AIM:The immunosuppressive microenvironment plays a crucial role in T-cell immunodeficiency in multiple myeloma (MM). Overexpression of T-cell immunosuppressive receptors, including programmed death-1 (PD-1) and T-cell immunoglobulin and mucin-domain-containing-3 (Tim-3), may be related to tumor immunosuppression and poor prognosis, and the malignant bone marrow (BM) microenvironment may contribute to such immunosuppression. The purpose of this study was to analyze the distribution of PD-1+ and/or Tim-3+ T cells in different T-cell subset in patients with MM.METHODS:The expression of PD-1 and Tim-3 with exhausted (CD244+ and CD57+ ) CD3+ , CD4+ and CD8+ T cells between BM and peripheral blood (PB) from 10 patients with untreated MM was detected by multicolor flow cytometry assay.RESULTS:A significant increase in both PD-1+ CD57+ and Tim-3+ CD57+ CD3+ T cells and PD-1+ Tim-3+ CD3+ T cells was detected in PB from patients with MM compared with 10 healthy individuals (HIs), and the alteration was mostly in the CD8+ T-cell subset. Significant higher percentage of PD-1+ CD3+ T cells was found in BM compared with PB from patients with MM. The level of PD-1+ Tim-3+ CD3+ , CD4+ , and CD8+ T cells was high in BM group compared with PB. Moreover, PD-1+ CD244+ or PD-1+ CD57+ CD3+ T cells, particularly PD-1+ CD244+ and PD-1+ CD57+ CD8+ T cells were significantly higher in BM than in PB. In addition, limited dynamic detection data from three MM cases who achieved complete remission after treatment showed that the numbers of either PD-1+ or PD-1+ Tim-3+ T cells in different T-cell subsets were decreased in both BM and PB.CONCLUSION:We characterized the distribution of PD-1 and TIM-3 concurrent with exhausted CD3+ , CD4+ and CD8+ T cells between BM and PB from patients with MM. Higher numbers of PD-1+ CD244+ or PD-1+ CD57+ CD3+ T cells in BM from patients with MM may contribute to mediate the BM immunosuppressive microenvironment. Although heterogeneous alterations in Tim-3+ T cells may represent a complex immunosuppressive pattern in MM. Overall, higher levels of PD-1+ CD244+ or PD-1/Tim-3+ CD57+ CD8+ T cells may be a major reason for lower T-cell activation and T-cell immunodeficiency in MM.

Objective To investigate the association between the T cell inhibitory receptor programmed death 1 (PD-1) and T cell exhaustion status in T cells from patients with de novo acute myeloid leukemia (AML) and AML in complete remission (CR). Methods Surface expression of PD-1 and the exhaustion and immunosenescence markers CD244 and CD57 on CD3+, CD4+ and CD8+ T cells from peripheral blood samples from 20 newly diagnosed, untreated AML patients and 10 cases with AML in CR was analyzed by flow cytometry. Twenty-three healthy individuals served as control. Results A significantly higher percentage of PD-1+ cells were found for CD3+ T cells in the de novo AML group compared with healthy controls. In addition, an increased level of PD-1+CD8+ T cells, but not PD-1+CD4+, was found for CD3+ T cells in the de novo AML and AML-CR samples. A higher percentage of CD244+CD4+, CD244+CD8+, CD57+CD4+ and CD57+CD8+ T cells was found in CD3+ T cells in samples from those with de novo AML compared with those from healthy controls. Strong increased PD-1+CD244+ and PD-1+CD57+ co-expression was found for CD4+ and CD8+ T cells in the de novo AML group compared with healthy controls. Conclusions We characterized the major T cell defects, including co-expression of PD-1 and CD244, CD57-exhausted T cells in patients with de novo AML, and found a particular influence on CD8+ T cells, suggesting a poor anti-leukemia immune response in these patients.

