Dominant clonotypes in the repertoire of peripheral CD4+ T cells in rheumatoid arthritis.

Clonal expansion of T cell specificities in the synovial fluid of patients has been taken as evidence for a local stimulation of T cells. By studying the T cell receptor (TCR) repertoire of CD4+ T cells in the synovial and peripheral blood compartments of patients with early rheumatoid arthritis (RA), we have identified clonally expanded CD4+ populations. Expanded clonotypes were present in the peripheral blood and the synovial fluid but were not preferentially accumulated in the joint. Dominant single clonotypes could not be isolated from CD4+ cells of HLA-DRB1*04+ normal individuals. Clonal expansion involved several distinct clonotypes with a preference for V beta 3+, V beta 14+, and V beta 17+CD4+ T cells. A fraction of clonally related T cells expressed IL-2 receptors, indicating recent activation. The frequencies of clonally expanded V beta 17+CD4+ T cells fluctuated widely over a period of one year. Independent variations in the frequencies of two distinct clonotypes in the same patient indicated that different mechanisms, and not stimulation by a single arthritogenic antigen, were involved in clonal proliferation. These data support the concept that RA patients have a grossly imbalanced TCR repertoire. Clonal expansion may result from intrinsic defects in T cell generation and regulation. The dominance of expanded clonotypes in the periphery emphasizes the systemic nature of RA and suggests that T cell proliferation occurs outside of the joint.     

多发性硬化患者外周血CD8+CD28-调节性T细胞的变化

目的 探讨多发性硬化(MS)患者外周血CD8+CD28-T细胞数量的变化及临床意义.方法 采用前瞻性病例对照研究,收集自2005年10月至2008年8月间在温州医学院附属第一医院神经专科病门诊或病房治疗的处于急性活动期的复发缓解型MS患者51例,均符合2005年修改的Mc-Donald诊断标准[2].所有住院患者均给予甲基强的松龙1.0 g/d,连续使用5 d后改为强的松60mg/d,12 d后减量,总疗程不超过6周,对14例MS患者进行治疗前后的动态观察.以20例健康体检者为对照组(NC组),MS组和NC组在年龄、性别构成上差异无统计学意义.采用流式细胞技术检测外周血CD8+CD28-,CD8+CD28+,CD8+及CD4+CD8-T细胞的百分比.两组间均数比较采用独立样本t检验,治疗前后比较采用配对样本t检验,相关性分析采用Pearson相关检验.结果 急性活动期MS组CD8+CD28-T细胞的百分比为(18.48±9.89)%,低于NC组的(24.48±4.86)%(P<0.01),但CD8+CD28+T细胞百分比为(12.23±4.31)%,高于NC组的(8.55±3.49)%(P<0.01),CD8+T细胞的百分比两组比较差异无统计学意义(P>0.05);MS组CD8+CD28-与CD4+CD8-T细胞的百分比呈负相关性(r=0.488,P<0.01);MS患者激素治疗后CD8+CD28-和CD8+CD28+T细胞卣分比与治疗前相比差异无统计学意义(均P>0.05),且治疗后CD8+CD28-T细胞百分比(16.22±4.25)%低于NC组(P<0.01),CD++CD28+T细胞百分比(13.04±4.23)%高于NC组(P<0.01).结论 CD8+CD28-T细胞数日的减少参与了MS的病理过程,并可能通过调节CD4+T细胞起作用;MS急性期激素治疗并不能恢复CD8+CD28-T细胞的数目,激素可能是通过其他途径发生作用.     

RA患者外周血CD4/CD8 T细胞比值及CD4+ CD25+ T细胞的检测及其临床意义

目的 观察活动期类风湿性关节炎（RA）患者外周血T细胞CD4／CD8比值及CD4^＋ CD25^＋T细胞（Tn细胞）的数量,探讨其在RA诊治中的价值。方法 用流式细胞术检测CD4^＋ CD25^＋T细胞及TR细胞的数量。结果 49例急性期RA患者的CD4／CD8比值和TR细胞数量显著高于正常人群组。28例RA患者治疗后的TR细胞数量显著高于治疗前,而CD4／CD8比值的变化无统计学意义。结论 RA治疗后症状改善患者,TR细胞数量明显上升,RA的发病和发展与该细胞数量密切相关。     

Survival, persistence, and progressive differentiation of adoptively transferred tumor-reactive T cells associated with tumor regression

