骨髓微环境与多发性骨髓瘤

 骨髓微环境不仅支持正常造血细胞的生长和分化,而且有助于肿瘤细胞的生长,它在多发性骨髓瘤细胞的增生、分化、凋亡中起重要作用。多发性骨髓瘤（MM）的瘤细胞定居在骨髓很少浸润骨髓以外的器官,与肿瘤细胞所处的骨髓造血微环境密切相关。骨髓瘤细胞与骨髓基质细胞及细胞外基质相互黏附,并产生细胞因子,进而影响肿瘤细胞的存活,而且还影响瘤细胞对治疗的反应。MM是一个进展性、致死性疾病,传统的化疗很易发生耐药。目前随着免疫学、分子生物学的发展及对骨髓微环境的研究,一些新型的以生物学为基础的药物不仅针对瘤细胞本身,而且以骨髓微环境为靶子,通过改变骨髓微环境而达到克服耐药的目的,从而延长骨髓瘤患者的无病生存期。     

Bone marrow contains melanoma-reactive CD8+ effector T cells and, compared with peripheral blood, enriched numbers of melanoma-reactive CD8+ memory T cells

Circulating melanoma-specific T cells can be frequently detected in patients with melanoma. Effective T-cell immunity and tumor surveillance, however, requires the presence of specific T cells in tissues populated by tumor cells. The bone marrow (BM) is a compartment frequently harboring micrometastatic tumor cells. Here, we compared directly ex vivo in peripheral blood (PB) and BM frequencies and differentiation phenotypes of T cells reactive with the melanoma-associated antigen tyrosinase and with autologous melanoma cells. Using intracellular cytokine and tetramer staining, we detected tyrosinase- and melanoma-reactive CD3+CD8+ T cells in the BM in similar or enhanced frequencies as in PB. Additional characterization of the differentiation subset using CD45RA and CCR7 revealed the presence of specific effector and memory T cells in the BM in all five patients analyzed. Remarkably, the frequency of tyrosinase- and melanoma-specific memory T cells was significantly increased in BM compared with PB. Thus, the BM may be an important compartment for tumor surveillance harboring a tumor-specific memory T-cell pool in addition to effector T cells.     

Cognate interactions between memory T cells and tumor antigen-presenting dendritic cells from bone marrow of breast cancer patients: bidirectional cell stimulation,survival and antitumor activity in vivo

Dendritic cells (DC) and T cells were generated from Ficoll separated bone marrow (BM) mononuclear cells of primary operated breast cancer patients according to new cell culture protocols. BM-DC were capable of functioning as professional antigen-presenting cells (APCs) and of inducing autologous antigen-specific memory T-cell responses to either tetanus toxoid recall antigen or to breast cancer antigens. Treatment with lipopolysaccharide (LPS) resulted in phenotypic and functional maturation of BM-DC. When BM-DC, pulsed with breast cancer-associated tumor antigens, were cocultured with autologous patient-derived BM-T cells to allow for cognate breast cancer antigen recognition and stimulation, apoptosis of T cells-which occurred in noncognate coculture systems-was inhibited. Furthermore, in cocultures allowing for antigen-specific cognate interactions, the expression on BM-DC of CD83, MHC class II, CD40 and CD86 molecules was upregulated and the cytokines IL-12 and IFN-alpha were produced in significantly elevated amounts. Adoptive transfer of breast cancer-reactive memory T cells together with APCs into human breast cancer-bearing NOD/SCID mice caused a regression of the tumor and prolonged survival of the animals. This was not the case when such animals had been treated by transfer of reactivated BM T cells without BM-DCs. Our findings suggest that cognate interactions between cancer patient-derived memory BM-T cells and tumor antigen-presenting BM-DCs are important for reciprocal cell stimulation, survival and therapeutic activity.     

Bone marrow microenvironment and the progression of multiple myeloma.