TCRζ is a transmembrane protein, a component of the T cell receptor (TCR)/CD3 complex, and plays a crucial role in T cell activation (1,2). Previous studies showed that T cell immunodeficiency might be related to decreased expression of TCRζ . The alternatively spliced isoforms of TCRζ 3' untranslated region (3' UTR) in patients with chronic myeloid leukemia (CML) was related to change of T cell activation gene expression pattern, and may be as a novel immunological marker for the evaluation of the CML immune status (3). Recently, we found that polymorphisms/mutations in the TCR ζ 3' UTR can regulate the expression level of TCR ζ (unpublished data). However, little is known the genetic alteration of TCRζ 3' UTR in T cells from patients with CML who showed T cell immunodeficiency.

目的系统分析急性髓性白血病（AML）患者接受异基因造血干细胞移植前后γδT细胞免疫功能重建情况。方法利用反转录-多聚酶链反应（RT-PCR）方法检测12例AML患者移植前后外周血γδT细胞TRGV和TRDV各亚家族的谱系分布情况,进一步应用基因扫描分析各亚家族基因的互补决定区3（CDR3）的克隆性增殖情况。同时,采用实时荧光定量PCR方法对患者外周血γδT细胞的3个TRGV亚家族基因表达水平进行定量检测。收集8例健康成人外周血样作为对照。结果移植前后患者的TRGV亚家族谱系表达以及T细胞克隆性增殖情况无明显变化。移植前患者TRDV亚家族谱系存在明显限制性利用,部分亚家族不表达;其中TRDV3、TRDV4和TRDV8亚家族出现克隆性增殖,而移植后患者γδT细胞的TRDV亚家族谱系限制性利用情况虽有所改善,但TRDV3、TRDV4、TRDV5、TRDV6和TRDV8亚家族仍存在克隆性增殖情况。移植前后患者TRGVⅡ亚家族的表达水平均显著低于正常对照组;移植后患者的TRGV亚家族表达模式（TRGVⅠ＞TRGVⅢ＞TRGVⅡ）发生了明显改变,以TRGVⅠ亚家族的表达占优势。移植后无发生GVHD患者的TRGVⅠ亚家族的表达水平显著高于发生GVHD患者的TRGVⅠ亚家族的表达水平。结论移植后TRGVⅡ亚家族的免疫重建恢复较慢,且移植后TRGVⅠ亚家族表达水平较高提示较好的γδT细胞免疫重建。移植后克隆性增殖的γδT细胞可能是机体体内相关抗原的刺激下产生的反应性增殖性T细胞。

AIM:To explore the impact of granulocyte colony-stimulating factor (G-CSF) on acute graft-ver-sus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in a murine model and its possible mechanisms.METHODS:Male C57BL/6 (H-2b) and BALB/c (H-2d) mice were used as the allogeneic and syngeneic donor mice , respectively .Moreover , female BALB/c mice were used as recipient mice .The recipient mice were conditioned by a single dose ( 8 Gy ) of total body irradiation ( TBI ) .The recipient mice were randomly divided into 7 groups:TBI group, Syn-BMST control group, post-Syn-BMST G-CSF administration (Syn-BMST+G-CSF) group, allo-BMT control group, post-allo-BMT G-CSF administration (allo-BMT+G-CSF)group, allo-BMST control group and post-al-lo-BMST G-CSF administration (allo-BMST+G-CSF) group.The mice in control groups and G-CSF administration groups&nbsp;were subcutaneous injected with 0.1 mL normal saline (NS) and 0.1 mL NS containing 2μg G-CSF per day from 1st day, respectively.The effect of G-CSF on aGVHD was evaluated by clinical manifestations and pathological changes , as well as survival time of the mice in different groups .The serum levels of IL-2, IL-4, IFN-γand TNF-αin allo-BMST and allo-BMST+G-CSF groups were detected by ELISA at 10th day.Flow cytometry was used to analyze the immunophenotypes of splenocytes at 10th day.RESULTS:The mice in TBI group were all died for hematologic failure on 9~15 d after TBI.No effect of G-CSF on the survival of the mice underwent Syn-BMST and transplantation of single allogeneic marrow cells was observed.The mean survival days in allo-BMST group and allo-BMST+G-CSF group were (34.8 ±4.5) d and (19.8 ± 6.1) d’respectively (P<0.01).Moreover, post-transplant administration of G-CSF increased the spleen total nucleated cells count (SpTNC), NK cells subset, and DC1/DC2 ratio in the spleen with over 99%of donor chimerism rate at 10th day.No difference in the levels of serum IL-2, IL-4, IFN-γand TNF-αbetween the 2 group at 10th day was found.CON-CLUSION:The administration of G-CSF after allo-BMST significantly aggravates mouse aGVHD .The expansion of NK cells stimulated by G-CSF may be involved in the mechanism of generating alloreactivity against host cells .These results imply there may be potential risk of evoking or aggravating acute GVHD if G-CSF is administered in the early stage of clini-cal allo-HSCT.

