chain associated with gp130/gp35–65 (early complex) as well as the μH chain/surrogate L chain complex. These results reconcile some of the apparently discrepant results on surface expression of the surrogate L chain obtained with human and mouse bone marrow pre-B cells, and show that a surrogate L chain/μH chain-containing pre-B cell receptor can be expressed also on the surface of mouse pre-B-II cells.     

T cell lymphoblastic leukaemia/lymphoma associated with a microenvironment of thymic asteroid B cells in the bone marrow

Naresh K N, May P C, Reid A G, Marks A J, Macdonald D & Kanfer E
(2010) Histopathology 57, 549–554
T cell lymphoblastic leukaemia/lymphoma associated with a microenvironment of thymic asteroid B cells in the bone marrow Aims: Asteroid B cells are a component of normal thymus. It is currently unclear whether these cells are identifiable in T cell lymphoblastic leukaemia/lymphoma (T‐ALL/LBL) of the thymus. The aim of this study was to identify asteroid B cells both in thymic and extrathymic tissue involved by T‐ALL/LBL. Methods and results: Thymic, lymph node (LN) and bone marrow trephine biopsy (BMTB) samples from eight patients with T‐ALL/LBL were reviewed. All had been investigated by immunohistochemistry and one by fluorescent in situ hybridization (FISH). The BMTB samples of two of eight T‐ALL/LBLs and LN sample in one of them showed the presence of asteroid‐shaped B cells with dendritic cytoplasmic processes. These B cells also expressed CD23 and the features were akin to the unique thymic asteroid B cells. Both patients had aggressive/resistant disease. Cytogenetic analysis in one showed a complex translocation involving the T cell receptor beta (TCRB) gene at 7q35 and a distal region of 9q known to harbour the NOTCH1 gene. Conclusion: This is the first report of T‐ALL/LBL documenting the presence of an asteroid B cell‐rich microenvironment at bone marrow and LN sites. In this small subset, T‐ALL/LBL cells are possibly dependent upon asteroid B cells, and whether targeting of asteroid B cells with anti‐CD20 monoclonal antibody in such cases will result in clinical benefit remains to be determined.     

E2A、EBF和PAX5在早期B细胞分化发育中的作用

早期B细胞分化发育受到各种转录因子调控,特别是转录因子E2A、早期B细胞因子(EBF)及PAX5的调控尤为重要。本文以E2A、EBF与PAX5三个转录因子为综述对象,逐一阐述其在早期B细胞分化发育中的作用,并对3个转录因子在早期B细胞分化发育中的协同作用进行了综述。本文将为"B淋巴细胞分化发育调控机理研究"提供参考。     

miR-122-5p靶向KDM2A对骨髓间充质干细胞成骨分化的影响

目的 探讨miR-122-5p靶向组蛋白去甲基化酶2A（histone demethylation protein 2A, KDM2A）对骨髓间充质干细胞成骨分化的影响。方法 体外培养人骨髓间充质干细胞（bone marrow mesenchymal stem cell, BMSC）,利用qRT-PCR和Western blot方法检测miR-122-5p和KDM2A对BMSC成骨相关指标（OPN、OCN和RUNX2）表达情况的影响;茜素红染色和碱性磷酸酶染色检测miR-122-5p和KDM2A对BMSC钙沉积和碱性磷酸酶活性的影响;双荧光素酶实验验证miR-122-5p和KDM2A之间的结合关系;miR-122-5p inhibitor和si-KDM2A共转染研究两者对BMSC成骨分化的影响。结果 miR-122-5p mimic和si-KDM2A能够促进BMSC成骨相关基因的表达水平、钙结节沉积及碱性磷酸酶活性,miR-122-5p和KDM2A两者之间存在靶向结合关系,且miR-122-5p能够靶向抑制KDM2A表达,进而促进BMSC成骨相关基因的表达水平、钙结节沉积及碱性磷酸酶活性。结论 miR-122-5p靶向抑制KDM2A表达,进而促进BMSC成骨分化。