The BM microenvironment in MM, in terms of adhesive features, is well organized to entrap circulating precursors with BM-seeking properties and is able to produce cytokines that offer them the optimal conditions for local growth and final differentiation. Likewise, the malignant B cell clone is equipped with adhesion molecules which enable the cell to establish close contacts with BM stromal cells. Furthermore a number of cytokines are released including IL-1 beta and M-CSF activating BM stromal cells to produce other cytokines, such as IL-6, that stimulate the proliferation of plasma cells. Finally, most cytokines produced locally, including IL-1 beta, TNF-beta, M-CSF, IL-3 and IL-6, also have OAF properties, explaining why the expansion of the B cell clone parallels the activation and numerical increase of the osteoclast population.     

Bone marrow microenvironment in cancer patients: immunological aspects and clinical implications

The bone marrow (BM) of cancer patients is considered an essential secondary lymphoid organ with substantial impact on tumor cell dissemination and tumor–immune responses. Recent advances in the understanding of BM/primary tumor crosstalk, homing processes, premetastatic niche formation, tumor cell dormancy, and ultimately, identification of the BM micromilieu cytokines, chemokines, and growth factors may provide the basis for the development of targeted therapeutic strategies potentially rendering primary cancers and cancer bone metastases more susceptible to chemotherapy. The present review aims to dissect the individual components of the BM microenvironment in cancer patients, compare it to the healthy BM, and discuss its impact on interactions between the tumor and the immune system.     

记忆CD8^＋T细胞与造血干细胞移植免疫

移植物抗宿主病(graft-versus-host disease,GVHD)是异基因造血干细胞移植(allogeneic haematopoietic stem celltransplantation,Allo-HSCT)的主要并发症,主要由供者T淋巴细胞介导,如何有效控制GVHD,从而提高移植患者的生存率和生活质量一直是研究热点。记忆CD8 T细胞来源于效应CD8 T细胞,至少分为两个亚群:中央型记忆T细胞和效应型记忆T细胞,两者在功能上和游走性方面均有不同。记忆CD8 T细胞依赖IL-2、IL-7和IL-15等细胞因子支持而稳定生存,当再次遇到能迅速地增生和分化为效应细胞并参与二次免疫应答,在骨髓移植后GVHD的维持中起着重要作用。记忆性干细胞表达CD44LoCD62Lhi,具有自我复制更新的能力,在受到刺激时可产生效应细胞和所有记忆性细胞亚群。供、受者抗原提呈细胞对异体反应性记忆T细胞的发生和调节起不同的作用。     

记忆CD8+T细胞与造血干细胞移植免疫

目的：观察5-氮-2＇-脱氧胞苷（5-aza-2＇-deoxycytidine，又称5-aza-CdR）对卵巢癌细胞SKOV3和3AO增殖凋亡及DNA错配基因hMLH1和hMLH2表达的影响。方法：以特异性甲基转移酶抑制剂5-aza-CdR0．5、5、50μmol／L处理人卵巢癌细胞SKOV3和3AO3d，继续常规培养7d后，采用MTT比色法观察细胞经药物处理前后的增殖活性，用流式细胞术分析5-aza-CdR对细胞凋亡影响，以半定量逆转录-聚合酶链反应（RT—PCR）检测细胞经5-aza-CdR处理前后DNA错配修复基因hMLH1和hMSH2 mRNA表达水平的改变。结果：人卵巢癌细胞SKOV3和3AO经5-aza-CdR处理后，与对照组比较，0．5、5、50μmol／L均能明显抑制肿瘤细胞生长，随着5-aza-CdR浓度增加，细胞增殖速度下降。SKOV3经5-aza-CdR0．5、5、50μmol／L处理后细胞的凋亡率分别为（10．59±1．57）％、（17．52±1．72）％、（34．10±1．45）％，3AO经0．5、5、50μmol/L5-aza-CdR处理后细胞的凋亡率分别为（11．11±2．21）％、（17．24±1．11）％、（26．53±2．00）％，与对照组相比均有统计学意义（P〈0．01）；且凋亡率与剂量成正相关（FSKOV3=227．6，PSKOV3〈0．01；F3AO=108．4，P3AO〈0．01）。经5-aza-CdR处理后的两株卵巢癌细胞中hMLH1和hMLH2的mRNA表达量有不同程度的增加（P〈0．01），且与药物存在剂量依赖性。结论：在人卵巢癌细胞株SKOV3和3AO中，5-aza-CdR可部分逆转hMLH1和hMLH2的失活，恢复其生长调控功能，抑制肿瘤细胞生长，并诱导细胞凋亡。     