The clonally expanded T cells identified in cancer patients that specific respond to tumor-associated antigen (TAA) have definite. We previously identified a high frequency of oligoclonal expansion of the Vβ21 T cell subfamily in the peripheral blood (PB) from BCR-ABL positive chronic myeloid leukemia (CML) and B cell acute lymphocytic leukemia (B-ALL) patients, and the TCR Vα13/Vβ21 gene modified T cell maintained anti-CML cytotoxicity. However, little is known whether there are any TCR Vβ specific for the mutant BCR-ABL protein. In this study, we analyzed the TCR β repertoire in PB from two cases with CML in blastic crisis (CML-BC) with ABL gene mutation, case 1 with B-cell resembling lymphoid blast crisis (LBC) contained single kinase domain mutation (T315I), and case 2 with AML resembling myeloid blast crisis (MBC) contained compound-mutant BCR-ABL1 (L387M and T315I). Using RT-PCR and genescan analysis, clonally expanded Vβ17 was identified in samples from both cases. The size of clonal peak in PCR products which indicate the Vβ17 CDR3 length, was all in 181 bp, this indicated that both samples contained Vβ17 clones with similar CDR3 rearrangement. Interesting, the same size clonally expanded Vβ17 T cells was also identified in case 2 at 51 days after HLA-matched sibling hematopoietic stem cell transplantation (HSCT), which is thought from donor origin rather than recipient origin. This result indicated the possibility that the expanded Vβ17 clones may respond to CML associated antigen, particularly for mutant T315I-ABL. Moreover, clonally expanded Vβ18 T cells was identified in case 1, while clonally expanded Vβ15 T cells was found in case 2, this is thought to be individual response to CML associated antigen. In conclusion, our findings showed a identical clonal TCR Vβ17 expansion in CML-BC cases with T315I-ABL mutation. Further investigation will be performed to characterize the function of Vβ17+T cell clone in T315I-ABL mutant CML-BC patients. This study was supported by grants from the NSFC (81270604 and 81400109)

Defective T cell receptor (TCR) signaling resulting in lower T cell function plays a crucial role in the pathogenesis of T cell immunodeficiency in leukemia. Previous studies have indicated that lower TCRζ levels are a common characteristic of patients with leukemia, and upregulating TCRζ could partially recover T cell function. In this study, we characterized the effect of the stimulating factor induction on the TCRζ, Zap-70, and FcɛRIγ levels, IFN-γ secretion, and the distribution and clonal expansion of TCR Vβ subfamilies in CD3(+) T cells sorted from peripheral blood from acute myeloid leukemia (AML) patients. The induction included single stimulating factor or a combination with different cytokines (IL-2, IL-7, IL-2+IL-7, IL-7+IL-12, CD3, CD3+CD28 antibody, CD3+CD28 antibody+IL-2, and CD3+CD28 antibody+IL-7) at 72 h. The results showed that increased TCRζ and Zap-70 levels with deceased FcɛRIγ in T cells after induction, and different responses to cytokine in T cell from different cases may indicate the heterogeneity of T cells and different immune statuses in different AML cases. Increased IFN-γ levels in T cells from AML patients were detected after induction in the IL-12+IL-7, CD3+CD28+IL-2, and CD3+CD28+IL-7 groups. Moreover, the number of TCR Vβ subfamily T cells expressed was increased; however, all of the TCR Vβ subfamily T cells in the AML patients could not be completely recovered after induction. In conclusion, the cytotoxicity and activation function of T cells could be enhanced after induction by different stimuli accompanied by an increase in TCRζ and Zap-70 and recovery of the TCR Vβ repertoire in AML patients.