Objective clinical responses have been observed in approximately 50% of patients who received non-myeloablative chemotherapy prior to the adoptive transfer of autologous melanoma-reactive tumor-infiltrating lymphocytes (TILs). Recent studies carried out through the use of antibodies directed against T-cell-receptor beta chain variable region (TRBV) products, as well as by direct sequencing of the expressed TRBV gene products, indicated that clinical responses in this trial were associated with the level of persistence of adoptively transferred T cells. In an attempt to further characterize T cells that persist in vivo following adoptive transfer, five dominant T-cell clonotypes were identified in TIL 2035, an adoptively transferred TIL that was associated with the complete regression of multiple metastases. The most highly persistent clonotype, which expressed the BV1 TR gene product, recognized the MAGE-6 cancer/testis antigen in the context of HLA-A23. This clonotype was detected in peripheral blood for over 16 months following adoptive transfer, expressed relatively higher levels of the co-stimulatory markers CD28 and CD27, and possessed telomeres that were long relative to other clonotypes present in TIL 2035 that showed only short-term persistence. The long-term persistent BV1 clonotype appeared to differentiate more slowly toward an end-stage effector in vivo than short-term persistent clonotypes, as manifested by the downregulation of CD28, CD27, and CD45RO and upregulation of CD57 and CD45RA expression on these T cells. These results indicated that the differentiation stage and replicative history of individual TIL clonotypes might be associated with their ability to survive and to persist in vivo, and progressive differentiation of the persistent clonotypes occurred following adoptive transfer.     

In vivo persistence of expanded clones specific for bacterial antigens within the human T cell receptor alpha/beta CD4-8- subset

We analyzed the T cell receptor (TCR) rearrangements of 100 TCR- alpha/beta CD4-CD8- (double negative [DN]) T cell clones from normal individuals. We found that in four out of six donors this subset contains expanded clones that often account for 0.5% and, in one individual, even 7% of all peripheral blood lymphocytes. By combining limiting dilution analysis and N region oligotyping of polymerase chain reaction amplified TCR cDNA, we could measure the clonal size and show that two of these expanded clones remain stable in size for up to 4 yr in peripheral blood. The expanded clones analyzed ex vivo are not cycling and CD45 RAhi ROlo, but express high levels of alpha 4/beta 1 integrins, suggesting that they may have reverted to resting cells after activation. One of these expanded DN clones proliferates in vitro in response to Escherichia coli presented by monocytes cultured in GM- CSF plus IL-4 and kills CD1a+ Molt-4 cells. In contrast to what was found in the alpha/beta DN subset, alpha/beta CD4+ T cell clones specific for a tetanus toxin epitope showed a very small clonal size (< 1 in 10(7)) and could not be reisolated after 2 yr. Taken together, these results indicate that large clonal size and persistence are distinctive features of alpha/beta DN cells specific for bacterial antigens. These cells may use antigen-presenting cells, restriction molecules, and selection routes different from those used by antigen- specific CD4+ T cells.     

调节性T细胞的新标记CD4~+CD25~+CD127~(low/-)在类风湿性关节炎中的应用

目的选择用膜表面标志CD4+CD25+CD127low/-作为检测调节性T(Treg)细胞的指标,探讨其在类风湿性关节炎(RA)中的可能临床应用价值。方法用流式细胞术检测正常人及RA患者外周血CD4+CD25high、CD4+CD25+FoxP3+和CD4+CD25+CD127low/-T细胞占CD4+T细胞的比例,分析CD4+CD25+CD127low/-与CD4+CD25+FoxP3+2群细胞比例之间的相关性。结果正常人及RA患者外周血CD4+CD25+CD127low/-T细胞比例与CD4+CD25+FoxP3+T细胞比例之间呈显著正相关(r=0.694、0.768,P均<0.01)。RA患者外周血CD4+CD25high、CD4+CD25+FoxP3+及CD4+CD25+CD127low/-T细胞比例均显著低于正常人(P均<0.01)。结论膜表面标志CD4+CD25+CD127low/-可以用来鉴定Treg细胞,RA患者外周血CD4+CD25+CD127low/-T细胞的明显减少可能是RA的发病机制之一。     