The subsets of dendritic cells and memory T cells correspond to indoleamine 2,3-dioxygenase in stomach tumor microenvironment

The abnormal distributions of memory T cells (Tm) and dendritic cells (DC) in stomach cancer are not well understood. Indoleamine 2,3-dioxygenase (IDO), produced by DC, may be an important enzyme affecting function and proliferation of Tm. In this study, IDO expression was examined by immunohistochemical staining. The subsets of Tm and DC were counted by flow cytometry. The percentages of CD4?+?Tm and CD4?+?central Tm (Tcm) were lower in tumor tissues than in normal tissues (P?P?P?=?0.009). The high expression of IDO was more frequently observed in tumor tissues (P?=?0.001). The percentages of CD4?+?Tm and CD8?+?Tm were positively associated with DC1 percentage and ratio of DC1/DC2 (P?P?=?0.025). The patients with high IDO expression had significantly lower CD4?+?Tm (P?=?0.012) and CD8?+?Tm percentages (P?=?0.033), but higher CD8?+?Tem percentage (P?P?=?0.019). The CD4?+?Tm and CD8?+?Tem percentages were significantly associated with clinical stage and lymph node metastasis; the high IDO expressions were significantly associated with deeper tumor invasion (P?=?0.016) and lymph node metastasis (P?=?0.038). Thus, DC subsets, Tm subsets, and IDO expression were correlated with each other. They were associated with the established clinicopathologic features, such as tumor size, depth of invasion, lymph node metastasis, and clinical stage.     

Bone marrow is a reservoir for CD4+CD25+ regulatory T cells that traffic through CXCL12/CXCR4 signals

CD4(+)CD25(+) regulatory T cells (Tregs) mediate peripheral T-cell homeostasis and contribute to self-tolerance. Their homeostatic and pathologic trafficking is poorly understood. Under homeostatic conditions, we show a relatively high prevalence of functional Tregs in human bone marrow. Bone marrow strongly expresses functional stromal-derived factor (CXCL12), the ligand for CXCR4. Human Tregs traffic to and are retained in bone marrow through CXCR4/CXCL12 signals as shown in chimeric nonobese diabetic/severe combined immunodeficient mice. Granulocyte colony-stimulating factor (G-CSF) reduces human bone marrow CXCL12 expression in vivo, associated with mobilization of marrow Tregs to peripheral blood in human volunteers. These findings show a mechanism for homeostatic Treg trafficking and indicate that bone marrow is a significant reservoir for Tregs. These data also suggest a novel mechanism explaining reduced acute graft-versus-host disease and improvement in autoimmune diseases following G-CSF treatment.     

肝癌微环境中CD4~+CD25~+调节性T细胞与T细胞免疫的关系

目的:探讨肝细胞癌组织中CD4 CD25 调节性T细胞(CD4 CD25 regulartory Tcell,Treg)与肿瘤微环境T细胞免疫的关系。方法:对52例肝细胞癌组织和癌旁组织用CD4、CD25双重酶标免疫组化染色和用CD8 EnVision法染色,对癌组织中Treg细胞和CD4 T、CD8 T、CD4 T/CD8 T比值进行相关性分析。结果:正常肝脏组织中未发现Treg细胞,肝癌和癌旁组织中Treg细胞单个高倍视野平均数分别为(7.6±2.84)、(5.2±1.67),两组比较有显著差异(P<0.01);肝癌及癌旁组织中CD4 T细胞单个高倍视野平均数分别为(18.2±3.57)、(25.9±3.36),两组比较有显著差异(P<0.01);肝癌及癌旁组织中CD8 T细胞单个高倍视野平均数分别为(49.9±6.61)、(49.5±6.43),两组比较无明显差异;肝癌及癌旁组织中CD4 T/CD8 T比值分别为(0.37±0.08)、(0.53±0.09),两组比较有显著差异(P<0.01);肝癌组织中Treg细胞的数量与其浸润性CD4 T淋巴细胞的数量及CD4 T/CD8 T比值呈显著负相关(P<0.01),而与浸润性CD8 T淋巴细胞的数量分布无明显相关性。结论∶Treg细胞在肝癌微环境中可能通过抑制CD4 T淋巴细胞的增殖来抑制肿瘤局部免疫。     