A20 is a dual inhibitor of NF-κB activation and apoptosis in the tumor necrosis factor receptor 1 signaling pathway, and both are related to tumorigenesis. A20 is frequently inactivated by deletions and/or mutations in several B and T cell lymphoma subtypes; however, knowledge of the role of A20 in B-cell acute lymphoblastic leukemia (B-ALL) remains limited. In this study, we characterized the A20 gene expression pattern, the expression level of its upstream regulating factor MALT1, and its downstream target NF-κB in adult B-ALL.

Previous studies indicated that upregulating TCRζ partially recovers T cell function in patients with leukemia. In this study, we characterized the cytokine profile of TCRζ-transfected T cells from acute myeloid leukemia (AML) patients by Quantibody®Array Glass Chip. Firstly, the significantly lower expression of TCRζ in CD3+/TCRζ+ cells from AML patients was found. Increased secretion of IL-2, IL-8, IL-10, IL-13, IFN-γ, TNF-α, GM-CSF, growth-regulated oncogene (GRO), MIP-1b, and regulated on activation, normal T cell expressed and secreted (RANTES) could be detected in T cells from AML patients after TCRζ upregulating. We concluded that upregulating TCRζ in T cells from AML can alter the secretion profile of cytokines and chemokine which are involved in T cell proliferation and activation.

T-cell immunodeficiency is a common feature in patients with leukemia, lower activation of T cells was one of reasons. The TCR zeta chain has emerged as a key subunit of the T-cell antigen receptor, which plays a central role in the signal-transducing events leading to T cell activation. The proliferation and activation of T cells may be inducted by T cell related cytokines. In this study, we explored the change of TCR zeta gene expression and the clonality of T cells after induction with different immune cytokines, including IL-2, IL-7 or IL-12. CD3+ T cells sorted from peripheral blood of 4 cases with AML were induced with different immune cytokines, including IL-2, IL-7, IL-12, anti-CD3 and anti-CD28 antibodies in vitro. The expression levels of TCR zeta gene and related genes in T cells before and after induction were then analyzed by fluorescence quantitative RT-PCR. The distribution and clonality of TCR Vβ subfamily T cells were analyzed by RT-PCR and Genescan techniques. Increasing expression levels of TCR zeta gene and zap-70 (TCR zeta chain associated-protein) gene in CD3+T cells from AML patients were found after induction with single stimulating factor or the combination with different cytokines, while the expression of FceRIγ (TCR ζ gene complementary factor) was down-regulated. We further compared the T cell clonality in CD3+T cells from AML patients after cytokine induction, eight to 22 TCR V β subfamilies could be detected in T cells from AML cases, most of them displayed polyclonal expansion. The number of expressed TCR Vβ subfamilies was increased without the change of clonality in T cells induced by CD3+CD28+IL7. In conclusion, TCR zeta gene and its related genes could be upregulated through induction with different cytokine combination such as IL-2, IL-7 and IL-12, therefore to improve the T cell activation in patients with AML. And the main effect of cytokines might to maintain the T cell clonality and nonspecific amplification of T cell clones. Further investigation can be designed to amplify the specific anti-AML TCR Vβ clones using AML associated antigens and such cytokine combination. Disclosures Shi:National Natural Science Foundation of China (no. 81100353, 81270604), the Fundamental Research Funds for the Central Universities (No. 21611447, 21612116), And Medical Science Foundation of Guangdong Province(A2011325). : Research Funding.

