胸腺基质细胞支持下骨髓CD34~+细胞向T细胞体外定向分化

目的：研究人骨髓CD34^＋细胞体外向T细胞定向分化的方法，为研究造血细胞淋系造血活性及T细胞发育和分化提供技术平台。方法：免疫磁珠法分离骨髓CD34^＋细胞，在骨髓基质细胞条件培养液构建的微环境下，在胸腺基质细胞的支持下，使其体外向T细胞定向分化，收集培养的非贴壁细胞，免疫荧光染色后经流式细胞术检测培养不同时间CD1^-CD3^＋细胞、CD3^＋CD4^＋CD8^-细胞及CD3^＋CD4^-CD8^＋细胞比例。结果：培养1周时，培养细胞中以不成熟的CD1^＋CD3^-细胞、CD1^＋CD3^＋细胞为主，可检测到少量CD1^-CD3^＋细胞，随培养时间延长，不成熟细胞比例逐渐减少，而成熟的CD1^-CD3^＋细胞比例逐渐增加；在CD3^＋细胞中，培养初期以不成熟的双阳性细胞CD4^＋CD8^＋为主，而成熟的单阳性CD^＋CD8^-细胞及CD4^-CD8^＋细胞占极小比例，随培养时间延长，双阳性细胞比例逐渐减少，而成熟的单阳性细胞比例逐渐增高；而无胸腺基质细胞支持的CD34＋细胞仅在培养初期检测到成熟细胞存在，而培养后4周基本检测不到成熟T细胞的存在。结论：在骨髓基质细胞及胸腺基质细胞的支持下，骨髓CD34^＋细胞可体外发育为成熟的CD1^-CD3^＋细胞及单阳性T细胞，其中胸腺基质细胞的支持对于造血细胞向T细胞的体外定向分化极其重要。     

Regulatory T cells control autoimmunity following syngeneic bone marrow transplantation

Sublethally irradiated, immunodeficient, C57BL/6 RAG-2 gene-deleted recipient mice reconstituted with T cell-depleted bone marrow (BM) grafts frequently developed diarrhea, lost weight and showed signs of autoimmunity, dying between 4 and 7 weeks after reconstitution. Mice died despite evidence of efficient donor-derived hemato-lymphoid reconstitution, and disease was associated with the presence of IgG anti-nuclear antibodies. Autoimmunity was initiated by T cells, but could be prevented by transfer of naturally arising regulatory T cells. In contrast, lethally irradiated, BM-reconstituted immunocompetent, C57BL/6 mice survived without signs of autoimmunity. Survival of immunocompetent mice was shown to be due to the presence of residual, extra-thymically located, radio-resistant, functional regulatory T cells. The importance of regulatory T cells was further shown by the reduced survival of immunocompetent BM recipients whose CD25+ T cells had been depleted prior to bone marrow transplantation. The implications of these results in the context of syngeneic graft-versus-host disease following BM transplantation are discussed.     

Human bone marrow contains a subset of quiescent early memory CD8+ T cells characterized by high CD127 expression and efflux capacity

Even today it is still not completely understood how CD8⁺ T‐cell memory is maintained long term. Since bone marrow (BM) is a niche for immunological memory, we sought to identify long‐lasting early memory CD8⁺ T cells in this compartment. To achieve this, we looked for CD8⁺ T cells that are able to efflux Rhodamine 123, a typical property of stem cells. Indeed, we identified a distinct subset of CD8⁺ T cells in BM, with the capacity to efflux and high CD127 expression. These CD127^hi effluxers are conventional CD8⁺ T cells exhibiting a broad TCR‐Vβ repertoire and are generated in response to viral peptides in vitro. CD127^hi effluxer CD8⁺ T cells have an early memory phenotype defined by preferential TNF‐α production and a Bcl‐2^hi, KLRG‐1^low profile. This population has long telomeres and shows constitutively low frequencies of Ki‐67 expression ex vivo, but has a high proliferative and differentiation capacity in vitro. However, IL‐15 downmodulates CD127 in CD127^hi effluxer CD8⁺ T cells in vitro. Consequently, the CD127^low effluxer subset may comprise cells recently exposed to IL‐15. Taken together, CD127^hi effluxer CD8⁺ T cells represent a novel population of early memory T cells resident in BM with properties required for long‐lived memory.     