PD-L1+ dendritic cells in the tumor microenvironment correlate with good prognosis and CD8+ T cell infiltration in colon cancer

BackgroundThe prognostic value of tumor‐associated dendritic cells (DC) in colon cancer remains poorly understood. This may be in part due to the interchangeable expression of immunostimulatory and immunoinhibitory molecules on DC. Here we investigated the prognostic impact of CD11c⁺ DC co–expressing the immunoinhibitory molecule PD‐L1 and their spatial relationship with CD8⁺ T‐cells in patients treated for stage III colon cancer.MethodsTissue microarrays containing representative cores of central tumor, leading edge, and adjacent normal tissue from 221 patients with stage III colon cancer were immunostained for CD8, CD11c, PD‐L1, and cytokeratin using immunofluorescent probes. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kaplan‐Meier estimates and Cox regression were used to assess survival.ResultsIntratumoral CD8⁺ cell density (HR = .52, 95% confidence interval [CI] .33‐.83, P = .007), stromal CD11c⁺ cell density (HR = .52, 95% CI .33‐.83, P = .006), intratumoral CD11c⁺PD‐L1⁺ cell density (HR = .57, 95% CI .35‐.92, P = .021), and stromal CD11c⁺PD‐L1⁺ cell density (HR = .48, 95% CI .30‐.77, P = .003) on leading‐edge cores were all significantly associated with good survival. CD8⁺ cell density was positively correlated with both CD11c⁺ cell density and CD11c⁺PD‐L1⁺ cell density in tumor epithelium and stromal compartments.ConclusionHere we showed that PD‐L1‐expressing DC in the tumor microenvironment are associated with improved survival in stage III colon cancer and likely reflect an immunologically “hot” tumor microenvironment. Further investigation into the expression of immunomodulatory molecules by tumor‐associated DC may help to further elucidate their prognostic value.     

Chronic B cell malignancies and bone marrow microenvironment

Chronic B-lymphoid malignancies depend upon supportive interactions within specific microenvironments. Follicular lymphoma (FL), chronic lymphocytic leukaemia (CLL) and multiple myeloma (MM) cells accumulate in the bone marrow (BM) where they receive survival or growth signals from by-stander cells. However, they deeply differ in their interaction with the microenvironment. We propose a model where FL and CLL recreate in the BM the microenvironment most suitable to their growth by 'importing' the normal cells that usually nurse them in secondary lymphoid organs. In contrast, MM takes advantage of the actual BM microenvironment by 'instructing' it through an abnormal activation state.     

Bone marrow transplantation: how important is CD34 cell dose in HLA-identical stem cell transplantation?

A recent analysis of Fred Hutchinson Cancer Research Center data has been undertaken to investigate the association of infused CD34 cell dose with various clinical outcomes after HLA-identical transplantation. Separate assessments for unrelated vs related donors and the use of bone marrow or mobilized G-CSF stimulated peripheral blood mononuclear cells (G-PBMC) have been incorporated. The three primary findings are: (1) Higher CD34 dose results in better neutrophil and platelet recovery in all settings. (2) Higher CD34 doses (8 x 10(6)/kg) are associated with the development of more chronic graft-versus-host disease when using related G-PBMC. (3) Higher CD34 dose is correlated with improved survival after unrelated donor bone marrow transplantation. These data suggest that the CD34 content of a graft can have a significant impact on clinical outcome after allogeneic transplantation, but defining an optimal dose is dependent on both the type of donor and the stem cell source.     