Background: Based on the dynamics of antigen recognition from T cell receptor complex and T cell activation, different function of T cells may present different morphology feature, at least at molecular level. Regulatory T (Treg) cells play a critic role in regulation the onset of graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Little is known about their morphology feature which may relate to the regulating function in GVHD.Results: In this study, we detected the biophysical architectural changes of specific Treg cells subsets after allo-HSCT using biological atomic force microscopy to characterize their biological characteristics and improve our understanding of cell structure-function relationships in GVHD. There were dramatic overall shape and surface membrane deformations of the Treg cells associated with patients at GVHD onset or without GVHD. The Treg cells at GVHD onset or without GVHD could be distinguished by the morphologic parameters of morphology, membrane nanostructure, and membrane pore. These AFM parameters of Treg cells morphology differed obviously between GVHD and without GVHD. The multiple irregular microspikes could be observed on the surface of Treg cells without GVHD.Conclusions: The remarkable biological morphology changes in the cell membrane structure of Treg cells after allo-HSCT might be related to the function of Treg cells which could inhibit the occurrence and development of GVHD.

T cells from patients with acute myeloid leukemia (AML) display antigen receptor–mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain, a subunit of the TCR/CD3 complex. Up-regulate TCR signaling activation by TCR zeta recombinant vector transfection was showed to reverse IL-2 production and increased the activation in T cells from patients with chronic myeloid leukemia. This study was undertaken to explore the possibility that increased TCR zeta gene expression stimulated with immune cytokines may upregulate T cell receptor signaling activation in T cells from patients with AML. CD3 + T cells sorted from peripheral blood of 8 cases with AML were induced with different immune cytokines, including IL-2, IL-7, IL-12, anti-CD3 and anti-CD28 antibodies in vitro. The result showed that TCR zeta chain protein was significantly upregulated after stimulation with IL-2 + IL-7 at 72 hours ( p p p Disclosures Chen: National Natural Science Foundation of China (no. 81100353, 81270604), the Fundamental Research Funds for the Central Universities (No. 21611447, 21612116), And Medical Science Foundation of Guangdong Province(A2011325): Research Funding.

<正>患者,男,9岁,因外周血造血干细胞移植后18个月呼吸困难于2011年10月2日入院。该患者起初因双髋关节疼痛伴发热3周于2009年10月12日首次入院。体格检查:T 37.1℃,HR75次/min,R 20次/min,BP 99/61 mm-Hg,发育正常,轻度贫血貌,皮肤黏膜未见出血点,浅表淋巴结未及,胸骨无压痛,肝肋下未及,脾肋下可及,关节无红肿畸形。血象:WBC 5.41×109.L-1,N 50.5%,L 27.2%,M 9%,Hb 105 g/L,Plt 29×109.L-1;骨髓穿刺干抽,外周血细胞形态学诊断为急性髓系白血病,FAB分型M5;腹部B超:脾脏肋下

Aim:To explore the safety of Busulfan-Fludarabine(Bu-Flud) regimen in patients given allogeneic hematopoietic stem cell transplantation(allo-HSCT) without prophylaxis of HVOD.Methods: Clinical data of 122 patients who received allo-HSCT conditioned with Bu-Flud regimen from December 2006 to June 2012 were analyzed,including 42 patients given low dose heparin for the prevention of HVOD,and 80 patients not given the prophylaxis of HVOD.The occurrence of HVOD was observed.Results: None of the 122 patients developed HVOD(0%),including unrelated donor transplantation,HLA haploidentical transplantation,patients with a history of hepatitis B and abnormal liver function prior to transplantion.Conclusion:It is safe for the patients who received allo-HSCT conditioned with Bu-Flud regimen without the prophylaxis of HVOD.

异基因造血干细胞移植(allo-HSCT)是原发性耐药和缓解后复发白血病患者唯一有希望治愈的治疗方法.对这些患者临床上通常争取在缓解后再进行移植.然而,是否所有患者都需要进行移植前抢救性化疗?是否可以立即移植?有关这一问题的临床资料不多.我们在临床上发现有部分患者即使采用抢救性化疗方案也难以获得缓解,此时如执意追求缓解的治疗目标则有可能错失移植机会,因此,对这一类患者我们多采用在未缓解状态下进行移植的治疗策略.我们对43例未缓解白血病患者行allo-HSCT的临床资料进行了分析,现报道如下.