Human bone marrow stromal cells hamper specific interactions of CD4 and CD8 T lymphocytes with antigen-presenting cells

Bone marrow stromal cells (BMSCs) may inhibit T-cell functions in vitro and thus have been proposed as immunoregulators to control in vivo graft-versus-host disease (GVHD) in haploidentical hemopoietic stem cell transplants. To better investigate this phenomenon, we used a defined experimental system in which responding T cells are antigen-specific and devoid of alloreactivity against BMSC from a different subject. Thus, we established antigen-specific human CD4 and CD8 T-cell lines as the readout system. Antigen-dependent proliferation was reduced with both T-cell subsets cultured on confluent BMSCs, and also on confluent human skin fibroblasts (HSF) inhibited T-cell proliferation with similar efficiency. Morphological observations of the cocultures showed impairment of physical interactions between T-cell and antigen-presenting cells in the presence of BMSC, with lack of formation of antigen-dependent clusters of T cells and antigen-presenting cells (APCs). In contrast, no effects were seen with BMSC-conditioned medium. Since suppression was seen only with confluent mesenchymal cells, this phenomenon may not be relevant in vivo, where BMSCs are at low frequency. In addition, if the reported suppressive effect of BMSCs on GVHD in vivo is confirmed, a different in vitro system should be envisaged to better understand and exploit the underlying mechanism.     

NKT cells,Treg, and their interactions in bone marrow transplantation

Bone marrow transplantation (BMT) is a potentially curative treatment for patients with leukemia and lymphoma. Tumor eradication is promoted by the anti‐tumor activity of donor T cells contained in the transplant; however, donor T cells also mediate the serious side effect of graft‐versus‐host disease (GVHD). Separation of GVHD from graft anti‐tumor activity is an important goal of research in improving transplant outcome. One approach is to take advantage of the immunomodulatory activity of regulatory NKT cells and CD4⁺CD25⁺ Treg of host and/or donor origin. Both host and donor NKT cells and donor Treg are able to prevent GVHD in murine models. In this review, we summarize the mechanisms of NKT cell‐ and Treg‐mediated protection against GVHD in mice while maintaining graft anti‐tumor activity. In addition, we also examine the interactions between NKT cells and Treg in the context of BMT, and integrate the data from murine experimental models with the observations made in humans.     

Separating antiviral and GVHD activities of donor T cells prior to bone marrow transplantation

Approaches to speed immune reconstitution following bone marrow transplantation (BMT) or peripheral-blood hematopoietic stem-cell transplantation (HSCT) could markedly reduce morbidity and mortality, particularly following partially major histocompatibility complex (MHC)-matched related donor (PMRD) transplants. However, it is critical to simultaneously eliminate the subpopulation of donor T cells that are alloreactive with the recipient and may produce graft-vs-host disease (GVHD). In this article, we discuss a number of promising cellular engineering approaches that could be applied to this problem, including the use of veto cells, regulatory T-cell subsets, and psoralen-treated donor lymphocytes. Emphasis is placed on whether these approaches can simultaneously transfer broad-spectrum immunity to the recipient without producing GVHD.     

Accumulation of allo-MHC cross-reactive memory T cells in bone marrow

The T cells in the bone marrow (BM) have recently been shown to be enriched with memory T cells. We investigated in this study the reactivity of minor-antigen specific memory cytotoxic T lymphocytes (CTLs) induced from the BM of in vivo primed mice using two different antigen systems. The antigen-specific CTLs could be efficiently induced from the BM of immunized mice. This CTL activity was not observed with naïve control mice, indicating that the activity was largely attributable to the memory T cells. Notably, these minor antigen specific CTLs showed cross-reactivity to allo-MHC antigens. Cold target inhibition analyses revealed that the same CTL populations were responsible for both anti-minor antigen and anti-allo-MHC reactivity. Taken collectively, these results not only confirmed functionally the enrichment of memory CTLs in the BM, but also indicated that such memory cells could cross-react with allo-MHC antigens. The possible role of these BM-resident memory T cells in the development of graft-versus-host disease (GVHD) is also discussed.     

Immunological memories of the bone marrow

Memory for antigens once encountered is a hallmark of the immune system of vertebrates, providing us with an immunity adapted to pathogens of our environment. Despite its fundamental relevance, the cells and genes representing immunological memory are still poorly understood. Here we discuss the concept of a circulating, proliferating, and ubiquitous population of effector lymphocytes vs concepts of resting and dormant populations of dedicated memory lymphocytes, distinct from effector lymphocytes and residing in defined tissues, particularly in barrier tissues and in the bone marrow. The lifestyle of memory plasma cells of the bone marrow may serve as a paradigm, showing that persistence of memory lymphocytes is not defined by intrinsic “half-lives”, but rather conditional on distinct survival signals provided by dedicated niches. These niches are organized by individual mesenchymal stromal cells. They define the capacity of immunological memory and regulate its homeostasis.     