类风湿关节炎继发肺间质病变患者激素冲击治疗前后 CD8+CD28-T 细胞的变化及意义

目的探讨类风湿关节炎（RA）继发肺间质病变（ILD）患者激素冲击治疗前后CD8+CD28-T细胞的变化与意义方法共入选23例RA-ILD患者（A组）,选取同期就诊的RA患者25例（B组）、非RA致ILD患者25例（C组）、25例健康人（D组）作为对照。对以上具有甲泼尼龙冲击适应症的A组患者进行治疗,治疗前后采用流式细胞术检测上述4组外周血CD8+CD28-T细胞及其细胞因子白介素10（IL-10）和转化生长因子β1（TGF-β1）的百分含量变化。结果与D组比较,A、B及C三组的CD8+CD28-T细胞、IL-10及TGF-β1均明显升高,含量由高至低均表现为A>C>B>D,且四组间任意两组上述三项指标均有统计学差异（P&;#60;0.05）。治疗后A组患者CD8+CD28-T细胞、IL-10及TGF-β1均较治疗前明显降（P&;#60;0.05）。结论CD8+CD28-T细胞在类风湿关节炎继发肺间质病变发病过程可能起到重要作用,而甲泼尼龙可显著抑制该细胞活性,有望作为结缔组织病致肺间质病变的病情观察及疗效评价指标。     

The functional CD8 T cell response to HIV becomes type-specific in progressive disease

High levels of HIV-specific CD8 T cells are demonstrable throughout HIV disease using laboratory assays that measure responses to consensus epitopes. In acute infection, the dynamics of the antiviral CD8 T cell response correlate well with the decline in viremia. However in chronic infection, although responses are detected against a broader spectrum of epitopes, virus-specific CD8 T cells are apparently unable to control viral replication. To investigate whether CD8 T cells responding to consensus epitopes may have lost their in vivo relevance in the chronic phase because of viral evolution driven by immune pressure, we compared the CD8 T cell response to CD4 T cell targets infected with either lab-adapted HIV(IIIB) or the patient's own virus. The magnitude of the IFN-gamma response declined with disease progression, especially to autologous virus. T cell receptor (TCR) clonotypes of HIV(IIIB) and autologous virus-responding cells were determined by sequencing TCR beta chain variable (TCRBV) genes. In two of three asymptomatic donors, the dominant clonotypes overlapped, whereas in five symptomatic patients, the TCR clonotypes responding to HIV(IIIB) virus were completely different from those responding to autologous virus. Moreover, in cytolytic assays, T cell lines derived from IFN-gamma(+) cells responding to lab-adapted or autologous virus cross-recognized target cells infected with either virus in asymptomatic subjects with shared TCR clonotypes but not in progressors with differing clonotypes. Therefore, in advanced-stage patients, viral-specific CD8 T cells recognizing consensus epitopes persist from an earlier response but no longer effectively recognize autologous virus.     

Contribution of CD4+, CD8+CD28+, and CD8+CD28- T cells to CD3+ lymphocyte homeostasis during the natural course of HIV-1 infection.

The relationship between the number of circulating CD4+ T cells and the presence of particular CD8+ T cell subsets was analyzed by flow cytometry on PBL from asymptomatic HIV-1-infected patients whose specimens were collected every 2 mo for a total period of 32 mo. Only slight variations were detected in the absolute number of lymphocytes and percentage of CD3+ lymphocytes, whereas both CD4+ and CD8+ T cell subsets showed wide intrapatient variation. Variations in the number of CD8+CD28+ cells paralleled those of the CD4+ T cell subset in each patient tested, while the presence of CD8+CD28- T cells correlated inversely with CD4+ and CD8+CD28+ T cells. These data show that changes in the number of circulating CD4+-and CD8+CD28+ T cells are strongly related to the presence of CD8+CD28- T cells in these patients. Insight into the significance of CD8+CD28- T cell expansion will allow us to understand the mechanisms and significance of the HIV-1- driven change in CD4+CD8+ T cell homeostasis and the basic immunopathology of HIV disease.     

Dendritic cell-lymphocyte clusters that form spontaneously in rheumatoid arthritis synovial effusions differ from clusters formed in human mixed leukocyte reactions.