<正>例1,男,31岁,因移植后11个月,腹痛1周伴皮肤疱疹4 d于2004年8月22日入院。患者2003年6月确诊慢性粒细胞白血病(慢性期),并于2003年9月26日行同胞全相合外周血干细胞移植。

>异基因造血干细胞移植(allo-HSCT)后非感染性肺部并发症是移植相关死亡的一个重要原因,包括特发性肺炎综合征(IPS)、弥漫性肺泡出血症(DAH)和药物相关肺毒性反应等,而allo-HSCT后肉芽肿性肺炎极其罕见。我们报告1例X线表现酷似』血行播散性粟粒性肺结核、经肺活检病理诊断为allo-HSCT后肉芽肿性肺炎。病例资料患者,男,40岁,于2006年12月在外院骨髓检查诊断为"急性淋巴细胞白血病(ALL)",免疫表型CD19、CD22、CD34、HLA-DR阳性;FISH检查BCR-ABL融合基因阴性。

Despite the success of imatinib and other tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable, and a number of CML patients die due to Abl mutation-related drug resistance and blast crisis. The aim of this study was to evaluate proliferation inhibition and apoptosis induction by down-regulating PPP2R5C gene expression in the imatinib-sensitive and imatinib-resistant CML cell lines K562, K562R (imatinib resistant without an Abl gene mutation), 32D-Bcr-Abl WT (imatinib-sensitive murine CML cell line with a wild type Abl gene) and 32D-Bcr-Abl T315I (imatinib resistant with a T315I Abl gene mutation) and primary cells from CML patients by RNA interference. PPP2R5C siRNAs numbered 799 and 991 were obtained by chemosynthesis. Non-silencing siRNA scrambled control (SC)-treated, mock-transfected, and untreated cells were used as controls. The PPP2R5C mRNA and protein expression levels in treated CML cells were analyzed by quantitative real-time PCR and Western blotting, and in vitro cell proliferation was assayed with the cell counting kit-8 method. The morphology and percentage of apoptosis were revealed by Hoechst 33258 staining and flow cytometry (FCM). The results demonstrated that both siRNAs had the best silencing results after nucleofection in all four cell lines and primary cells. A reduction in PPP2R5C mRNA and protein levels was observed in the treated cells. The proliferation rate of the PPP2R5C -siRNA-treated CML cell lines was significantly decreased at 72 h, and apoptosis was significantly increased. Significantly higher proliferation inhibition and apoptosis induction were found in K562R cells treated with PPP2R5C -siRNA799 than K562 cells. In conclusion, the suppression of PPP2R5C by RNA interference could inhibit proliferation and effectively induce apoptosis in CML cells that were either imatinib sensitive or resistant. Down-regulating PPP2R5C gene expression might be considered as a new therapeutic target strategy for CML, particularly for imatinib-resistant CML.

The aim of this study was to detect the expression level of eIF4E gene in patients with non-treated, remission and non-remission/relapse acute myeloid leukemia (AML), and other non-malignant haematologic diseases so as to analyze and reveal the relationship of eIF4E gene expression with AML progression. SYBR Green I RT-PCR was used to assay the expression level of eIF4E mRNA extracted from bone marrow mononuclear cells in 30 patients with AML (6 in M2, 5 in M3, 8 in M4, 10 in M5, 1 in M6) and 20 patients with non-malignant hematologic diseases. The β2-microglubin(β2M) was used as internal reference and the formula 2(-ΔCt)×100% was applied to calculate the expression level of eIF4E gene. The results showed that the eIF4E expression level (7.098 ± 5.544)% in patients with non-treated and non-remitted/relapsed AML was significantly higher than that in patients with remission (0.964 ± 0.312)% (P < 0.01) and non-malignant hematologic diseases (0.248 ± 0.163)% (P < 0.01). There was no difference between latter two group patients, even though the expression level of eIF4E gene in patients with M4 and M5 was higher. As compared with non-malignant hematologic diseases, the expression level of eIF4E gene of patients with remission patients showed no significant difference. It is concluded that the over-expression of eIF4E gene has been found in patients with AML, and its level obviously decreases along with remission of disease, thus the eIF4E gene may be a surveillance parameter for disease progression.