Tat mammaglobin fusion protein transduced dendritic cells stimulate mammaglobin-specific CD4 and CD8 T cells

Summary Proteins can be efficiently introduced into cells when fused to a protein transduction domain, such as Tat from the human immunodeficiency virus. We recently reported that dendritic cells transduced with a Tat fusion protein containing the extracellular domain of Her2/neu (Tat-Her2/neu) induced CD8 cytotoxic T lymphocytes (CTL) that specifically lysed Her2/neu-expressing breast and ovarian cancer cells. In the current study we further investigated the mechanism of protein transduction, utilizing the breast cancer-associated protein, mammaglobin-A, which is expressed in about 80% of breast cancers. Using a Tat-mammaglobin fusion protein, we tested the ability of Tat-mammaglobin transduced dendritic cells to stimulate antigen-specific CD4 and CD8 T cells. Low levels of serum considerably improved protein transduction as determined by Western blot, and also improved presentation of antigenic peptide as evidenced by functional studies using antigen-specific T cells. Confocal microscope analyses of antigen-presenting cells (APC) incubated with Tat-mammaglobin showed localized distribution in addition to diffuse distribution in the cytosol. In contrast, mammaglobin lacking Tat showed only a localized distribution. Simultaneous incubation with both proteins resulted in overlapping localized distributions, suggesting Tat fusion proteins are processed through both the MHC class I and class II pathways. Indeed, stimulation of T cells with Tat-mammaglobin transduced dendritic cells led to an expansion of mammaglobin-specific CD4 T helper-1 lymphocytes along with CD8 CTL. We conclude that Tat-mammaglobin transduced dendritic cells can induce both CD4 and CD8 mammaglobin-specific T cells. These findings could be further exploited for the development of a mammaglobin-based vaccine for breast cancer.     

CD+4CD+25调节性T细胞与肿瘤免疫

 近期研究发现一个有独特免疫调节功能的T细胞亚群：CD+4 CD+25调节性T细胞,不仅能抑制自身免疫性疾病发生,还参与肿瘤免疫的调节。这群细胞具有免疫无能和免疫抑制特性,与肿瘤免疫逃逸有密切的关系。肿瘤环境中CD+4 CD+25调节性T细胞增加,导致肿瘤免疫失调,去除这群细胞可有效诱导肿瘤免疫,为肿瘤治疗提供了一种新的思路。     

Bone marrow CD34+ cell count is predictive for adequate peripheral progenitor cell collection

Several studies have investigated the influence of clinical and biological variables on mobilisation of peripheral blood progenitor cells (PBPCs). The aim of this study was to evaluate the role of steady-state bone marrow (BM) CD34+ cells as a predictive parameter of PBPC yield. We studied 90 patients with multiple myeloma (MM) (41 patients), non-Hodgkin's lymphoma (NHL) (25 patients) or acute myeloid leukaemia (AML) (24 patients), mobilised with chemotherapy and growth factor. The median time from first treatment to mobilisation was 5 months. Only one patient was previously exposed to alkylating agents. The median BM CD34+ count at mobilisation was 833 microl(-1) (range: 1.4-15.540) corresponding to 1.51% of mononuclear cells (range: 0.02-8.6). Sixty-six patients (73%) reached the optimal target of 4 x 10(6) kg(-1) CD34+ cells with 1 (18 patients), 2 (42 patients) or 3 leukaphereses (6 patients). Eleven patients (12%) mobilised less than 4 x 10(6) kg(-1) CD34+ cells and 13 (15%) failed mobilisation. Among the laboratory and clinical parameters evaluated at the time of mobilisation, only BM CD34+ count was a predictive factor for adequate collection (P = 0.04), particularly in MM patients (P = 0.003). In this setting, a BM concentration of CD34+ cells lower than 66 microL(-1) was associated with a higher probability of inadequate collection.     