Lymphocytes cluster about dendritic cells (DC) spontaneously in 48 h cultures of rheumatoid arthritis synovial fluid (RA SF) mononuclear cells and in peripheral blood autologous or allogeneic mixed leukocyte reactions. In the latter case, the clusters are predominantly CD4+ T cells (T4/T8 greater than 5) and with time progress in blastic cells that express IL-2 (Tac) and/or transferrin (T9) receptors. In contrast, the clusters in RA SF cultures have a T4/T8 ratio of less than 1 and a majority of the T8 cells coexpress the Leu 7 marker. T cells in these clusters remain inert and with time the clusters disintegrate. Addition of IL-1, IL-2, or IFN-gamma alone or in combination had no effect on RA SF clusters but T cells became blastic when exposed to 10% RA SF. Mixing experiments using RA SF DC with normal T cells and RA T cells with normal DC show that both RA SF DC and T cells are immunofunctional. In addition, clusters of RA SF from a patient with active tuberculosis proliferated vigorously to PPD. Therefore, the unique RA SF cluster profile may reflect the memory nature of the RA SF T cells resulting in a paucity of T cells that are responsive to autologous stimulation. However, an immunosuppressive role for the double-labeled (CD8 and Leu 7) cells has not been excluded.     

Highly efficient expansion of human CD4+CD25+ regulatory T cells for cellular immunotherapy in patients with graft-versus-host disease

CD4+CD25+ regulatory T cells (T(REG)) are engaged in the regulation of murine and human immune responses as well as graft-versus-host disease (GvHD) after allogeneic stem-cell transplantation. Despite their suppression of GvHD they do not impair graft-versus-tumor activity in the mouse, which makes T(REG) especially attractive candidates for cellular immunotherapy. T(REG) comprise only 5% to 10% of CD4+ T cells in peripheral blood and are naturally anergic, which prevented their use as therapeutic suppressor cells in the context of autoimmune or alloimmune reactions so far. We therefore developed an in vitro expansion protocol for human T(REG), breaking their anergy with anti-CD3/anti-CD28-coupled paramagnetic beads and a combination of interleukin (IL)-2 and IL-15. Highly purified human T(REG) can be expanded 285-fold to 1000-fold within 20 days and keep their phenotype as well as all their suppressor functions even in the context of stimulation with mature allogeneic dendritic cells. However, we demonstrate that FoxP3 is not a reliable marker for human T(REG) as it is transiently inducible in CD4+CD25- cells upon activation with cytokines or via their T cell receptor. In addition, we successfully expanded CD4+CD25+ cells from patients after allogeneic stem-cell transplantation with or without GvHD and show that different suppressor functions might be lost independently, demonstrating that human T(REG) biology is likely more complicated than previously thought.     

Human papillomavirus 16 E6-specific CD45RA+ CCR7+ high avidity CD8+ T cells fail to control tumor growth despite interferon-gamma production in patients with cervical cancer

We defined the nature of the cellular immune response in 5 women with human papillomavirus (HPV) 16+cervical carcinoma at a single time point when surgery was performed for treatment. To monitor the differences in T-cell recognition, 2 approaches of tetramer-guided technology were employed: (i) the in situ localization of major histocompatibility complex class I peptide complexes in the tumor lesions and (ii) the ex vivo sorting of HLA-A*0201-restricted and HPV16 E6-reactive T cells. CD8 T cells from the periphery (peripheral blood lymphocytes), the tumor (tumor-infiltrating lymphocytes), and T cells harvested from draining lymph nodes (T-LN) were analyzed. HPV16 E6 tetramer-sorted lymphocytes from the different anatomic sites recognized an HLA-A*0201-restricted E6 peptide irrespective of the type of antigen-presenting cells used for stimulation as determined by interferon-gamma production: autologous tumor cells, HLA-A*0201 surrogate antigen-presenting cells pulsed with the nominal peptide, and an HLA-A*0201-matched human dendritic cell line transgenic for HPV16 E6. Further analysis showed that the HPV16 E6-reactive CD8 T cells were of high avidity defined by blocking with an anti-CD8-alpha specific monoclonal antibody. We found that HPV16 E6-reactive T cells reside preferentially within the CD45RA+ CCR7+ T-cell subpopulation of tumor-infiltrating lymphocyte, peripheral blood lymphocyte, and T-LN in cervical cancer patients, suggesting that successful immune surveillance of HPV16+ tumor cells in cervical cancer patients is impaired. The CD45RA+/CCR7+ phenotype of HPV antigen-reactive T cells may serve as an indicator of dysfunctional T cells, despite effective interferon-gamma production in response to HPV antigens.     