Maintenance of CD8+ memory T lymphocytes in the spleen but not in the bone marrow is dependent on proliferation

It is current belief that numbers of CD8⁺ memory T lymphocytes in the memory phase of an immune response are maintained by homeostatic proliferation. Here, we compare the proliferation of CD8⁺ memory T lymphocytes, generated by natural infections and by intentional immunization, in spleen and bone marrow (BM). Fifty percent of CD8⁺ memory T lymphocytes in the spleen are eliminated by cyclophosphamide within 14 days, indicating that numbers of at least 50% of splenic CD8⁺ memory T lymphocytes are maintained by proliferation. The numbers of CD8⁺ memory T lymphocytes in the BM, however, were not affected by cyclophosphamide. This stability was independent of circulating CD8⁺ memory T cells, blocked by FTY720, showing that BM is a privileged site for the maintenance of memory T lymphocytes, as resident cells, resting in terms of proliferation.     

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。     

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.     

Low pre-infection levels and loss of central memory CD4+ T cells may predict rapid progression in SIV-infected pigtail macaques

CD4⁺ T lymphocyte subsets are targeted to different degrees by SIV infection. We studied central memory, effector memory, naïve, and regulatory T cell levels longitudinally in 11 SIV_mac251-infected pigtail macaques. Depletion of CD28⁺CD95⁺ central memory CD4⁺ T cells, but not other populations, correlated with both SIV viral load and disease progression. A low pre-infection level of central memory CD4⁺ T cells was also predictive of rapid disease progression. If confirmed in larger studies, our results suggest stratifying macaques for baseline central memory CD4⁺ T cells would be useful in defining both the pathogenesis of SIV disease and SIV vaccine efficacy.     

Contribution of 4–1BBL on radioresistant cells in providing survival signals through 4–1BB expressed on CD8+ memory T cells in the bone marrow

The persistence of memory lymphocytes is a critical feature of adaptive immunity. The TNF family ligand 4–1BBL supports the antigen‐independent survival of CD8+ memory T cells. Here, we show that mice lacking 4–1BB only on αβ T cells show a similar defect in CD8+ T‐cell recall responses, as previously shown in 4–1BBL‐deficient mice. We show that 4–1BB is selectively expressed on BM CD8+ but not CD4 + memory T cells of unimmunized mice. Its ligand, 4–1BBL, is found on VCAM‐1+ stromal cells, CD11c+ cells, and a Gr1^lo myeloid population in unimmunized mice. Adoptive transfer of in vitro generated memory T cells into mice lacking 4–1BBL only on radioresistant cells recapitulates the defect in CD8 + T‐cell survival seen in the complete knockout mice, with smaller effects of 4–1BBL on hematopoietic cells. In BM, adoptively transferred DsRed CD8+ memory T cells are most often found in proximity to VCAM‐1+ cells or Gr1+ cells, followed by B220+ cells and to a much lesser extent near CD11c+ cells. Thus, a VCAM‐1+CD45^? stromal cell is a plausible candidate for the radioresistant cell that provides 4–1BBL to CD8+ memory T cells in the BM.     

大鼠成体骨髓干细胞在肾小管上皮细胞再生中的作用

目的：观察肾小管上皮细胞的更新、再生有无来源于骨髓干细胞。方法:建立性别不匹配大鼠间的骨髓移植和肾脏移植模型，通过激光切割技术，将肾小管上皮细胞切割下来，提取其基因组DNA，用针对大鼠Y染色体上性别决定基因（Sry基因）的引物进行PCR反应，以观察提取的DNA中有无Sry基因。结果:据PCR反应结果，在2种模型所取标本的肾小管上皮细胞中，大部分可见Sry基因的存在。结论:骨髓干细胞可能是肾小管上皮细胞再生的一个来源。     

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.     

Regulation of allergen-induced bone marrow eosinophilopoiesis: role of CD4+ and CD8+ T cells