Human CD8 and CD4 T cell epitopes of epithelial cancer antigens

Recent human tumor immunology research has identified several genes coding immunogenic peptides recognized by CD8 cytotoxic T lymphocytes (CTLs) in melanoma tumors. Very recently, CD4 T cell antigenic epitopes were also determined in certain melanoma tumors. The use of these peptides in conjunction with human immunotherapy could prove to be of great benefit. However, such peptides in clinically common tumors of epithelial cell origin, such as of the stomach, colon, lung, etc., have not yet been determined extensively. We describe for the first time an HLA-A31 (A*31012)-restricted natural antigenic peptide recognized by the CD8 CTL TcHST-2 of gastric signet ring cell carcinoma cell line HST-2. We also identified the HLA-DRB1*08032-restricted peptide recognized by the CD4 T cell line TcOSC-20 of squamous cell carcinoma OSC-20 derived from the oral cavity. The antigenic peptide of HST-2, designated F4.2, is composed of 10 amino acid residues with two anchor motif residues necessary for binding to HLA-A31 molecules. The synthetic F4.2 peptide enhanced the reactivity of TcHST-2 against HST-2 cells. Furthermore, introduction of an expression minigene coding F4.2 peptide to HLA-A31(+) cells conferred cytotoxic susceptibility to TcHST-2 on the cells. Some stomach cancer lines into which the HLA-A31 gene had been introduced, such as MKN28-A31-2, were lysed by TcHST-2, suggesting the presence of F4.2 peptide in at least some HLA-A31(+) stomach cancers. Furthermore, F4.2 peptide induced an F4.2 peptide-specific CTL response in at least 30-40% of HLA-A31(+) peripheral blood lymphocytes from gastric cancer patients, suggesting that F4.2 peptide could be used as a cancer vaccine for gastric tumors. The natural antigenic peptide of OSC-20 was also determined using acid extraction and biochemical separation and by mass spectrometry. Consequently, OSC-20 peptide was designated as the 6-1-5 peptide, an HLA-DRB1*08032-restricted 16-mer peptide with two possible anchor motifs. It has an amino acid sequence identical to that of human !-enolase, suggesting that it was derived from the processed parental !-enolase protein. We are presently attempting to determine the genes that code tumor rejection antigens recognized by HLA-A24- and A26-restricted T cells, including those of pulmonary and pancreatic carcinomas. The search for these antigenic peptides may lead to the identification of immunogenic peptide antigens that would be suitable for clinical use in commonly occurring epithelial cancers.     

Therapeutic limitations in tumor-specific CD8+ memory T cell engraftment

Background  

Adoptive immunotherapy with cytotoxic T lymphocytes (CTL) represents an alternative approach to treating solid tumors. Ideally, this would confer long-term protection against tumor. We previously demonstrated that in vitro -generated tumor-specific CTL from the ovalbumin (OVA)-specific OT-I T cell receptor transgenic mouse persisted long after adoptive transfer as memory T cells. When recipient mice were challenged with the OVA-expressing E.G7 thymoma, tumor growth was delayed and sometimes prevented. The reasons for therapeutic failures were not clear.     

Antigen-specific CD4+ T-cell help is required to activate a memory CD8+ T cell to a fully functional tumor killer cell

Although the importance of CD4+ T-cell help for generation of an effective CD8+ effector cytotoxic T cell (CTL) response is well established, the role of T-cell help in the activation of memory T cells to become fully functional tumor killer cells is undefined. Using synthetic peptide immunizations corresponding to the major CTLs and T-helper epitopes of ovalbumin, adoptive transfers of ovalbumin-specific memory CTLs (mCTLs), and ovalbumin as the tumor-specific antigen in a mouse tumor model, we have determined that T help is essential for the activation of mCTLs to kill tumors. Our data show that T-helper cells specific for the tumor-associated antigen are required for the reactivation of mCTLs by antigen presented indirectly from tumor. In contrast, effector CTLs do not need T help to kill tumors. These results have implications for induction of tumor immunotherapy by immunization.