类风湿性关节炎的发生发展与外周血T淋巴细胞亚群及CD4+TH1/TH2细胞功能亚型的关系

背景类风湿性关节炎作为一种自身免疫病其免疫学发病机制目前尚未完全明确.T淋巴细胞、尤其是CD4+ TH1/TH2细胞在类风湿性关节炎发生发展中可能有重要作用.目的探讨主要参与细胞免疫的CD4+ TH1细胞和辅助体液免疫应答的TH2细胞在类风湿性关节炎发生发展过程中的作用.设计病例-对照,对比观察.单位武警医学院免疫学教研室.对象选择1999-03/2000-03在天津医科大学总医院内科就诊的类风湿性关节炎患者15例为患者组,男2例,女13例,其中12例受检者类风湿因子阳性,3例为阴性.同期选取健康体检者或本单位工作人员健康者30人为对照组,男4人,女26人.纳入对象均对实验目的知情同意.方法①采用酶联免疫斑点法对两组对象的外周血单个核细胞中CD3+T细胞(总T细胞)、CD4+T细胞和CD8+T细胞进行检测,普通光学显微镜油镜下计数200～500个淋巴细胞,计算出阳性细胞的百分率.②采用酶联免疫斑点法检测活化的分泌细胞因子的TH细胞;细胞内有红色斑点的细胞为阳性细胞,分泌γ-干扰素的细胞为TH1细胞,分泌白细胞介素4的细胞为TH2细胞;普通光学显微镜油镜下计数200～500个淋巴细胞,计算出TH1细胞、TH2细胞的百分率及TH1/TH2细胞的比值.③采用t检验或x2检验比较数据间差异性.主要观察指标两组对象外周血T淋巴细胞亚群(包括CD3+T细胞、CD4+T细胞、CD8+T细胞)及CD4+TH1/TH2细胞定量分析结果.结果类风湿性关节炎患者15例和健康者30人均进入结果分析.①两组外周血的总T细胞(即CD3+T细胞)、CD4+T细胞及CD8+T细胞的百分率差异不明显(P＞0.05).②患者组外周血的TH1细胞的百分率明显高于对照组[(24.44±5.25)%,(14.93±3.82)%,P＜0.05];而其外周血TH2细胞的百分率及TH1/TH2细胞的比值与对照组相近(P＞0.05).结论TH1细胞介导的细胞免疫可能与类风湿性关节炎的发生发展有关.     

Phenotypic and functional lymphocyte recovery after CD34+-enriched versus non-T cell-depleted autologous peripheral blood stem cell transplantation

To determine the effect of CD34+ selection on immune recovery after high-dose chemo/radiotherapy in the setting of autologous stem cell transplantation (ASCT), we analyzed quantitative and qualitative lymphocyte reconstitution for up to 1 year post-transplantation in 27 consecutive adult patients receiving either CD34+-enriched or unmanipulated autologous stem cell (SC) grafts. Pretransplant immunological parameters were identical for both treatment groups. Total lymphocyte counts as well as CD3+ T cells provided a similar course of recovery in both cohorts, returning to baseline values within the first 3 months. There were no significant differences in the reconstitution kinetics of CD4+, CD8+, CD45RA+, and CD45RO+ T cells. CD4+ and CD45RA+ T cells between the two groups were significantly decreased within the first 6 months, returning to pretransplant baseline values by 1 year. Although within the first 3 months the majority of CD3+ cells were activated as demonstrated by expression of HLA-DR, we observed a significant loss of CD25+ T cells in both groups within the first 6 months. B cell numbers returned to baseline values within 3 months but in vivo B cell function measured by serum immunoglobulin M (IgM) and IgA levels did not recover as early as 6 months post-transplantation. T cell function measured by proliferation in response to the lectins phytohemagglutinin (PHA) and Concanavalin A (ConA) and to alloantigens in the mixed lymphocyte reaction (MLR) was significantly impaired, but tended to return to pretransplant baseline values by 1 year. Although preliminary, our results provide strong evidence that T cell depletion (TCD) by CD34+ enrichment using the CellPro device does not result in delayed phenotypic immune reconstitution after autologous peripheral blood stem cell transplantation (PB-SCT). Even in the absence of a high thymic T cell regenerative capacity in adults, T cell numbers and subset distributions were restored within the time frame studied. T and B cell function, however, remained significantly impaired for a prolonged period of time (>6 months after SCT) with a more profound defect in patients autografted with CD34+-enriched SC.     