BACKGROUND: The mechanisms of the distant stimulation of the bone marrow (BM) after airway allergen exposure remain largely obscure. T cells have been implicated in allergic airway inflammation but their role in allergen-induced BM eosinophilopoiesis is poorly understood. The aim of this study was to determine the role of CD4(+) and CD8(+) T cells in allergen-induced BM eosinophilopoiesis. METHODS: Ovalbumin (OVA)-sensitized wild type (WT), CD4 knockout (CD4-/-) and CD8 knockout (CD8-/-) mice were exposed intranasally to OVA or saline. Bromo-deoxyuridine (BrdU) was used to label newly produced cells. Bone marrow, blood and bronchoalveolar lavage (BAL) were sampled 24 h after the final exposure. Immunostaining for newly produced eosinophils (i.e. BrdU(+)/MBP(+)) and BM eosinophil progenitor [CD34(+)/CD45(+)/interleukin-5 (IL-5)Ralpha(+)] cells was performed. RESULTS: The number of newly produced BM eosinophils (BrdU(+)/MBP(+) cells) was significantly reduced in allergen exposed CD4-/- or CD8-/- mice compared with allergen exposed WT mice, which was followed by a subsequent decrease in newly produced blood and airway eosinophils. Furthermore, BM eosinophil progenitors were significantly reduced in allergen exposed CD4-/- and CD8-/- mice compared with WT mice. Finally, serum IL-5 and Bronchoalveolar lavage fluid eotaxin-2 levels were abolished in allergen exposed CD4-/- mice to levels seen in saline exposed WT mice. CONCLUSIONS: These data suggests that both CD4(+) and CD8(+) T cells have a regulatory role in allergen-induced BM eosinophilopoiesis, whereas CD4(+) T cells are obligatory for allergen-induced airway eosinophilia. The subsequent traffic of eosinophils to the airways is likely to be at least partly regulated by a CD4(+) T-cell-dependent local airway eotaxin-2 production.     

FVB小鼠骨髓源树突状细胞的培养

目的 建立FVB小鼠骨髓源树突状细胞体外培养和扩增方法,观察其形态和特征.方法 在无菌条件下提取FVB鼠骨髓细胞,分离单个核细胞,用IL-4和GM-CSF联合协同诱导下培养,加用磷酸脂多糖刺激.应用光镜下观察树突状细胞的形态,流色细胞仪检测鉴定其生物学特征.结果 联合培养小鼠骨髓细胞3d后,可见细胞形态发生改变,细胞形...     

反应性浆细胞增多症患者骨髓浆细胞百分比改变

目的分析反应性浆细胞增多症(RP)患者骨髓各阶段浆细胞的分布情况,为反应性浆细胞增多症临床诊断提供依据。方法每例骨髓片经瑞氏染色后计数200个骨髓有核细胞。骨髓各阶段浆细胞百分比总和≥4.0%时,诊断为RP。结果 46例患者诊断为RP。原浆细胞、幼浆细胞、浆细胞百分比中位数分别为0.50%,4.50%,1.50%;其最大值分别为4.0%,19.0%,4.0%;浆细胞百分比总和最大值26.0%。结论 46例RP患者骨髓中浆细胞百分比并不高,幼浆细胞较多见,原浆细胞也并非罕见。     

年轻骨髓间充质干细胞可通过细胞融合改善年老骨髓间充质干细胞功能

目的:诱导年轻与年老小鼠骨髓间充质干细胞(BMSCs)融合,探讨融合后年轻BMSCs对年老BM-SCs功能的影响。方法:年轻(2-3月龄)及年老(18-24月龄)C57BL/6小鼠BMSCs在红色荧光染料PKH26标记后分别与年轻及年老绿色荧光蛋白转基因C57BL/6小鼠BMSCs通过聚乙烯二醇(PEG1500)诱导,建立细胞融合模型。通过流式细胞仪鉴定细胞融合率和细胞表面特异性抗原的表达。免疫荧光显微镜观察融合细胞形态及细胞核特性。通过细胞计数比较5组细胞(年轻组Y,年老组O,年轻融合组Y-Y,年轻年老融合组Y-O,年老融合组O-O)在2d、4d、6d、8d的细胞增殖能力。诱导细胞向成骨细胞及脂肪细胞分化,比较5组细胞的分化能力。结果:通过PEG诱导,可获得30.45%±4.13%的融合细胞,3组不同年龄融合组的细胞融合率无显著差异。融和细胞表达间充质干细胞表面特异性抗原CD44、Sca-1,而不表达造血干细胞表面特异性抗原CD34、CD117、CD31、CD45。在2d、4d、6d、8d,Y-O组细胞数量增加百分率比O-O组均显著增高。代表成骨细胞分化能力强弱的茜素红染色阳性率和代表脂肪细胞分化能力强弱的油红O染色阳性率,Y-O组比O-O组均显著增高,具体数值分别为:(25.46%±1.52%)vs(13.85%±1.69%),P0.01;(12.99%±2.61%)vs(6.03%±1.71%),P0.05。结论:年轻BMSCs可通过细胞融合改善年老BMSCs增殖及分化功能。