A clinical-scale expansion of mobilized CD 34+ hematopoietic stem and progenitor cells by use of a new serum-free medium

BACKGROUND: The autologous transplantation of CD 34+ cells expanded ex vivo in serum-free conditions dramatically reduces post-myeloablative neutropenia in myeloma patients. In our cell therapy unit, cells for this clinical assay have been expanded under GMP with serum-free Irvine Scientific (IS) medium with stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor (MGDF; 100 ng/mL, respectively). Because this clinical-grade IS medium is no longer available, a new serum-free medium, Maco Biotech HP 01 (Macopharma), was evaluated. STUDY DESIGN AND METHODS: Purified CD 34+ cells (Isolex 300i, Baxter) from mobilized peripheral blood samples of myeloma patients were thawed, washed, and cultured, as for previous clinical assays. Twenty million CD 34+ cells were resuspended per 1 L of SCF-, G-CSF-, and MGDF-supplemented medium (HP 01 or IS), introduced into 3-L culture bags (AFC), and cultured for 10 days in 5 percent CO(2), at 37 degrees C, and at 100 percent humidity. RESULTS: A higher amplification of total nucleated cells (NCs) and colony-forming cells (CFCs) was obtained with HP 01 medium than with IS medium (42+/-16.6-fold vs. 20.5+/-5.9-fold for NCs and 26.7+/-7.4-fold vs. 15.5+/-2.5-fold for CFCs, respectively), whereas an increase in CD 34+ cells (3.5+/- 1.2-fold for HP 01 vs. 2.7+/- 1.5-fold for IS) was not significant. IS medium partially maintained SCID-repopulating cells (SRC), whereas the culture in HP 01 medium fully maintained the stem cell activity for 10 days. A higher frequency of CD 41+ cells after expansion in HP 01 than in IS medium was also observed. CONCLUSION: Maco Biotech HP 01 medium is suitable for clinical-scale expansion of CD 34+ cells with the SCF, G-CSF, and MGDF cytokine cocktail, permitting an intensive amplification of CFCs and maintenance of SRCs.     

粒系集落刺激因子对外周血T淋巴细胞亚群的影响及与CD34+细胞动员效果的相关性

本研究观察粒系集落刺激因子(G—CSF)作为造血干细胞动员剂对外周血T淋巴细胞亚群的影响及与CD34^ 细胞动员效果的关系。对26例行自体造血干细胞移植患在G—CSF动员前后收集外周血标本，用流式细胞术检测动员前后CD3^ 、CD3^ CD4^ 、CD3^ CD8^ 、CD3^ CD4^ CD8^ 及CD3^ CD4^-CD8细胞绝对数量的变化并与外周血CD34^ 细胞的动员效果进行相关性分析。结果表明：GCSF动员后外周血CD3^ 、CD3^ CD4^ 、CD3^ CD4^ CD8^ 及CD3^ CD4^-CD8细胞的绝对数量分别增加2.23，2．62，2.99及10．96倍，而CD3^ CD4^ CD8^ 细胞的变化无统计学意义(P=0．243)。各亚群细胞的变化与CD34^ 细胞动员效果比较，仅CD3^ CD4 CD8细胞的变化与CD34^ 细胞动员效果间具有良好的相关性，r=0．796，P=0.000。结论：G—CSF将造血干细胞由骨髓动员到外周血的同时，使外周血中T细胞亚群的绝对数量发生不同程度的变化。在各T淋巴细胞亚群中CD3^ CD8^-细胞的增加与CD34^ 细胞的动员效果间具有统计学意义的相关性。     

脐血体外同时诱导扩增T,NK和CD34+细胞

体外研究表明,人脐血含有比骨髓细胞更原始更早期的造血干细胞群。移植后所引起的GVHD发生率及程度都比骨髓及外周血要低,但其相应的GVL效应也较低,容易复发。单份脐血所含有的造血细胞量仅可以满足一定体重以下的儿童患者需求,对需移植的大部分成人患者则要进行体外扩增。脐血体外扩增后进行干细胞移植是一种新的设想和尝试,移植物中免疫细胞的组成和功能是决定干细胞移植能否成功重建造血与免疫、以及平衡GVHD和GVL的重要因素。为了研究脐血体外同时诱导扩增T、NK和CD34 细胞的可能性,本实验无菌采集健康正常足月产新生儿脐血,并分离出单个核细胞,在不同的细胞因子组合作用下用IMDM培养液培养14天。在培养0、3、7、14天时收集细胞,用FCM分析扩增前后脐血干/祖细胞及T、NK免疫细胞含量。结果表明,与无细胞因子的对照组相比,所有细胞因子SCF,IL3,IL6,IL7,IL2组合组别均能显著扩增脐血中的单个核细胞。所有细胞因子组合组别均能显著增加脐血中的CD34 细胞比例,使其含量从新鲜脐血中的1.6%升高到最高D组的11.9%。第7天时CD34 细胞平均增加10至50倍不等。新鲜脐血中CD3 T细胞平均为(18.7±4.3)%,在无细胞因子的对照组中CD3 T细胞下降明显,而在含细胞因子的组别中CD3 T有不同程度的增加,扩增最高的组别中CD3 T细胞是新鲜脐血的2倍。在新鲜脐血中含(3.6±1.9)?56 细胞。CD56 细胞数量仅在含细胞因子IL2的组别中有显著增加,其它组则无明显变化。结论:脐血中T细胞、NK细胞在一定细胞因子组合下,可与干/祖细胞同时在体外被扩增和诱导分化。     

Immunization with analog peptide in combination with CpG and montanide expands tumor antigen-specific CD8+ T cells in melanoma patients

Analog peptides represent a promising tool to further optimize peptide-based vaccines in promoting the expansion of tumor antigen-specific cytotoxic T lymphocytes. Here, we report the results of a pilot trial designed to study the immunogenicity of the analog peptide NY-ESO-1 157-165V in combination with CpG 7909/PF3512676 and Montanide ISA 720 in patients with stage III/IV NY-ESO-1-expressing melanoma. Eight patients were immunized either with Montanide and CpG (arm 1, 3 patients); Montanide and peptide NY-ESO-1 157-165V (arm 2, 2 patients); or with Montanide, CpG, and peptide NY-ESO-1 157-165V (arm 3, 3 patients). Only the 3 patients immunized with Montanide, CpG, and peptide NY-ESO-1 157-165V in arm 3 developed a rapid increase of effector-memory NY-ESO-1-specific CD8+ T cells, detectable ex vivo. The majority of these cells exhibited an intermediate/late-stage differentiated phenotype (CD28-). Our study further demonstrated that our vaccine approach stimulated spontaneous tumor-reactive NY-ESO-1-specific CD8+ T cells in 2 patients with advanced disease, but failed to prime tumor-reactive NY-ESO-1-specific T cells in 1 patient with no spontaneously tumor-induced CD8+ T-cell responses to NY-ESO-1. Collectively, our data support the capability of the analog peptide NY-ESO-1 157-165V in combination with CpG and Montanide to promote the expansion of NY-ESO-1-specific CD8+ T cells in patients with advanced cancer. They also suggest that the presence of tumor-induced NY-ESO-1-specific T cells of well-defined clonotypes is critical for the expansion of tumor-reactive NY-ESO-1-specific CD8+ T cells after peptide-based vaccine strategies.     

Changes in CD4+ T lymphocyte subsets in circulating blood and synovial fluid following filtration leukocytapheresis therapy in patients with rheumatoid arthritis.

The purpose of this study is to determine the changes in CD4+ T lymphocyte subsets in the circulating blood and synovial fluid following filtration leukocytapheresis (LCP) therapy for patients with rheumatoid arthritis (RA). A Cellsorba column packed with polyester fibers was used for the removal of circulating leukocytes. For patients with RA, filtration LCP or sham procedures were performed 3 times with 1 week intervals between procedures. T lymphocyte surface markers in the peripheral blood and synovial fluid were measured by flow cytometry. The proportions of activated CD4+ T cells (CD4+DR+, CD4+CD25+, and CD4+CD71+) and CD4+CD29+ T cells increased significantly in the peripheral blood, but the counts of these cells were significantly reduced in the synovial fluid after 2 treatment sessions in the LCP group. No significant changes were observed in the proportion of these cells in the control group. Our findings suggest that filtration LCP may cause a redistribution of activated T cells from affected joints into the circulating blood.